Production and Characterization of Extremolytes from...

Production and Characterization of Extremolytes from

Indigenous Radio-resistant Microorganisms and their

Evaluation for Potential Biotechnological Applications

By

Wasim Sajjad

Department of Microbiology

Quaid-i-Azam University

Islamabad, Pakistan

2017

Production and Characterization of Extremolytes from

Indigenous Radio-resistant Microorganisms and their

Evaluation for Potential Biotechnological Applications

A thesis

Submitted in the Partial Fulfillment of the

Requirements for the Degree of

DOCTOR OF PHILOSOPHY

IN

MICROBIOLOGY

By

Wasim Sajjad

Department of Microbiology

Quaid-i-Azam University

Islamabad, Pakistan

2017

DECLARATION

The material contained in this thesis is my original work and I have not presented

any part of this thesis/work elsewhere for any other degree.

Wasim Sajjad

DEDICATED

TO

My Ammi, Abbu &

Late Grand Father

CERTIFICATE

This thesis, submitted by Mr. Wasim Sajjad is accepted in its present form by the

Department of Microbiology, Faculty of Biological Sciences, Quaid-i-Azam

University, Islamabad as satisfying the thesis requirement for the degree of Doctor

of Philosophy (PhD) in Microbiology.

Examiner: _______________________________

Examiner: _____________________________

Supervisor: ______________________________

Dr. Aamer Ali Shah

Chairperson: ___________________________

Dr. Rani Faryal

Dated:

CONTENTS

S. No Chapter no Title Page no

1 List of Abbreviations i

2 List of Tables iii

3 List of Figures iv

4 Acknowledgements vi

5 Abstract viii

6 Chapter 1: Introduction 1

7 Chapter 2: Review of Literature 14

8

Chapter 3:

Isolation and Characterization of Ultra Violet

Rays (UVR) Resistant Bacteria from Desert Soil

Samples of Pakistan

78

9 Chapter 4: In-Vitro Cytotoxic and Antioxidant Activities of

Extremolytes from Radio-Resistant Bacteria

112

10 Chapter 5: Radio-protective and antioxidative activities of

astaxanthin from newly isolated radio-resistant

bacterium Deinococcus sp. strain WMA-LM9

132

11 Chapter 6: Ectoine: a Compatible Solute in

Radiohalophilic Stenotrophomonas sp. WMA

LM19

Strain to Prevent Ultraviolet-Induced Protein

Damage

165

12 Future prospectives 196

13 I Appendices 197

II Turnitin Origiility Report 216

III Published Research Article 219

List of Abbreviations

A Adenosine

ATP Adenosine-5’-triphosphate

AUG 30 Augmentin 30µg

BSA Bovine serum albumin

BLAST Basic Local Alignment Search Tool

BLASTN BLAST search using a nucleotide query

bp Base pairs

C Cytosine

°C Degree celsius

CAT Catalase

CTX 5 Cefotaxime 5µg

DNA Deoxyribonucleic acid

dNTP Deoxynucleoside triphosphate

DPPH 2,2-Diphenyl -1-picrylhydrazyl

DTNB 5,5’-Dithio-bis 2-nitrobenzoic acid

DEPPD N,N- Diethylparaphenyldiamine

DNPH 2, 4- Dinitrophenyl hydrazine

DNS Dinitrosalicylic acid

EDTA Ethylenediaminetetraacetic acid

et al. et alii/alia, and others

e.g. Exempli gratia, for example

Fe Iron

Fig. Figure

FTIR Fourier transform infrared spectroscopy

GR Glutathione reductase

G Guanine

HPLC High-performance liquid chromatography

i.e. id est, that is

IMI 10 Imipenem 10µg

LCMS Liquid chromatography mass spectroscopy

MDA Malondialdehyde

MDR Multidrug resistant

NBT Nitrobluetetrazolium

NMR Nuclear magnetic resosnance

NaCl Sodium chloride

NCBI National Center for Biotechnology Information

OD Optical density/ Absorbance

PCR Polymerase chain reaction

pH Power of Hydrogen

POD Peroxidase

rRNA Ribosomal ribonucleic acid

Rf Retardation factor

ROS Reactive oxygen species

rpm Rotation per minute

SDS Sodium dodecyl sulfate

SDS PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis

SOD Superoxide dismutase

SXT 25 Trimethoprim-sulphamethoxazole 25µg

T Thiamine

TBA Thiobarbituric acid

TCA Trichloroacetic acid

TGC 15 Tigecycline 15µg

TGY Tryptone glucose yeast extract

TLC Thin layer chromatography

TPP Tripolyphosphate

U Uracil

List of Tables

No. Title Page No.

2.1 Ultraviolet radiation (UVR) resistance in variety of radio-resistant micro-

organisms.

21

2.2 Ultraviolet radiation (UVR)-inductive microbial metabolic products and

their therapeutic implications.

22

3.1 Shows culture code, Sampling site, Colony morphology and Gram

staining of UVR isolates from desert soil.

92

3.2 Biochemical and physiological characteristics of UVR isolates from Lakki

Marwat and Bahawalpur desert soil.

94

3.3 16S rRNA sequence homologues, closest related species, %

survivability, gene bank accession number and query coverage of

Ultraviolet radiation (UV subtype –B) resistant isolates from desert

samples.

95

3.4 Effect of the metal ions (in ppm) on growth of UVR resistant selected

bacteria from desert samples on TGY agar plates.

98

4.1 Anti-microbial activity of partially purified extracts from UV resistant

microbes.

124

List of Figures

No. Title Page

No.

2.1 Diagrammatic representation of origin of different types of radiation and

their effects on extremophiles

20

2.2 Carotenoid biosynthetic pathway showing biosynthetic enzymes and

intermediate product with Deinoxanthin as the final product.

28

2.3 Different pathways for DNA double-strand break repair 34

2.4 Two stages of genome reconstitution in Deinococcus radiodurans 35

2.5 Main pathways involved in the processing of DNA ends. 39

2.6 Screening strategy for bio-active compounds from extremophiles. 43

2.7 Structure of Scytonemin, a novel dimeric molecule (molec. wt. 544) of

indolic and phenolic subunits

46

2.8 Production and hypothetical pathway in which scytonemin inhibits

PLK1

50

3.1 Metal analysis (in ppm) of soil samples collected from deserts 93

3.2 Survivability of total UVR resistant isolates from desert soil at varying UV-

B exposure.

93

3.3 Phylogenetic analysis UV resistant bacterial strains by maximum likelihood

method

96

3.4 Effect of UVB on total cell protein content in mg/ml. 97

3.5 Protein oxidation to quantify carbonylated protein and lipid peroxidation

assay for TBARS in UV treated isolates from desert soil.

99

4.1 The fluorescent quenching spectrum of methanolic extracts 1mg/ml from

UVR bacteria

120

4.2 The cytotoxic effect of partially purified fractionated extracts on HeLa cell

line by using MTT assay.

122

4.3 Comparative analysis of IC50 values of different fractionated extracts of

UV resistant microbes.

123

4.4 Anti-microbial assay of partially purified extracts from radio resistant

bacteria.

123

5.1 Astaxanthin synthesis from isophorone, cis-3-methyl-2-penten-4-yn1-ol

and a symmetrical C10-dialdehyde has been discovered and is used

commercially.

135

5.2 Structure of deinoxanthin from Dienococcus radiodurans. 136

5.3 Structure of phenolcs and flavonoids 136

5.4 Survivability of strain WMA-LM9 from desert soil at varying UV-B

exposure.

145

5.5 Comparison of intracellular protein carbonylation level between radio-

resistant Deinococcus sp. strain WMA-LM9 and E.coli (10536)

148

5.6A Flash chromatography of the extract using different solvent system of

Hexane Dichloromethane water and methanol.

149

5.6B Different fractions collected upon flash chromatography for bioassay

guided fractionation.

149

5.7 HPLC chromatogram/positive ESI-MS spectrum of carotenoid extract 150

5.8 1H NMR spectra of purified compound LM9F1 150

5.9 13C NMR spectra of purified compound LM9F1. 151

5.10 Astaxanthin chemical structure from NMR peaks using ChemDraw

software.

151

https://en.wikipedia.org/wiki/Isophorone



5.11 Anti-oxidant activity of carotenoid extracted from strain WMALM9. 153

5.12 Inhibitory effect of carotenoid from strain WMA-LM9 of different protein

oxidation in-vitro.

154

5.13 Role of carotenoids in prevention of oxidative damage to pUC18 plasmid

DNA after exposure to oxidative agents.

155

6.1 Structure of some compatible solutes from extremophiles 167

6.2 Survivability of strain WMA-LM19 from desert soil at varying UVB

exposure.

176

6.3 Neighbor joining phylogenetic tree based on 16S rRNA gene sequence

analysis, showing the position of isolate WMA-LM19

177

6.4 HPLC chromatogram/positive of the polar extract for WMA LM19 177

6.5 ESI-MS spectrum of purified ectoine extract 179

6.6 1H NMR spectra of purified compound LM19F2. 179

6.7 13C NMR spectra of purified compound LM19F2. 180

6.8 Chemical structure of ectoine from NMR spectroscopy. 180

6.9 DPPH and OH Free radical scavenging assay using ascorbic acid as positive

control

183

6.10 Assay of reducing power and Fe chelation of ectoine using ascorbic acid

and EDTA as positive control

183

6.11 Protein oxidation and lipid peroxidation inhibition activity offered by

ectoine

184

6.12 Membrane damage preventing assay by ectoine using human RBCs and

Lecithin as positive control

185

6.13 Analysis of protein protection offered by ectoine on SDS-PAGE. 186

Acknowledgements

Praise to ALMIGHTY ALLAH, whose blessings enabled me to achieve my goals.

Tremendous praise for the Holy Prophet Hazrat Muhammad (Peace Be upon Him), who is

forever a torch of guidance for the knowledge seekers and humanity as a whole.

In front of you lies the result of my PhD research at the Quaid-i-Azam University Islamabad,

Pakistan. A period in which I have learned so many things: not only about radio-resistance

and bacteria, but even more about ourselves and each other. This research proposal

represents that period, our first –humble- steps on the stage of science. It was a time full of

surprises: the uneasy but cosy introduction, the path from our starting point to the radio-

resistant microbes, and above all, the taste of a genuine scientific research project.

All of this would not have been possible without the help of numerous smart people. I would

therefore like to thank the following people sincerely for the time and effort they put into our

project: Prof. Dr. Fariha Hasan, Dr. Javid Iqbal Dasti, Dr. Samiullah Khan, and. Dr. Arshad

Jhangir for the extremely useful suggestions and guidelines during our research work.

I do not find enough words to express my heartfelt gratitude for Dr. Kerry McPhail, Associate

Professor, Department of Pharmaceutical Sciences Oregon State University, USA. She

supervised me during my studies in Oregon State University during International Research

Support Initiative Program (IRSIP). This experience would not have been as valuable without

the guidance, support and inspiration provided by her. I am impressed by her scientific

thinking and politeness. I am also thankful to Celick, David, Richard and Nazir Muhammad,

Postdoctoral Research Associates at Department of Pharmaceutical Sciences, for their care

and immense help during my entire stay at Oregon State University.

I would also like to thank Higher Education Commission, Pakistan, for providing me grant

under the Project “IRSIP” to work in multicultural dynamic environment with internationally

recognized scientists.

A non-payable debt to my loving Ammi (Mushtari BiBi), Abbu (Muhammad Pervaiz),

brothers (Shahid Iqbal, Fawad Alam, Muhammad Abbas and Waqas Ahmad) and sisters for

bearing all the ups and downs of my research, motivating me for higher studies, sharing my

burden and making sure that I sailed through smoothly. Completion of this work would not

have been possible without the unconditional support and encouragement of my loving family

members. I would like to acknowledge my Uncle Iqbal Ahmad Tajik and cousins Irshad Alam.

Mukhtiar Alam (My school mentor), M. Idrees, Shahab Ahmad for their support.

I extend my greatest depth of loving thanks to all my friends and laboratory mates (seniors

and juniors) especially Muhammad Rafiq, Muhammad Irfan, Imran Khan, Sahib Zada, Abdul

Haleem, Arshad, Abdul Haq, Ghufran, Matiullah, Waqas, Wasim Hukam, Manzoor, Umair,

Akhtar Nadhman, Faiz, Saad, Shahid, Barkat, Faiz, Shama, Maliha, Nazia Anum and

Mahvish, Adnan Khan, Asim Shah, Adil Nawaz, Izhar for their help throughout my study.

Special thanks to a cluster of people, always with me in my ups and down during PhD. These

special people are Manzoor Ahmad, Salman Khan, Sunniya Ilyas, Sundas Qadir, Maryam

Zeest Hanif and Misbah, MSc students Anum, Saiqa and Shaista. I especially want to express

a heartfelt gratitude to Fariha Aman Nazir, whose sincere encouragement and

understanding attitude are assets worth cherishing forever.

There is, however, one person who stands out among all these people: Dr. Aamer Ali Shah,

my mentor. I would like to thank him for the time he spent at our meetings and assessment

evenings, for the input he gave me during the process and especially for the fact that he,

despite my project being way out of his research area, has always helped me throughout the

entire period.

Finally, I express my gratitude and apology to all those who provided me the opportunity to

achieve my endeavors but I missed to mention them personally.

Wasim Sajjad

Summary

Radio-resistant microorganisms have the puzzling ability to withstand with high range of

extreme conditions including high doses of ultraviolet radiation and desiccation. Radiation in

general has the ability to cause extensive damage to various cell components such as nucleic

acids, proteins and membranes. Nevertheless, metabolic products such as extremolytes

facilitate the survival of microorganisms in such extreme conditions. These extremolytes are

not essential for the organism in terms of its normal growth. However, it provides a

competitive edge to the organism in terms of survivability against stress and assist in

regulating reproductive responses. Thus far, the use of radio-resistant extremophiles as a

source of natural products has largely been ignored in modern biotechnology. The current

study focused on the isolation of radioresistant bacteria from extreme environment. The

phylogenetic diversity of ultra-violet (UV) resistant bacteria from deserts soil, was

investigated by culture and molecular based analysis. The bacterial strains were observed for

their tolerance to UV doses, salt concentration, and heavy metals. The effect of UV radiation

on cellular protein and lipids was also investigated. The secondary intracellular bio-active

compounds extracted from these radio resistant bacteria, were assayed for cytotoxic,

antioxidant and antibacterial activities in order to evaluate their potential to be considered

as therapeutic and radio protective agents. Based on higher UV resistance and production of

higher amount of UV absorbing compounds, two strains designated as WMA-LM9 and WMA-

LM19 were selected for further studies. Two different compounds were extracted in a

solvent system and purified by highperformance liquid chromatography (HPLC) on a C18

analytical column. The compounds were characterized as mono-esterified astaxanthin and

ectoine by 1H, 13C nuclear magnetic resonance (NMR) and mass spectrometry (MS). These

pure compounds were tested for antioxidant activity, total flavonoids and phenolic content,

radio protective potential in correlation to the prevention of protein oxidation and DNA

strand breaks in-vitro. The antioxidant and radio-protective properties of the pure

compounds were evaluated by hydroxyl scavenging, 2,2-diphenyl-1picrylhydrazyl (DPPH)

reducing, lipid peroxidation inhibition assays and protein radio-protection assays by sodium

dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis.

A total of 09 UV resistant bacteria were isolated and identified through biochemical tests and

16S rRNA gene sequencing. Based on the results obtained, bacterial strains were assigned

four phyla: Firmicutes, Proteobacteria, DeinococcusThermus and Actinobacteria. High UV

survivability was observed in case of genus Deinococcus followed by Firmicutes. The bacteria

were found to grow at wide temperature and pH range, resistant to high salt concentration

as well as various metal ions. The bacterial strains exhibited minor damages to protein and

lipids as a result of exposure to UV radiation as compared to Escherichia coli (ATCC 10536).

The purified carotenoid pigment, Astaxanthin from Deinococcus sp. strain WMA-LM9, also

showed a higher inhibitory action against oxidative damage to collagen, elastin and bovine

serum albumin than a standard compound, β-carotene. It also inhibited breaks to DNA

strands, as indicated by the results of the DNA damage prevention assay. Another

compatible solute, Ectoine, purified from strain WMA-LM19, exhibited strong Fe2+ chelation

in comparison to EDTA (38.58± 0.84%). The OH- radical scavenging efficiency of ectoine

(53.68 ± 0.48%) was estimated in terms of % inhibition of deoxy D-ribose degradation in a

non-site-specific assay using a concentration of 10.0 μg/ml. Maximum reduction in DPPH

(~60.45 ± 1.185%) was observed at 10 μg/ml ectoine concentration. Ectoine effectively

inhibited oxidative damage to proteins and lipids in comparison to the standard ascorbic

acid. Furthermore, a high level of ectoine-mediated protection of bovine serum albumin

against ionizing radiation (1500-2000 Jm-2) was observed, as indicated by SDS-PAGE analysis.

The results indicated that these radio-resistant microbes harbor a sophisticated phenotypic

character and molecular repair mechanisms that can prolong their survival in extreme

radiations. The current research work concluded that the radio-resistant strains from

extreme environment have great potential to produce potent metabolites with a wide range

of antioxidant and cytotoxic activities. The extremolytes showed a good radio-protective

effects against radiation-mediated cell damage, and were considered as a potential mitigator

to overcome oxidative damages in extreme environment. These extremolytes can potentially

be harnessed as radio-protective drugs and can also be used as sunscreen to block UV

radiations.

1. Introduction

This environment manifests itself as any of two versions; moderate or extreme.

Environments with a limited number of nutrients and water supply, such as deserts, or

others with highly saline waters such as lakes with high salinity constitute examples of such

extreme environments. The term extremophile was put to paper roughly a quarter century

ago (MacElroy, 1974). Extremophiles are the microbes that can survive in harsh or extreme

condition of pressure, salinity, temperature or in concentrations of other chemicals that

would kill other microbes or creature. Although a varied number of definitions for the term

exist, they all refer to organisms that can thrive in such environments that are otherwise

uninhabitable to other organisms. Twenty years ago extremophiles were considered cheap

and useless organisms and were explored by very few scientists, but nowadays they are used

as a source of novel compounds and extremozymes (Schulze-Makuch et al.,2015).

Radio-resistant microbes constitute a group of extremophiles that have the ability to

survive environments with a high level of radiation (Singh et al.,2013). Radio-resistant

microbes too, in accordance with the definition of extremophiles, thrive under conditions

which are otherwise hostile to other organisms; in this case, the extreme conditions are high

levels of radiation. The phenomenon of tolerance to radiation is known as radio-resistance.

All three domains of life, namely Bacteria, Archaea and Eukaryotes can exist as radio-

resistant extremophiles. These radioresistant bacteria carry reactive oxygen species (ROS)-

scavenging enzymes and exhibit diverse metabolic properties (Yu et al.,2015). The search for

radio-resistant species and mechanisms resulting in higher, radio-resistance in certain

several species, all of which are competent to withstand huge amounts of radiation.

Although the earliest list included organisms from three distinct domains (Farmilo et

al.,1973), including the following four species.

Chroococcidiopsis sp.: This is the most primitive cyanobacteria. It can survive in harsh

environmental conditions, both in high and low temperature. It can also survive in high

salinity and ionizing radiation. They have the potential to withstand gamma radiation as high

as 15 kGy without undergoing mutation (Billi et al., 2000).

Deinococcus radiodurans: One of the best known radio-resistant specie. This bacterium has

the unique ability to repair damaged DNA and is recorded as the maximum resistant life

form (as of May 2012) in the Guiness Book of Records. They possess two primary factors to

tolerate lethal radiations i.e. carotenoids and efficient DNA repair pathways.

Rubrobacter radiotolerans: This is a relatively unknown bacterium, resist UV rays by two

different protecting mechanisms. This bacterium partially known to have superoxide

dismutase and carotenoids producing ability to survive in extreme environments (Ferreira et

al.,1999; Terato et al.,2011).

Thermococcus gammatolerans: This archaea clustered in order of Thermococcales and

without apparent lethality, specie can withstand a dose of 3 kGy, but the exposure to a

higher dose of radiations can only slightly reducing its viability (Tapias et al.,2009).

Radiations are the energy in the form of electromagnetic waves. Elements with

unstable isotopes decay spontaneously and can ultimately form other toxic elements. They

radiate radiations (ionizing or may non-ionizing) during this process. These radiations have a

great potential to eject electrons from the outermost orbital of the atoms. All this results in

the formation different ions (with having great ionization power) which can charge the other

molecules (Kumar et al., 2010). UV radiation (UVR), can form different dimers in DNA by

changing its molecular bonding and structure between DNA strands. Radiation in general has

the ability to cause extensive, complex and lethal damage to various cell components such

as nucleic acids, proteins and membranes. Free radicals produced by such radiation as it

passes through the cell, directly or indirectly, are responsible for the damage caused to the

cellular components (Santos et al., 2013). These can undergo different metabolic damages

due to superoxide’s generation if exposed to the right amount of radiation, such as

ultraviolet (UV) rays, Gamma Rays or X-rays, owing to the induction and the consequent

presence of a variety of cytotoxic and mutagenic DNA lesions. Examples of damage at the

DNA level, brought on by radiation be composed of single and double-strand breaks,

impaired purine and pyrimidine bases, subtraction of bases and cross-linkage formation

between adjacent protein molecules and DNA (Upadhyay et al., 2005). The most extreme

effect of irradiation is protein oxidation and lipid peroxidation in biological membranes,

especially mitochondrial membranes (Tian et al., 2009).

The two different mechanisms that render organisms radio-resistance:

protection and repair. In the first case the antioxidant defense system protects the cell vital

constituents from direct or indirect damage by ionizing radiation. In the second case DNA

damage is followed by well developed a repair mechanism which mends DNA lesions quickly

and efficiently. Examples of this mechanism exist in several distinct organisms. Earth

molecules receives a numerous amount of radiation continuously marked from the Earth’s

crust and space. These organisms need to refine a huge radio-resistant, but on the hot and

dry deserts the dose is significantly above the average. The survival of radio-resistant

microbes to tolerate radiation is based on the development of sophisticated radio-resistant

mechanisms. In biology and medicine, the consequences of proteome damage on the

maintenance of life are highly underestimated (Krisko and Radman, 2013). Premature aging

and cancer are the consequences of protein and lipid modifications brought on by stress or

other environmental factors. UVR resistant microbes enjoy a largely effective protection

against proteome damage. However, this protection does not extend to their genome. The

well protected proteome not only counters the effects of extreme radiation damage, by

facilitating molecular repair but its production for extremolytes play an central role in the

survival.

In light of the above findings, various therapeutic agents have been studied that may

reverse the effects of radiation injury in the target organism. The effects of these agents are

as follows:

1. Reduction in free radical induced oxidative stress

2. Repair of oxidative damage and recovery enhancing

3. Modifications in the immune system (biological-response modifiers) (Ghose

1983; Nair et al., 2004)

Nevertheless, metabolic natural products such as extremozymes and extremolytes

facilitate the survival of microorganisms (Gabani and Singh, 2013). Thus far, the use of radio-

resistant extremophiles as a source of natural products that may be used as drugs has not

been studied enough. However, from the limited research that has been conducted on

products obtained from radio-resistant microbes, we now know that in addition to being

used as tools across a range of potencies, radio-resistant possess a range of beneficial

properties such as: lowering cholesterol, immunosuprresion, antiparasitic abilities,

herbicidal, used as diagnostic tools, antibacterial, antifungal properties and antiviral

activities.

Extremolytes are secondary organic molecules with low molecular weight possessing

a range of frequently very potent biological activities. These metabolites are not essential for

the organism in terms of its normal growth, development or reproduction. They do,

however, give a competitive edge to the organism to help in its survival against other inter-

species competitors, provide protection against stress and assist in regulating reproductive

responses (Mandal and Rath, 2015). Success has been achieved in the search for radio-

resistant extremophiles that yield extremolytes which have potential as therapeutic and

protective agents. So far, the isolation of several UVR-protective compounds obtained from

UVR-resistant extremophiles has been successful, including: ectoine a UV protectant (Beblo-

Vranesevic et al., 2017), scytonemine, mycosporine-like amino acids (MAAs),

bacterioruberin, pannarin, melanin and many others (Ferroni et al., 2010). Some of these

compounds have shown to be instrumental in the treatment or prevention of a myriad of

diseases, many of which had no known cure prior to the discovery of these compounds.

These extremolytes obtained from various extremophiles including cyanobacteria,

act as antioxidants and have offered protection against oxidative stress in human cells,

proteins and lipids (Rastogi et al., 2009; Singh et al., 2013; WaditeeSirisattha et al., 2016).

Manganese-metabolite mixtures, yielded from the environmentally resilient D. radiodurans

species, display excellent antioxidant properties that have been studied with effective role

in offering protection to different cell lines against oxidative damage. These organisms, thus

appear promising in delaying the process of aging, working against other age-related

diseases as well as preventing cancer. The challenge that remains in the near future is to

utilize these extremophilic antioxidants or extremolytes against aging and cancer-related

DNA as well as protein modifications (Slade and Radman, 2011).

The attention of several industrial sectors, especially the cosmetics and

pharmaceutical industries, has turned to these extremolytes because of the association that

exists between their biological activities and the various health benefits offered (chronic

disease prevention, activity against carcinogenic agents etc.). Although their industrial

significance has been significantly exposed, much light remains to be shown on their

significance as therapeutics. The unique metabolism of these microbes, in addition to being

responsible for great diversity amongst the microbes, also facilitates the ability of these

microbes to survive in harsh environments (Ferrer et al., 2007; Gostincar et al., 2010). The

demand for carotenoids in the food, feed and pharmaceutical industries is ever-growing and

can be met by employing microorganisms using biotechnology (Galano et al., 2010).

Radio-resistant extremophiles hold great importance as biological materials and as

an enzyme source in regards to their biotechnological applications globally, in addition to

their role in structural and biochemical biodiversity (Jane and Alan, 2004; Podar and

Reysenbach, 2006). The discovery of radio-resistant extremophiles and their associated bio

products and their industrial use show great promise in advancing mankind’s advances. The

development of innovative or better drugs has been facilitated by an increased

understanding of the roles that these microorganisms play in these processes, coupled with

our ability to manipulate their activities using molecular biology techniques.

Genome stability is known to be one of the important factors for UV resistant

bacteria to survive in extreme environments, but in response to sever UV radiations they are

also capable of producing different primary and secondary metabolites (Makarova et al.,

2001; Bagwell et al., 2008; Goosen and Moolenaar, 2008; Singh and Gabani, 2011). The

secondary metabolites are produced for their own defense in these extreme environments

and are yet to be investigated for mechanistic intervention (Carreto and Carignan, 2011).

The recent biotechnological applications may aid to pinpoint the adapted microbial

approach of self-engineering to oppose under extreme UVR. Therefore, it is necessary to

determine the diversity of UVR- resistant microorganism from the natural environment on

earth to explore the physiological mechanism adapted by microorganism to withstand

extreme UVR. It could be justified that the hot and dry environment with limited water

availability and nutrients would divulge a diversity of UVR resistant extremophiles with

regulated proteins/enzymes. Therefore, we pursue to segregate and characterize the diverse

variety of microorganisms resistant to UVR isolated different desert soil of Pakistan. The

protection based radio-resistant bacteria that were found in a dry and hot desert of Pakistan

was mainly focused in this report. By comparison with Deinococcus radiodurans that is

considered as model specie in radio-resistance studies, the question raised was whether all

of these UVR resistant bacteria contain any carotenoids and compatible solutes like

structure that are strong anti-oxidant found in extremophiles. Modern biotechnological

techniques could play a vital role in induction or activation of biosynthesis of bioactive

compounds cellular metabolites or radio responsive pigments to provide an opportunity for

the other organism to survive under radiation rich environment. Genes that produce the

metabolites that can be protective against radiation could be induced by growing UV

resistant organism in the presence of UV-light (Singh and Gabbani, 2011).

The hot and dry environment with high UV radiation has largely been ignored by

scientists in Pakistan. To the best of our knowledge, there is no report on production of

extremolytes from radio-resistant microbes in Pakistan. The research work presented in this

thesis includes 6 chapters (including this introduction and literature review as chapter 1 and

2). The second chapter describes the diversity, and adaptation mechanisms of radio-

resistant bacteria in high radiation as mentioned in separate sections of literature review

(see chapter 2). The current study focuses: (a) Isolation and characterization of UV resistant

bacteria from the extreme environment of Pakistan. Effect of UVB on cell proteins and lipids

was also investigated in comparison to E. coli, (UV sensitive ATCC strain 10536) that has

been subjected to UV radiations (see chapter 3). (b) Extraction of extremolytes and its partial

purification which is then subjected to various bio-assays such as measurement of cytotoxic

and antioxidant activities. The main focus here was to screen out the potent UV absorbing

and radio-protective compounds (see chapter 4). The main subjects were Deinococcus sp.

strain WMA-LM9 and Stenotrophomonas sp. strain WMALM19, isolated from desert soil

samples of Pakistan and characterized via 16S rRNA sequencing (c) The fifth chapter (paper

3) deals with the extraction and purification of astaxanthin from Deinococcus sp strain

WMA-LM9. A purified astaxanthin was quantified and assayed for different anti-oxidative

and radio-protective activities. (d) We further selected another radio-halophillic strain

Stenotrophmonas sp. WMALM19. A compatible solute, ectoine was extracted from this

radio-halophillic bacterium Stenotrophomonas sp. WMA-LM19. It was purified and its

potential role as radio-protective and anti-oxidative agent in vitro has been discussed in

chapter 6 (paper 4). Deinococcus and Stenotrophomonas species possess very strong

molecular repair mechanisms which have been investigated previously by many scientists.

The protection mechanism against free radical provides resistance to microbe by producing

antioxidant carotenoids and compatible solutes ectoine that offers protection against UVB.

This study will come up with implications such as the discovery of a unique

environment for UVR microbes in the context of Pakistan, source of novel extremozymes

and extremolytes as a source of therapeutic agents with potential industrial applications.

These results will also open the exiting standpoints on investigating bacterial lenience to

desiccation, radiation and survey in the deserts. This report tries to put one of these

mechanisms in the spotlight that is the production of different compounds with radio-

protective and anti-oxidative activities. These compounds can act as a primary defense

mechanism and play positive part in the survival of radio-resistant microbes in extreme UV

radiations and thus can prolong their survivability. The experimental design and the

outcome of this research proposal will contribute information on the radio-resistance of the

specified microorganisms.

1.1. Research Hypothesis

The UV resistant organisms produce carotenoids and UV absorbing compounds that

protect the bio-molecules from excessive damage due to UV exposure and extend their

survival. These compounds however act as superoxide quencher that can detoxify the

unstable toxic molecules which leads to tissue protein and lipid damage. The extremolytes or

the bioactive compounds produced by radioresistant microbes can prolong the survival of

these extremophilc bacteria in a high UV exposure. These compounds can act as melanin in

human that prevent the entry of UV radiation to body and thus acting as a primary line of

defense in UV exposure to prevent the cells from excessive damages. Based on our research

hypothesis, we want to investigate anti-oxidative strength of extremolytes and carotenoids

produced during cell stress.

1.2. Research Questions

2. Do UVR microbes possess carotenoids or other bio-active compounds?

3. How they help in cell survival under extreme stress conditions like high UV dose,

desiccation and high salt concentration?

4. Could we consider these bio-active compounds as potential candidates for biomedical

applications?

1.3. Aim and Objectives of the Study Aim

The aim of this study was to isolate radio-resistant microorganisms from soil as well as to

investigate the role of extremolytes in their survival to UV radiation.

Objectives

The aim has been accomplished through the following objectives.

1. Isolation and characterization of radio-resistant bacterias from the desert soil.

2. Extraction and characterization of the UV absorbing compounds that might be involved in

their resistance to UV radiation.

3. Chemical and structural analysis of these metabolites using LC-MS/MS, proton and carbon

NMR.

4. Bio-assays of these metabolites in order to confirm their role in cellular protection under

UV radiation stress.

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2. Literature Review

2.1. Extremophiles

Diverse physical conditions give rise to diverse environment, where living

organisms thrive. This environment can be either moderate or extreme. Temperature

range of 20-40°C, pH near to neutral, atmospheric pressure of 1 atmospheric and

sufficient level of water, nutrients and salts is normally considered as moderate

environment. On contarary to moderate environment, there lies hostile and unusual

environment i.e. hot springs, deserts, acidic and saline environment. Microbes that have

the ability to persist in geochemically extreme ecological surroundings are called

extremophiles. They adapt theirselves to these harsh conditions. Extremophiles can even

be isolated from stratosphere and troposphere where temperature range of -20 to -40°C

exists (Shivaji et al., 2004). Similarly, thermophiles, acidophiles and alkaliphiles possess

the ability to survive and replicate at relatively hot, acidic and alkaline conditions.

Barophiles require high pressure for their sustainibility and can utilize low and high

organic matter as a substrate. Radio-resistant microbes can with stand an exposure to

intense radiations and chemical mutagens (Satyanarayana et al., 2005). Members of

extremophiles are prevalent in different genetic linages of all three domains i.e. eucarya,

archea and bacteria (Averhoff and Müller 2010).

2.2. Ionizing Radiations

Energy in the form of electromagnetic waves is called radiation. The isotopes of

some elements are not stable due to which they deteriorate rapidly. In the process of

decay, they are transformed into another element. During conversion they discharge

ionizing radiation. These ionizing radiation possess the ability to knock out electrons from

their respected orbits around another atom, which in turn charges the remaining particle.

As a result, an ion is formed. These radiations act as a mutagen as they interact with

molecules of the cell and alters their chemical composition hence causing mutations. For

example these radiations oxidize DNA which results in programmed cell death and in

some cases it leads to cancer, if the process of repairs fails (Peter et al., 2012).

2.3. Types of Radiations

Ionizing radiations includes gamma rays, x–rays, radio waves and UV radiations.

These radiations harm bio molecules such as proteins, DNA, RNA by causing an oxidative

damage. Sunlight contains the most basic form of radiation that is the ultra violet

radiation (UVR). The UVR has a wavelength of 10-400 nm (Figure 2.1). Its energies ranges

from 3-124 eV (Hockberger, 2002). The UVR that spreads the earth’s surface is currently

increased due to constant ozone layer depletion. The largest organ of the body is skin,

which is directly exposed to variety of detrimental environmental factors, UVR is one of

them (Barzilai, 2010). UVR penetrates into the epidermal layers where it produces

reactive oxygen species (ROS), which results in single and double DNA strand breaks

(Singh and Gabani, 2011). Variety of responses ranging from inflammation,

immunosuppression, and gene mutation can occur. The carcinogenesis occurs by

alteration of p53 gene, which in turn is altered by the induction of cyclobutane pyrimidine

dimer by mutation, thus disrupting the normal cell cycle (Klein et al., 2010). Another type

of ionizing radiation is gamma radiation (GR). It is not generated by the sun. The main

source of nuclear decay is GR . Exposure to radiations from nuclear facilities is associated

with acute impact on human health. Fever, Fatigue, weakness, dizziness, hair loss,

stomach problems may occur due to prolong exposure. The prolong exposure in some

cases may lead to leukemia and leucopenia (Kurnaz et al., 2007). Microbes which are able

to withstand such extreme radiations are called radio-resistant or radio extremophiles

(Gabani and Singh, 2013)

2.4. Diversity of Radio-resistant Microorganisms

Different organisms show resistance to radiations because of their evolutionary

adaptation. In order to withstand the increased doses of radiations, these organisms

certainly have established a massive radio-resistance. Besides Chernobyl, these organisms

have cosmopolitan distribution. Radioactive lakes, beaches and quarries are present in

Japan, Brazil, China and India. Undeniably, minor doses of background radiations fall on

almost every part on Earth. In search for additional examples bacteria were discovered

having comparable abilities. Billi et al., (2000) reported different species of radio-resistant

microorganisms that were able to tolerate elevated levels of radiations. The preliminary

listing documented the organisms from three different domains (Table 2.1). Of all the

species, Chroococcidiopsis specie, Deinococcus radiodurans, Rubrobacter radiotolerans

and Thermococcus gammatolerans are the well-studied (Billi et al., 2000; Tapias et al.,

2009; Terrato et al., 2011).

Among the radio-resistant microbes many are spore formers. However, others

growing vegetatively are not efficient radiation resistant. Several are pathogenic which

are deprived of systems due to which they cannot be manipulated genetically.

Enterococcus faecium and Alcaligens are pathogenic radiation resistant bacteria.

Deinococcus are noticeably unusual (Daly, 2000). Among the extreme radio-resistant

microorganisms, the species of Deinococcus genus predominates. In eubacteria

phylogenetic tree, it is one of the oldest lineages. In 1956, first Deinococcus strain R1 was

isolated. Isolation of D. radiodurans (SARK) as an air contaminant from Ontario hospital

led to the discovery of second strain. From arsenic- contaminated aquifer, arsenic

resistant Deinocuccus indicus was isolated (Suresh et al., 2004). Isolation of Deinococcus

saxicola, Deinococcus frigens and Deinococcus marmoris was reported from continental

Antarctica (Hirsch et al., 2004). From Sahara desert Deinococcus deserti was isolated (De

Groot et al., 2005) and isolation of Deinococcus navajonensis, Deinococcus hopiensis, and

Deinococcus pimensis from single soil sample of Sonoran desert (Rainey et al., 2005). In

an approach to isolate unusual radiation resistant bacteria, a novel Deinococcus specie

from contaminated tryptone glucose yeast was isolated (Shashidhar and Bandekar, 2006).

High levels of gamma radiations are found to be withstand by Thermococcus littoralis and

Pyrococcus furiosus which belong to hyperthermophilic archaea (Satyanarayana et al.,

2005).

On this planet, the photosynthetic prokaryotic cyanobacteria are believed to be

among the most ancient organisms. They thrive in anerobic environment and have

evolved different mechanisms to reists extremely high light and UV radiations and

desiccation (Sorrels et al., 2009). Although the harmful effects of these radiations are

immense, yet the microorganisms are able to survive under elevated levels of radiations.

Under super lethal effects of UVR, Deinococcus radiodurans are capable of survival (Yun

and Lee, 2009). Harmful effects of UVR are also found to be tolerated by endolithic

cyanobacteria (Rastogi et al., 2010). Due to efficient DNA repair mechanisms,

cyanobacterium strain Chroococcidiopsis displays resistance to ionizing radiation (Singh

and Gabani, 2011). Among the hyperthermophilic archaea, radio-resistance is widely

observerd. Thermococcus gammatolerans which belongs to hyperthermophilic archaea

shows immence resistantance to radiations similar to that of Deinococcus radiodurans

(Confalonieri and Sommer, 2011). Species of phylum Deinococcus – Thermus i.e.

Deinococcus geothermalis is found to be extremely resistant against infra-red and UV

radiations (Makarova et al., 2007). The organism was reported first to be isolated from

geothermal springs i.e. from aerobic environment and later from deep oceanic sub

surface i.e. from the environment with complete absence of oxygen (Liedert et al., 2012).

Rhodanobacter and Desulfuromonas ferrireducens were found to be present in areas with

high levels of radionuclides. Radio-resistant microorganisms survive under the harmful

radiations and have well- organized DNA repair mechanism, due to which they are easy to

yeild primary and secondary protective metabolites (Gabani and Singh, 2013).

2.5. Adaptation of UVR Microbes to UV Radiation

Ancient organisms like cyanobacteria have advanced variety of mechanisms to

preserve from the harmful UV radiation. Avoidance of stress, defense against stress and

repair mechanisms are the three generalized responses of radio-resistant to stress

conditions

2.5.1. Avoidance of UV Stress

Limited information is present of the UV influence on cyanobacterial vertical

migration, contrary to the fact that by gliding a number of cyanobacteria are motile.

Avoidance of elevated solar radiations through daily vertical migrations have been

reported in species of Oscillatoria and Spirulina. In Microcoleus chthonoplastes, vertical

migration has shown to be induced by UV andphotosynthetically active radiation (PAR)

(Bebout and Garcia-Pichel 1995). It has also been reported that by moving downward into

the mat communities, high solar radiations are escaped by motile cyanobacteria

(Quesada and Vincent, 1997). However the influence of migration on primary productivity

of mats still needs to be understood completely.

2.5.2. Defense against Stress

Production of UV-absorbing compounds by cyanaobacteria is considered as one of the

important mechanisms to avoid UV photodamage. Cyanobacteria and other lower

organisms have been observed to posses mycosporin like aminoacids that can effectively

absorb harmful UV rediations, ultimately photo-protecting them from UVR. Such amino

acids like mycosporine have maximum absorption between 310 and 360 nm (Scherer et

al., 1988; Karentz et al., 1991; Ehling-Schulz et al., 1997). Substituted cyclohexenones are

linked to amino acids and amino alcohols in the conformation of mycosporine amino

acids. The mycosporine amino acids themselves are water soluble and are supposed to be

originated from shikimate pathway (FavreBonvin et al., 1987). Cyanobacteria have a

number of microsporin like amino acids (MAAs). However the particular specie and the

location of pigments in them determine the relative protection provided by MAAs against

UV-B damage. Only 10 to 26% photons are absorbed in some cyanobacterial species by

the MAAs located in the cytoplasm. This is significant yet limited protection against

harmful radiations (Garcia-Pichel and Castenholz, 1993). In Nostoc commune, MAAs that

are located in the extracellular glycan plays a crucial role in photo protection. Two out of

the three photons are absorbed by the pigments (Böhm et al., 1995). Colonies are

exposed to high solar radiations, two pigments which are UV–A/B absorber were found

with absorption limits in rage 312-335 nm (Scherer et al., 1988). One was located in the

extracellular glycan. Mycosporine was the first reported that is covalently joined with

oligosaccharide (Peat et al., 1994; Böhm et al.,, 1995). By absorbing the harmful

radiations, the pigment provides protection to the organism. Additional protection by

radical quenching may be provided by 312 chromophore of one pigment which is

supposed to be a MAA-glycoside (Dunlap and Yamamoto, 1995). UV-absorbing

compounds have vital role in N.commune photo protection. They have ability to survive in

quiescence for long time and can withstand prevalent series of rewetting and desiccation,

during which the mechanism of repair are totally ineffectual (EhlingSchulz et al., 1997).

Table 2.2 presents ultraviolet radiation (UVR) induced microbial therapeutic implications

and their metabolic products.

Cyanobacteria possess another pigment known as Scytonemin which have UV-

shielding properties. Scytonemin located in the cyanobacterial sheath with maximum

absorption of 370 nm in vivo. Its molecular mass is 544 Da. It is a yellowbrownish ,dimeric

pigment with a lipid soluble properties. Indolic and phenolic subunits lay the base of its

structure. Condensation of tryptophan and phenylpropanoid derived subunits have a role

in the formation of this pigment (Proteau et al., 1993). UV-A irradiations strongly induce

its synthesis in comparison to UV-B irradiations which have limited potential. Because of

aforementioned reasons it is suggested to serve as UV-A sunscreen (Garcia Pichelet al.,

1992; EhlingSchulz et al., 1997). In the course of evolution different approaches were

selected that contributed to an organisms radio-resistance. These involved the

modification of cellular metabolism and detoxification of radiation byproducts. Oxidative

stress protection, efficient DNA repair mechanism,repairment of DNA double strand, and

helps cell recovery from the injuries induced by radiations (Confalonieri and Sommer,

2011).

Figure 2.1: Diagrammatic representation of radiations and their effects on origin of

different types of extremophiles (Source: Prashant Gabani and Om V. Singh, 2013).

Table: 2.1. Presenting resistance to Ultraviolet radiation (UVR) in diversity of

radioresistant micro-organisms.

http://link.springer.com/article/10.1007/s00253-012-4642-7/fulltext.html#ContactOfAuthor2

http://link.springer.com/article/10.1007/s00253-012-4642-7/fulltext.html#ContactOfAuthor2

Table: 2.2. Showing the therapeutic implications along microbial metabolic products due

to Ultraviolet radiation (UVR)-inductive.

2.5.3. Oxidative stress protection

In D. radiodurans, the ROS (reactive oxygen species) scavenging system consists

of anti-oxidant enzymes and non-enzymatic antioxidants. In D. radiodurans enzymatic

antioxidants like catalase (CAT) and superoxide dismutase (SOD) play an crucial role in

resistance. Markillie et al., 1999; Makarova et al., 2001). Reactive oxygen species (ROS)

harmful for cells and cause damages, but some antioxidant enzymes like superoxide

dismutase and catalase act as first line of defense and guard the cells against oxidative

stress. They protect the cell from damage due to the mechanism is, the enzyme

superoxide dismutase acts as a catalyst in the conversion of oxygen superoxide to

hydrogen peroxide. Then by the enzymes catalase or peroxidase, hydrogen peroxide is

transformed into water (Luis et al., 2002). Induction of three predicted superoxide

dismutases and three catalases resulted in D. radiodurans after exposure to ionizing

radiations (Omelchenko et al., 2005). Superoxide dismutase are particularly encoded by

the aerobic organisms such as Halobacterium or a few anaerobes Methanosarcina of the

phylum Archaea (Brioukhanov et al., 2006). In the anaerobic radio-resistant archaea such

as Thermococcus gammatolerans, superoxide reductase (SOR) is in place of superoxide

dismutase (Grunden et al., 2005). During the processing of ROS, superoxide reductase is

oxidized and by rubredoxin it needs to be reduced. By NADPH rubredoxin oxidoreductase

(NROR) , the rubredoxin is in turn re-oxidized. By the action of rubrerythin the hydrogen

peroxide is converted into water (Molina-Heredia et al., 2006).

An increased amount of Mn+2 to Fe+2 ratio provides shelter against oxidative

stress. Through the Fenton reaction that is (H2O2 + Fe2 +2OH+ Fe3+) the Mn+2 to Fe+2

ratio provides heightened capacity to stop the synthesis of iron dependent ROS (Imlay,

2003). Mn mechanism of action is similar to the enzymes catalase and superoxide

dismutase (Seib et al., 2004). It has no relation to DNA stoppage of double strand breaks.

D. radiodurans was analyze by growing the organisms in defined medium deprived of

Mn+2 supplements (Daly et al., 2004). Mn+2 Intracellular concentration is 300 times higher

in the most radio-resistant species and that of Fe+2 is three times lower in comparison to

the most sensitive species. The level of protein oxidation was shown to be inversely

correlated to the Mn+2 to Fe+2 concentration ratio. With elevated Mn+2 to Fe+2 ratio, no

protein oxidation was detected (Dalyet al., 2007)(Kish et al., 2009). After exposure to

irradiations, a ferritin/Dps like genes expressions can be reduced by P.furiosus. The

protein coded by this gene limited the production of hydroxyl radicals by chelating iron

through the Fenton reaction (Williams et al., 2007). To neutralize the protein oxidization,

the decrease in the iron levels could be a common approach between some radio

resistant bacteria and archaeon. However, Mn to Fe ratio cannot be conclude that radio

resistant mutant E.coli strains are similar to that of wild type (Harris et al., 2009).

Protection against oxidative stress is also provided by some other ions. It was

found that cell survival of Halobacterium NRC-1 to ionizing radiation was enhanced, with

high concentrationof NaBr (1.7) (Kish et al., 2009). The levels of protein oxidation differs

among the radio-resistant D. radiodurans and that of radio-sensitive E. coli. However,

the number of DNA breaks formed in their DNA are similar to each other, even if the

action of ROS indirectly resulted in 80% of DNA damage (Gerard et al., 2001). As cell

require protein protection for DNA repair after exposure to irradiations, this observation

led Daly and coworkers to propose that a main feature of radio resistance could be

protein protection (Daly et al., 2007). Recently it was also indicated that hydrophilic

proteins with low complexity (LC) regions have a role in recovery of DNA damage in D.

radiodurans (Kriško et al., 2010).

2.5.4. Non-enzymatic antioxidants

Among the non-enzymatic antioxidants, stored Mn in D. radiodurans was

recommended to aid the bacterium, battering against oxidative stress. This is

accomplished through non-enzymic dismutation of superoxide anion radicals (Daly et

al.,2004; Ghosal et al., 2005). D. radiodurans also produce an antioxidant metabolite

Pyrroloquinoline-Quinone (PQQ). This metabolite is a ROS scavenger and has the rate

constants analogous to other eminent antioxidants (Misra et al., 2004). It was found that

Mn and PQQ protected the proteins from oxidative stress in D. radiodurans.

Among non-enzymatic antioxidants, the carotenoids (Krinsky and Johnson, 2005)

which are the efficient scavengers of ROS, chiefly of singlet oxygen (Fraser et al., 2004).

Carotenoids are natural pigments, normally tetra-terpenoids, including C40 hydrocarbon

backbones (carotenes) and its oxygenated derivatives (xanthophyll’s). They are

manufactured by microorganisms and plants. D. radiodurans also synthesize quiet large

number of carotenoids (Armstrong, 1997). The bacterium possesses 13 genes, which play

a role in the biosynthesis of carotenoids. The characteristic red color of the bacterium is

due to the presence of these carotenoids. However, some carotenoids are colorless such

as phyotene, thus a colorless mutant might synthesize some carotenoids. In the

carotenoid synthesis pathway, the major product formed is the deinoxanthin. Compared

to other carotenes such as lycopene and -carotene, deinoxanthin has a stronger

scavenging ability on hydrogen peroxide and superoxide and in D. radiodurans. The

deinoxanthin contributes to cell resistance under oxidative stress condition. Under in

vitro condition, it also inhibits protein oxidation at lower concentrations than other

carotenoids.

2.6. Carotenogenesis Genes and Related Biosynthetic Pathway

2.6.1. Synthesis of Phyotene

First carotenoid is the phyotene that is produce in the biosynthesis pathway of

bacteria. It is colorless and is formed of two molecules of geranyl geranyl pyrophosphate

(GGPP) by condensation (Armstrong et al., 1990). Another C5 compound. Isoprenoids,

quinones, terpenes, sterols and carotenoids can be obtained from IPP (Ohto et al.,1999).

Phyotene is formed by the action of phyotene synthase (CrtB) on GGPP. In D.

radiodurans, CrtB gene (DR0862) was determine through gene mutation and carotenoid

product analysis (Tian et al., 2007; Zhang et al., 2007).

2.6.2. Lycopene Synthesis through

Desaturation of Phyotene

Phyotene is transformed into lycopene through four desaturation steps. Bacterial

type phyotene desaturase (CrtI) catalyze these steps. These steps can also be catalyzed by

plant type phyotene (Takaichi and Mochimaru 2007). In the genomes of Deinococcus-

Thermus species only, CrtI homologs have been reported. In T. thermophilus strains HB27

and HB8, the carotenoid biosynthesis gene cluster codes CrtI. This CrtI is grouped with

CrtB and GGPP synthase. Additionally the function of CrtI was proved and described in D.

radiodurans (Bruggemann et al., 2007; Henne et al., 2004).

2.6.3. Lycopene Cyclization

Among acyclic and cyclic carotenoids ,carotenoid biosynthesis is diverse. CrtL or

CrtY type lycopene β-cyclase catalyze the cyclization of lycopene on one or both C-ends of

lycopene (Armstrong et al., 1997). In D. radiodurans an asymmetrically acting lycopene β-

cyclase (CrtLm DR0801) was recognized via coexpression of CrtLm with CrtEIB genes from

Pantoea stewartii in E. coli (Tao et al., 2004). It was observed to be involved in the

catalysis of monocyclic carotenoids production. Accumulation of lycopene occurred in D.

radiodurans, due to mutation in CrtLm gene homolog. It was confirmed that, this gene

(CrtLm ) codes for lycopene cyclase in the natural host (Tian et al., 2008).

2.6.4. C-end and Ring End

Modification

In monocyclic or dicyclic carotenoids, modification occurs on the C-end and ring

end. These modifications comprise of hydroxylation, ketolation, desaturation, acylation

and glycosylation. These modifications results in emergence of different structures and

types of carotenoids found among Deinococcus-Thermus. In D. radiodurans and M.ruber,

the C-end modification of monocyclic carotenoids,such as the main carotenoids need

carotenoid 10,20 hydratase and carotenoid 30,40desaturases in order to attain C-10,20

hydration and C-30,40 dehydrogenation of gcarotene, correspondingly. A CruF-type

carotenoid 10,20-hydratase was documented in D. radiodurans and D.geothermalis (Sun

et al., 2009 A), mostly raised in photosynthetic bacteria. The C-30, 40 desaturation of g-

carotene derivatives cannot take place. A C-30, 40 desaturase (CrtD) was discovered in D.

radiodurans (Tian et al.,2008). As in the mutant or wild type of CrtLm, the C-30, 40

desaturated lycopene products of earlier intermediates not originated, this suggested

that the CrtD in D. radiodurans they are unlike the acyclic carotenoids detailed CrtDs that

are found in purple bacteria and Bradyrhizobium ORS278 (Steiger et al., 2000).

For ketolation of C-4 or C-2 on b-ring of carotenoids in D. radiodurans or

M.ruber, the presence of carotenoid ketolases are necessary. In D. radiodurans and

Actinomycetale, Rhodococcus erythropolis, a CrtO-type carotenoid ketolase was

documented (Tao et al., 2004). In an E. coli expression system that stores -carotene, the

Deinococcus CrtO have capacity of symmetric catalyze addition to two keto groups to -

carotene into finally developing canthaxanthin. (Sun et al., 2009 B). In D. radiodurans, a

carotenoid biosynthetic pathway was suggested. Based on the charterization of

carotenoid enzymes biosynthetic as well as intermediary product analysis (Figure 2.2)

(Bing and Yuejin, 2010).

Figure 2.2: Carotenoid biosynthetic pathway (Source: Sun and Hua, et al., 2009) showing

biosynthetic enzymes and intermediate product with Deinoxanthin as the final product.

Bold line indicate enzymes that been confirmed in Deinococcus radiodurans. The dotted

line shows enzymes that have not been identified and involved in Deinoxanthin (final

product) biosynthesis from this bacterium.

2.7. Toxic Oxygen Specie Removal

2.7.1. Role of Pigments

By photo dynamically producing reactive oxygen intermediates,oxidative stress is

known to be caused by UV-A and UV-B (Cunningham et al., 1985; Shibata et al., 1996). A

strategy like elimination of toxic oxygen specie can be used for mechanism of defence.

Studies have shown that in plants, carotenoids have a crucial role in protection against

UV-B radiations (Middleton and Teramura, 1993). Carotenoids have tremendous amount

of antioxidant activity as they elliminate singlet oxygen, chlorophyll triplet and prevent

lipid peroxidation (Edge et al., 1997). In response to UV-A and UV-B radiations, it has been

revealed that an increase in the carotenoid/Chla ratio occurs, which supports the role of

carotenoid as an significant anti-oxidant specie (Paerl, 1984; Ehling-Schulz et al., 1997;

Quesada and Vincent, 1997). Changes in the carotenoid patterns of cyanobacteria such as

N.commune have been examined in response to UV-B irradiations. Outer membrane

bound myxoxanthophyll along with echinenone were proposed to function as UV-B

photoreceptors (Ehling-Schulz et al., 1997).

2.7.2. Role of Enzymes

Level of reactive oxygen species has shown to be decreased by scavenging

enzymes like peroxidases and superoxide dismutase. Ascorbate peroxidase and catalases

are the scavenging enzymes known to be produced by cyanobacteria (Miyake et al.,

1991). UV-B irradiations have been reported to induce the production of superoxide

dismutase’s and ascorbate peroxidases in plants and in microalga. However, in

cyanobacteria the significance of enzymes with radical scavenging activities needs further

investigation (Lesser, 1996; Rao et al., 1996).

2.7.3. Extracellular Polysaccharides

Synthesis

UV damage can also be prevented by the extracellular polysaccharide synthesis. It

provides shelter against phagocytosis, desiccation and lysis by viruses have been

documented to be provided by bacterial extracellular polysaccharides (EPS) (Tease and

Walker, 1987; Hill et al., 1994). In cyanobacteria, the mechanism of action of EPS

containing sheath is such that it creates a buffer zone between the cell and environment.

It has been noted that the yielding of extracellular glycan in N. commune is stimulated by

UV-B irradiations. It was shown that the in UV-B irradiated cultures the production of EPS

was three times higher in comparison to that of control cultures. In effect path for the

absorpable lengths of radiations are lengthier with thicker sheaths. It has been proposed

that in N. commune synthesis of EPS is roused, so that UV-B absorbing oligosaccharides

matrix (mycosporines) which are located in the sheaths (Ehling-Schulz et al., 1997).

2.8. Molecular Level Adaptation to UV Stress by Efficient Repair Mechanism

An improved targets synthesis mechanism or damaged targets repair of without

de novo synthesis are the possible replacements to overcome the UV–damage to the

targets. The mechanism of DNA repair is common in all cell types. However, in E. coli it

has been extensively studied. Several phenomena like excision repair photo reactivation

and repair after replication (SOS) in E. coli are involved in the recognition and repair of

UV-induced photoproducts (Walker, 1985). In the course of photo reactivation, the UV-

Aand blue light activate the enzyme DNA photolyase which coverts cyclobutane type

pyrimidine dimers into monomers (Pang and Hays 1991). Excision repair is not dependent

on light. The process involves numerous enzymes. First damaged part of the DNA is

nicked, finally the process of re-synthesis fills the gaps. Photo reactivation and excision

repair, both the mechanisms have been documented to be found in cyanobacteria

(O'Brien and Houghton 1982; Levine and Thiel, 1987; Kooiman et al., 1990). In

cyanobacteria, the Rec-A like genes have shown to complement a rec-A deletion in E. coli

(Owttrim and Coleman, 1987). Increased UV-C resistance was shown by complemented

rec-A strains. In the SOS repair mechanism, the first step is RecA protein activation by

DNA damage. The SOS genes are expressed (SOS regulon) when the RecA cleaves the

LexA repressor (Walker, 1985). UV-C irradiation is used in most studies associated with

DNA damage repair. However recently in Pseudomonas aureginosa, the UV-A and UV-B

irradiation induction of recA expression has also been reported (Kidambi et al., 1996).

2.8.1. Enhanced Degradation of

Protein and Resynthesis

To overcome the damage caused by UV radiations, enhanced protein

degradation and resynthesis plays a crucial role. It helps in rapid replacement of

UVsensitive proteins. In Scynechocystis sp PCC6803, in response to UV-B irradiations, an

increased turnover of photosystem ii (or water-plastoquinone oxidoreductase) reaction

system proteins D1 and D2 was reported. The replacement of newly produced D1 and D2

with that of UV-damaged D1 and D2 in the thylakoid was investigated (Sass et al., 1997).

In the degradation of UV-B damaged D1 protein, the role of specific cleavage site has also

been documented (Barbato et al., 1995). In the past few years the turnover of D1 protein

has been examined in detail. It has been noticed that in most of the stressed conditions it

is being regulated. In response to environmental stresses, its turnover is generally

adaptive response.

2.8.2. Efficient Repair of DNA Double

Strand Break

It is believed that the most hazardous and tough types of DNA breaks are the

ones which are in double strands of the DNA. However, hundreds of double stranded

breaks are tolerated by the organisms like D. radiodurans and T. gammatolerans (Battista

et al., 1999). It was shown that after being exposed to γ radiations of 6,800 Gy, the repair

of DNA double strand breaks was robust and a number of fragments were joined to

reconstruct an intact piece of genome when the cell were incubated up to 3 hours in a

medium supplemented with numerous nutrients (Servant et al., 2007; Tapias et al., 2009).

2.8.3. Mechanism Involved in the

Repair Process

The damaged copy of DNA is also kept by the cell in the mechanism involved in

the repair like single strand annealing (SSA), homologous recombination, extended

synthesis dependent strand annealing (ESDSA) (Figure 2.3). Contrary to this non

homologous recombination does not require extra copy to be preserved to join two

fragments (Hefferin and Tomkinson, 2005).

2.8.3.1. Homologous Recombination In the organisms like bacteria and Saccharomyces cerevisiae that is yeast, the

homologous recombination is the chief pathway eleborated in the repair of DNA double-

strand breaks (Lee et al., 1999; Wyman and Kanaar, 2006). This mechanism incorporates

an intact homologous DNA molecule at the damaged site to reinstate the correct DNA

sequence (Figure 2.3). Generation of single stranded 3´overhang for the formation of

nucleoprotein filament is the first step is the breakage of DNA double strand and repair by

homologous recombination. The enzymes involved in this filament formation are RadA in

archaea, RecA in bacteria and Rad51 in the cell of eukaryotes (Seitz et al., 1998). Invasion

of homologous overlapping fragments; followed by the DNA strands exchange nascent

DNA heteroduplex extension and resulting cross over resolution are respectively the next

steps involved in the homologous recombination.

2.8.3.2. Single Strand Annealing (SSA) It has been suggested that in irradiated Deinococcal cells, single strand annealing

also occurs at the very beginning besides homologous recombination. Its early occurrence

corresponds to the repair observed in the Deinococcal cells (Daly and Minton, 1996). To

accomplish this process it is obligatory that two copies double break of like chromosome.

Single stranded overhangs are produced soon after the sectioning of DNA ends in the

reaction catalyzed by exonuclease. Annealing can only takes place if there are

complementary sequences in the overhangs. By the process of DNA synthesis, the single

strand gaps are being filled, in the re-constructed chromosome.

2.8.3.3. Extended Synthesis Dependent Strand Annealing (ESDSA) ESDSA was proposed by Zahradka et al., (2006) as an alternative to SSA model.

This model of ESDSA was the justification of their findings that, in irradiated cells the DNA

fragments assembly corresponds with DNA synthesis and is much higher in comparison to

un-irradiated growing cultures. In the damaged cells, an enormous number of single

strand DNA is present. Both the old and newly synthesized DNA segments are present in

newly assembled genome. The DNA synthesis occurs as the single stranded ending of the

lower fragment occupies a partially overlapping fragments. However, differing to an

aforementioned model, in classical way of double stranded DNA replication, through the

displacement of Dloop, generation of single stranded fragments occurs (Figure 2.4). And

this is the same as that observed in the transcription process. In transcription when the

RNA polymerase progresses on the DNA template, it results in the liberation of newly

synthesized RNA from the transcription machinery. The extension of strand can ensue to

DNA template end. Defined reconstruction of long double strand DNA intermediates is

accomplished by the annealing of those newly synthesized single stranded DNA tail, which

contain complementary sequences. To reform the circular chromosome, recombination

of these long intermediates takes place (Slade et al., 2009). Though many enzymes play

crucial role in the synthesis step, but in the steps like an invasion prime DNA synthesis

and direct intermediates maturation of chromosome through conventional

recombination requires RecA enzyme. Previously observed in D. radiodurans resistance

to radiations, the ESDSA plays a key role. P. abyssi massive DNA synthesis begun after 2

hours of their exposure to gamma radiations of dose 2,500 Gy. This suggested the role of

ESDSA in Archaea (Jolivet et al., 2003). However the existence of ESDSA in archaea, is yet

to be studied (Confalonieri and Sommer, 2011).

2.8.3.4. Non-Homologous End Joining

In eukaryotes joining of non-homologous end is the key pathway for repair of

DNA double strand breaks (Weller et al., 2002). In bacteria it has been recently classify

and characterized (Shuman and Glickman, 2007). Under ionizing radiations and

desiccation conditions a protein PprA, specific from the family Deinococcacea is

exceedingly induced (Tanaka et al., 2004). This protein specifically binds to

doublestranded DNA having strand breaks. By doing so, it constrains the degradation of

DNA by the enzymatic activity of exonuclease and facilitates DNA ligation by DNA ligase

enzyme (Narumi et al., 2004; Murakami et al., 2006). Increased radio sensitivity was being

observed in cells lacking the protein PprA. However, in D. radiodurans DNA double-strand

break through non-homologous end joining is yet to be investigated (Confalonieri and

Sommer, 2011).

Figure 2.3: Different routes for DNA break on repair figure (source: Blasius et al., 2008).

During DNA both strand break repair, green color strand demonstrates newly synthesized

DNA. Red and black color strands are showing two copies belonging to same

chromosomal region.

Figure 2.4: Two steps of genome reconstitution in Deinococcus radiodurans (Cox, Keck

and Battista, 2010). Once the DNA is degraded by several stress mechanisms.

Nucleases activity yields 3′-OH single stranded tails (may be 100 bp of longs). The 3′OH

end search for the homologous strand, the process is mediated by recombination protein.

The strand form a loop called D or displacement loop (extended synthesisdependent

single-strand annealing (ESDSA). The complementary strand act as a template to for DNA

synthesis. Replication bubble dissociates and hetero-duplex formation occurs, now the

newly form strand with 3′-OH end serve as a template for newly synthesized strands and

the remaining gaps are filled by ligases.

2.8.3.5. DNA Ends Processing All of the three foremost pathways that are involved in the DNA double-strand

break repair require single stranded DNA tails in both bacteria as well as in archaea

(Figure 2.5). Variety of helicases and nuclease are involved in this process (Yeeles and

Dillingham, 2010). In the DNA ends re-sectioning and in the loading of RecA recombinase

on SSB covered single stranded DNA tails, the key role is being performed by E .coli

RecBCD complex and with their helicases, ATP-dependent nucleases. In E. coli, under

certain circumstances if the RecBCD pathway is inactivated, an alternate pathway

becomes functional that supports the repair of recombinational DNA double strand break

(Horii and Clark, 1973; Mahdi and Lloyd, 1989; Sakai and Cox, 2009). To resect DNA

duplex, the E. coli RecF requires the RecQ helicase and RecJ exonuclease. And in order to

load RecA on SSB covered single stranded DNA, RecF requires RecO and RecR (Handa et

al., 2009).

Organisms like D. radiodurans, D. geothermalis and D. deserti don’t have ability

to synthesized RecB and RecC enzymes. These organisms encode all the RecF alternative

pathway components. ESDSA type mechanism is observed in D.radiodurans for the repair

of DNA double-strand breaks .RecFOR protein plays effective role in repair process and

RecJ protenin facilitates cell’s vitality (Bentchikou et al., 2010). Moreover it was reported

that cells were resistant to γ-irradiation even if they were lacking a very crucial helicase

that is the RecQ required in repair process involving the RecF pathway of the E. coli. In

addition to this, these cells presented a wild type repairing capability of DNA double-

strand break. Contrary to this, the UvrD mutants exhibited strikingly low level of radio-

resistance and the fragment assembly was also at much slower rate which indicates role

of UvrD in the double stranded DNA ends processing.

Despite the close association of archea and bacteria, archea’s metabolic

machinery [DNA replication, transcription and translation, repair and recombination of

DNA] is more closely related to eukaryotes. For the repair of DNA double-strand breaks,

eukaryotic cells use two chief pathways that is the homologous recombination and non-

homologous end joining. Both of these pathways are dependent on the cell cycle phase as

well as on the nature of DNA ends. To produce recombinogenic single stranded tails, the

―licensing‖ of DNA ends is the central step on which the pathway choice is being made.

The key regulators of DNA end processing are the conserved complexes MRX/MRN

comprising of protein Rad50, Mre11 and Xrs2/Nbs1 [in yeast or human] (Mimitou and

Symington 2009). Proteins homologous to Rad50 and Mre11 are coded by archaea,

however, it lacks proteins similar to that of Xrs2/Nbs1. Rad50 and Mre11 in combination

with two other genes that code for a nuclease [NurA] and a helicase [HerA/MlaA] form a

well conserved operon in the archaea that occurs in hyperthermophilic regions

(Constantinesco et al., 2002; Constantinesco et al., 2004; Manzan et al., 2004).

In vitro studies showed that the Pyrococcus furiosus proteins NurA and HerA

form a complex that’s displayed helicase or nuclease activity. They also reported this

process was the results of stimulation in Rad50/Mre11 presence (Hopkins and Paull,

2008). Short 3´ ends are being produced by Rad50/Mre11 complex before enlistment of

NurA/HerA complex. This leads to degradation of DNA at 5´ prime end and results in the

generation of 3´ protruding ends. These ends produced may be overrun by RadA. In vitro

studies involving Sulfholobous acidocaldarious have shown that exposure of cells to the

1000 Gy gamma rays dose resulted in Mre11 recruitment on DNA (Quaiser et al., 2008).

The study further revealed the interaction of HerA, Rad50 and Mre11 in

formation of complex on chromosome and their possible role in DNA repair mechanism.

However, NurA was neither detected on chromosome nor was found to form a complex

with Rad50/Mre11/HerA after the ionizing radiations. To explore the in vivo role of NurA

in the DNA repair process in thermophilic archaea further research is required. In a

polyploid archaeoan, Haloferax volcanii, which does not encode NurA and HerA proteins,

the Rad50 and Mre11 roles were also investigated in vivo (Delmas et al., 2009). Dissimilar

to yeast Saccharomyces cerevisiae, higher resistance to DNA damaging agents was shown

by this archaeoan on deletion of rad50 and mre11. In H.volcanii cells that lack the Rad50

and Mre11 proteins, for the repair of DNA double strand breaks, homologous

recombination act as prime pathway. Whereas in wild type it’s not the same. In wild type

it is similar to that of mammalian cells in which NHEJ mainly perform the repair of DNA

double strand break. However, for the cells to remain viable after DNA damaging

treatment, the presence of RadA proteins is necessary (Delmas et al., 2009). After the

reassembling of most of the particles through NHEJ process, the role of RadA –dependent

HR would be late in the restoration of an intact circular chromosome. The complete set of

archaeal protein involved in the NHEJ pathway still needs to be identified. Similar to that

of bacteria, to favor homologous recombination, eukaryotic cells also requires accessory

proteins. In irradiated cells it was observed that Rad52 and Rad55/57 facilitated the

loading of the Rad51 recombinase on DNA single-stranded tails, covered by the SSB like

complex RPA.

The presence of a motor protein Rad54 that translocate along the

doublestranded DNA has been studied. The function of this motor protein is the

promotion of chromatin remodeling along with strong stimulation of Rad51 DNA strand

exchange activity. In yeast S. cerevisiae, the rad51, rad52 and rad54 are among the most

ionizing radiation sensitive single mutant (Mazin et al., 2010). In archaea to support

homologous recombination, proteins form by sequence homology with RadA act as

accessory proteins. These accessory proteins comprise of RadA2, RadB, RadC, KaiC /aRadC

and Sms proteins (Haldenby et al., 2009). In H. volcanii, reduction of cell growth,

increased sensitivity to UV, and defects related to recombination are associated with the

deletion of RadB. In P. furiosus, in vitro studies of RadB showed that this protein possess

the ability to interact with DNA, however, it lacks the capability to catalyze the DNA

strands exchange that is required for homologous recombination. To compensate for this

other homologs of the eukaryotes such as Rad54 are present, which facilitates the

process of homologous recombination. SsoRad54 from Sulfolobous solfataricus was

suggested to be a translocase as it was able to interact with the DNA double strand under

in vitro conditions. It was also revealed that SsoRad54 stimulates DNA strand exchange by

interacting with the RadA protein (Haseltine and Kowalczykowski, 2009).

Figure 2.5: Main pathways involved in the processing of DNA ends. For detail see the

text.

2.9. Structure of Nucleoid

In bacteria condensed structures are formed by the combination of chromosome

with proteins termed as nucleoids. High degree of genome condensation was observed in

the nucleoids radio resistant genera Deinococcus and Rubrobacter. Moreover their

nucleoids remained unaltered even after exposure to high dose of gamma radiations

(Levin-Zaidman et al., 2003; Zimmerman and Battista, 2005). In order to efficiently repair

radiation induced multiple DNA double stranded breaks, restricted DNA ends diffusion

could be a possible repair mechanism. In D. radiodurans, it was also anticipated that four

to ten copies of genome was prearranged (Minton, 1994). This pre-alignment helped

effective error free repair [homologous recombination and ESDSA] of DNA double strand

break by assisting the search for homology. Nevertheless the pre arrangement of the

homologous chromosome copies is yet to be demonstrated . In E. coli large amount of

histone like protein HU was present, and it was linked with the bacterial nucleoid. This HU

effect the survival of cell after exposure to the gamma radiations (Boubrik and Rouviere,

1995). In D. radiodurans HU protein plays a crucial role by maintaining cell’s viability. It

was shown that when this protein was expressed on temperature sensitive plasmid,

progressive cellular depletion of HU at critical temperature range resulted in nucleoid de-

condensation and fractionation, earlier to lysis of cell. These observations indicated that

in D. radiodurans, HU proteins play a key important role in structure of nucleoid (Nguyen

et al, 2009).

Pyrococcus and Thermococcus archaea which belongs to Euryarchaeota lineage,

and also the methanogenic archaea possess histone like proteins. Their histones like

proteins show structural homology to those present in the eukaryotes (Sandman and

Reeve, 2006). Archaeal nucleosome is different from eukaryotic one, as it does not exist in

octamer form. The archaeal histones like protein form dimers, which in turn join to form

tetramer which eventually binds to the DNA. In Euryarchaea species as well as in

methanogenic archaea, small proteins have been characterizes that may have a role in

chromosome compaction (Pavlov et al., 2002). Sulfolobous a specie form Crenarchaeota

lineage, differs in the structure of chromatin from that of found in Pyrococcus and

Thermococcus. And until now there is been no specie of them, that encodes histones.

AlbA and Sul7d are the two other DNA binding proteins that is used by these organisms to

achieve DNA compaction (White and Bell 2002). In eukaryotes, alterations in the

chromatin architecture directs the lesions to be easily accessed by DNA repair machinery.

Chromatin modulation regulates he process of of transcription which is catalysed by, two

significant enzymes histone acetylases and histone acetylases display a key important role

(Hager et al., 2009). Similar to that of eukaryotic organisms, in archaea have adjusted the

chromatin compaction. In Sulfolobous the enzymes Pat and Sir2 through the process of

acetylation and deacetylation, modulate the DNA binding affinity of AlbA (Bell et al.,

2002; Marshet al., 2005). As a result of deacetylation of AlbA, the repression of

transcription occurs. Due to lack of complete data on global structure on archaeal

genome during DNA repair, the role of nucleoid structure in radio resistance of archaea

still needs to be answered (Confalonieri and Sommer, 2011).

2.10. Biotechnological Applications of UVR Microbes

2.10.1. Therapeutic Applications

Extremolytes are special organic natural compounds of extremophiles with great

biotechnological potentials. They are indeed reserves of microbial metabolic processes.

They do not take part in the growth, development or reproduction of the organism, yet

their lack of presence results in the long-term damage to organism’s survivability,

productivity and aesthetics (Figure 2.6 shows a screening strategy of novel bio-active

compounds from radio-resistant microbes). The industrial significance of these

extremolytes is being widely explored; however, their therapeutic implications are yet to

be discovered. It is due to the presence of unique metabolism, to sustain under extreme

environmental conditions and also a major cause of their diversity (Thomas and

Dieckmann, 2002; Ferrer et al., 2007; Gostinčar et al., 2010). Advances in the analytical

tools in the discipline of genomics, proteomics, and metabolomics have made it possible

to identify the genes and proteins that may contribute in the regulation of the

extremolytes under the extreme environment (Singh 2006; Ferrer et al., 2007; Hammon

et al., 2009; Singh, Klisch et al., 2010). In search for various anti-cancerous agents,exciting

developments have been made by investigating the marine organisms inhabiting the

extreme conditions (Singh and Gabani 2011). Some of them are discussed below:-

2.10.2. Role of Carotenoids in

Circumventing Diseases

Carotenoids are assumed to yeild certain health advantages. Reduction in the

rate of numerous illness is also attributed to the presence of carotenoids. They do so by

protecting cells and organisms from damaging substances (Vertuani et al., 2004). Several

studies have indicated the protective role of Deinoxanthin in avoidance of certain

diseases. These include eye diseases and certain types of cancers (Krinsky et al., 2005).

The principal mechanism behind their ability to lessen the rate of diseases is the

scavenging ability of ROS(Fan et al., 2012).

Among the number of degradation products made as a result of lipid

peroxidation, malondialdehyde (MDA) is also one of the degradative products being

produced (Girotti et al., 1998). It causes cell membrane disruption and also damages the

DNA. The chain reactions elaborate in lipid peroxidation are inhibited by the cells via

scavenging of free radicals. Thus scavenging of free radicals is one of the main anti-

oxidative mechanisms involved in protection. In an in vitro study showed that carotenoid

rich extracts from D. radiodurans possess strong ferric ion reducing ability and lipid

peroxidation inhibition capability. It was also investigated that pre-treatment with

deinoxanthin rich extract from Deinococcus radiodurans (EDR), reduced the elevated

levels of AST, ALT and ALP in serum. Thus indicating the hepatoprotective and therapeutic

capabilities of deinoxanthin rich extracts of D. radiodurans (Cheng et al., 2014).

Figure 2.6: Screening strategy of bio-active compounds from extremophiles. In general

bio-active compounds are of two forms: cell bound and non-cell bound. The cell bound

bio-active compounds can be recovered by cell lyses and solvent extraction. While the

non-cell bound compounds are excreted to the broth and can be recovered by different

solvents. The second stage include of 16S rRNA sequencing genes and bio-active

compound analysis through HPLC complementary methods.

2.10.3. Mycosporine Like Amino Acids

Mycosporine are photo protective compounds due their ability of maximum UV

absorption. MAA absorbs UV in the range between 310-360 nm. They possess high molar

extinction co-efficient [e=28,100-50,000 per Mcm]. Photostability, resistance to several

abiotic stressors, and capability to dissipate absorbed radiations efficiently without

reactive oxygen species (ROS) production are some of the properties, attributed to the

presence of MAA’s (Conde et al., 2000; Whitehead and Hedges, 2005). They are colorless,

small i.e. upto 400 Da, intracellular, (Singh et al., 2008). They occur in a variety of

microorganisms including cyanobacteria and eukaryotic algae. Pyrimidine dimers are

formed in the DNA on exposure to UVR. This dimerization leads to lethal concequences.

The cell cycle may go out of control if these mutations are found in p53, which eventually

leads to cancer. MAA’s are known to resist dimer formation, ultimately protecting DNA

damage induced by UVR.

A new mycosporine from the lichenized ascomycete Collemacristatum, which

exhibited protection against membrane destruction induced by UVB (Singh et al., 2013).

Apart from this, the UVB absorbing mycosporine was also prevented pyrimidine dimer

protection and was also shown to protect against erythema in cultured human

keratinocytes (Singh and Gabani, 2011). Protective effects of MAA’s from harmful UVR

were also observed on fibroblast cells of human skin (Torres et al., 2006; Oyamada,

Kaneniwa et al., 2008). MAA’s are being used in the cosmetic industry for the formulation

of UV sunscreens as they are regarded as UVR absorbers. Studies have indicated their use

in the prevention of cancer such as melanoma (De la Coba et al., 2009). Prevention of

sunburns, corneum stratum, malphigian thickening of dermal and hypodermal, structural

and morphological changes in the biopsies of non-photoprotected skin was reported in a

study in which the formulation consisted of MAA [Prophra-334 and shinorine i.e. P-

334+SH]. UV induced illness in mice skin was also prevented by the MAA formulation.

Moreover, it was also reported to be associated with the maintenance of anti-oxidant

defense system of the skin (De la

Coba et al., 2009). Palythine, asterina and palythinol are among the various MAA’s

suggested to have a photo-protective properties.

Many microalgae species such as G. galatheanum and G. venificum are known to

possess palythine (Llewellyn and Airs, 2010). Maristentorin occurs in Maristerntor

dinoferus, a heterotrich ciliate. The Maristentorin has a potentially similar biological role

to stentorian and blepharismim and this includes the UV irradiation defense also. For the

low photo protection, Usurijene is well known. usurijene is converted to palythine by its

cis-trans photo isomerization. Palythine is more photostable compared to usurijene

(Mukherjee et al., 2006) (Conde et al., 2003). Despite of these compound’s promising

qualities,the through therapeutic consequences of MAA’s as drug in human is quiet

expected (Singh and Gabani, 2011).

2.10.4. Scytonemin

It was first reported by Nageli in 1849. He described it as yellow green

pigmentation in cyanobacterial sheaths (Garcia Pichel and Castenholz, 1991). Scytonemin

unique dimeric indole-phenolic structure (Proteau et al., 1993). It is exclusively produced

by the cyanobacteria (Figure 2.7). It is considered as a true sunscreen (Cockell and

Knowland, 1999)

It is found in almost 30 sheathed cyanobacterial species of various geographic

sites and environments. Scytonemin absorbs UV radiations strongly with an in vivo λmax

= 370 nm. Scytonemin prevents the UV-A radiations for reaching the interior of the cell by

85-90% (Proteau et al., 1993). Upto 5% of cultured cyanobacterial dry weight is

constituted by this pigment and this percentage may exceed in the natural collections

(Karsten 1988). Scytonemin mostly appears in the topmost layers of microbial mats. The

microbial mats are thick microbial communities, found in the extreme environments and

mostly grow in the areas exposed to light. In filamentous heterocystous cyanobacteria,

Nostoc punctiforme, gene cluster important for the biosynthesis of Scytonemin was

identified (Soule et al., 2007). A sequence of studies were carried out, in order to

understand the regulation of biosynthetic gene expression (Sorrels et al., 2009; Soule et

al., 2009).

Figure 2.7: Structure of Scytonemin, indolic and phenolic subunits (novel dimeric

molecule) (mol. wt. 544) recognized for sheaths surrounding cyanobacteria cells.

Soule et al., (2009) recommend the cellular compartmentalization of Scytonemin

biosynthesis in cyanobacteria, in a working model for UVR

neutralization. In N. punctiforme, a distinctive set of eighteen gene cluster

[NpR1276NpR1259] was observed. This gene cluster was involved in scytonemin

biosynthesis. In the model it was proposed that trp and typ genes are induced by UVA

irradiations, as a result of which tryptophan and p-hydroxyl phenyl pyruvate monomers

are produced from chorismate. Two genes aroG and aroB were shown to boost the

central metabolism machinery. Moreover these genes also controlled the rate limited

enzymes. Whereas, in the cytoplasm certain precursors were also suggested to be

processed by ScyA, ScyB, ScyC, and NpR1259. These intermediates were then transported

to the periplasmic space by the mechanism which is still not known. To produce reduced

forms of Scytonemin, these intermediates were subjected to different reactions by the

enzymes in the periplasmic space. The periplasmic enzymes comprised of DsbA ScyD,

ScyE, ScyF and TyrP. The Scytonemin that was then secreted was auto-oxidized, having

yellow-brown color (Soule et al., 2009).

In humans it has been speculated that the oncogenes are controlled by the polo-

like kinases known as ATP-competitive inhibitor [PLK’s] and bind with ATPbinding sites in

response turning off their activity. Because of this reason, the deep cavities in proteinase

kinases ATP-binding domains are potent targets in search for

ATP-competitive inhibitors. Out of four PLK’s, fond in human’s PLK1 has been consider as

a confirmed mitotic cancer target for many years (Figure 2.8). By using flash plate

screening assay, the group revealed scytonemin ability to treat hyper proliferative

disorders. Because of their investigation, scytonemin was idenify as a non-specific ATP

competitor (Stevenson et al., 2002). It was shown in a recent study that those cells are

very sensitive to the PLK1 inhibition, in which mutant Ras acts as an oncogene. This is due

the fact that in these cells the key event appears to be the mitotic stress (Luo et al., 2009).

It was observed that scytonemin mediated PLK1 expression inhibition, resulted in the

induction of apoptosis in cancer cell and other osteosarcoma cells (Stevenson et al., 2002;

Duan et al., 2010). Owing to its anti protein kinase activity it is been proposed that

scytonemin may act as an antiinflammatory and anti-proliferative, drug by providing

novel pharmacphore for the development of protein kinase inhibitors (Singh and Gabani,

2011).

In the presence of scytonemin and its derivatives mediated activation of SAPK.s,

the cell could possibly survive (Singh and Gabani, 2011).

2.10.5. Ecotine

It is cyclic amino acid derivative of aspartate. Ecotine chemical structure is 1, 4, 5,

6-tetrahydro-2-methyl-4-pyrimidinecarboxylic (Galinski et al., 1985). It has low molecular

weight. It is neutral, non-ionic, strong water binding molecule (Galinski 1993). Ecotine

occurs in halophilic bacteria. The halophilic organisms thrive in extreme environmental

conditions as they can grow in intensive sun irradiations, high temperature and extreme

dryness (Galinski and Trüper, 1994). In nature, by synthesizing Ecotine, these organisms

protect themselves. They do so by ecotine synthesis and enrichment within the cell, in

order to protect their biopolymers against dehydration due to elevated temperatures,

high salt concentrations and low water activity. The ecotine synthesis is encoded by a

gene cluster ectABC (Nakayama et al., 2000). Ecotine is reported to be produced for

commercial usage. It is used as cosmetic additive, extensive applications as biological and

enzyme preparation, in pharmaceutical companies and in other fields as well. The

formation of ecotine from aspartate in halophilic organisms occurs in three steps. It can

be produced from H. elongata by continuous fermentation in which through the process

of microfiltration, culture broth is isolated from the biomass. The ecotine containing

filtrates acts as starting materials for the purification process. The purification processes

include, electro dialysis, chromatography and crystallization (Lentzen and Schwarz, 2006).

Ecotine protects skin from UVA induces cell damage in a number of different ways.

Ecotine protected human keratinocytes cells from damage on exposure to UVA

irradiations (Buenger and Driller, 2003). In the same study, it was discovered that ecotine

prevented the release of UVA-induced second messenger, activation of transcription

factor AP-2, expression of intracellular adhesion molecule 1 (part of immunoglobulin

super family) as shown in figure 2.8.

It was also found that ecotine prevented the mutation of mitochondrial DNA.

From this it could be concluded that ecotine mediated mechanism plays a role in

stabilization of membranes structures that leads to increase resistance to possible

damages induced by UVA irradiations. Bunger et al., studied the role of ecotine in the

reducing the formation of sunburns caused as result of exposure to UV radiations (Bunger

et al., 2001). Ecotine of 1% pretreated Langerhans cells in an in vivo study showed

substantial protective effects of ecotine on Langerhans cells which were sensitive to UV

stress. The Langerhans cells induce T-cells response, thus exerts an immunoprotective

effect, along with protecting the skin from potential damage of UV (Lentnez et al., 2000).

By inducing eminent levels of Hsp70 exhibit the cytoprotective effects of ecotine

triggered by bacterial lipopolysaccharides. Ecotine mediated UVR neutralization is

beneficial in the prevention of water loss in dry atopic skin, thus ecotine can stop the

aging of skin (Buommino et al., 2005; Singh et al., 2011).

2.10.6. Bacterioruberin

Rubrobacter radiotoleransis is exceptionally resistant to ionizing radiations which

have lethal effects on the organisms. Its resistance to ionizing radiations is even greater to

that of D. radiodurans, a well-known radio-resistant bacterium. Another red-pigmented

bacterium, Halobacterium salinarium containing the bacterioruberin was also shown to

be extremely resistant to UVR and hydrogen peroxide (Asgarnai et al., 2000). It was

documented that the organism show intensive resistant to lethal activities of DNA

damaging agents including (ionizing) radiations and UV light. This indicated a direct

correlation between the prevalence of bacterioruberin and DNA repair mechanism (Shah

et al., 1998). Bacterioruberin plays effective role against DNA damage by ionizing

radiations hence its potential use as a therapeutic agent in humans can not be

undermined (Singh et al., 2011).

2.10.7. Sphaerophorin and Pannarin

Secondary metabolites of pharmaceutical importance are produced by

UVtolerant organism’s (Muller et al., 2001). Among these organisms are lichens which are

symbiotic associations of fungi, algae and cyanobacteria. Secondary metabolites having

antioxidant activity obtained from the lichens have been considered as sunscreens for

protection against UV irradiations. Protection against intense UVR have been reported in

Chilean lichens (Russo et al., 2008). Protective effects on plasmid DNA was shown by both

compounds. Additional research on the molecular mechanism involved in sphaerophorin

and pannarin needs to be investigated (Singh et al., 2011).

2.10.8. Circimventing Cancer and

Aging

The extreme resistance of D. radiodurans to ionizing radiations and other

oxidative agents is attributed to extremely efficient protection against oxidative agents

and extremely efficient DNA repair. These abilities enable them to avoid or lessening

DNA, RNA and protein damage and also the oxidative stress that is closely linked with

cancer and aging. Increased DNA and protein oxidation due to ROS, decrease in the

strength of antioxidant defenses and DNA repair and also the deposition of end products

of oxidative are the sole causes of aging and cancers (Beckmann and James 1998). Delay

or prevention of aging and cancer could be accomplished by the interventions designed to

prevent accumulation of DNA damage and the by the oxidized protein production. In

aging and cancer research, the central point is to recognize the factors that reverse the

cancer development and aging process and also the designing of acceptable therapeutic

strategies. In this concern, approaches of combating oxidative stress of D. radiodurans,

may open new opportunities (Slade and Radman, 2011).

Figure 2.8: Scytonemin Production with exposure of UVR. Scytonemin absorbs UVRB

using as sunscreen product for survival of the cell scytonemin. Figure presents a

hypothetical pathway, scytonemin downregulate stress support pathways by inhibits PLK1

resulting in cancer cells destruction via apoptosis. C. Ectoine blocking cell cycle by

inhibiting secondary messengers induction, transcription factor (AP-2) activation, and

mutations of mitochondrial DNA and eventually leading to inhibition cancer induction by

UV radiation or additional severe circumstances.

2.11. Radiation Resistant Extremolytes and their Biotechnological Implications

Apart from their role in therapeutics, the radio-resistant extremolytes also

possess many biotechnological applications. In the bioenergy sector, their use has not

been comprehensively studied. It has been now extremely important to find an

inexpensive and efficient process for the production of bioethanol through the

degradation of complex carbohydrates (Gabani and Singh 2013). Two novel strains of

C.cellulansUVP1 and B. pumilis UVP4 were isolated by (Gabani et al., 2012). These strains

were able to withstand radiations of 1.03×106J/m2 and 1.71×105 J/m2UVC respectively,

and have ability to degrade cellulose under fluctuating physical and chemical conditions

i.e. under high salt content, high temperature and acidic pH was shown by both B.

pumilus and C. cellulans. Extremolytes related to radio-resistance are reported to be

associated in the nuclear waste bioremediation and was noticed that a Ni-Fe hydrogenase

from Desulfovibrio desulfuricans reduced Tc (VIII) (Luca et al., 2001). Shewanella

putrefaciens and Geobacter sulfurreducenspossess an enzyme ctype cytochrome, this

enzyme has the ability to reduce soluble radioisotopes of uranium into insoluble species

(Lloyd et al., 2003). A new Halomonas specie strain isolated having ablity to eliminate

technetium from solution by converting it into insoluble form (Fujimoto and Morita,

2006). It was reported that Geobacter sp. and Rhodoferax ferrireducens through the

enzymatic mechanism has the ability to reduce radioisotopes (Kim et al., 2012).

By further reduction, the product formed can be converted in multi-component

insoluble specie (Van Hullebusch et al., 2005). When grown in the presence of uranium,

tellurium, and plutonium, sulfate-reducing bacteria such as Microbacterium flavescens

showed capability to yeild, siderophores, organic acids and extracellular metabolites.

Apart from being reduced to insoluble forms, radio-resistant extremophiles absorb these

radionuclides. Effective uranium radioisotope adsorption was showed by brown marine

algae Cystoseria indica (Seyrig 2010). Other microorganisms like Citrobacter freudii and

Firmicutes were also reported to possess the ability to effectively adsorb radio nucleoids.

Wu et al., (2006) developed a process for the bioremediation of increased concentration

of uranium radioisotopes. This method involved the addition of ethanol.

For the bioremediation of heavy metals from acidic and neutral water,

radioresistant organism D. radiodurans have proven to be very effective. It was reported

that D. radiodurans possess a tremendous ability to remove uranium solution, i.e. upto

70% of 1Mm input uranium solution was removed by it (Misra et al., 2012). Furthermore,

D. radiodurans was also found to bio remediate phthalate esters. These esters are

extensively used in production of cosmetics, plasticizers and perfumes (Liao et al., 2010).

Through desired genetic engineering of the organism, the bioremediation ability can be

multiplied to include even more substrates (Gabani et al., 2013).

2.11.1. Engineering of Deinococcus

radiodurans for Purpose of

Bioremediation

Right after the demonstration that D. radiodurans can grow in the presence of

ionizing radiations at 6000 rad/ hour as compared to most radioactive DOE (department

of energy U.S) waste sites in 1997, research intended to develop D. radiodurans for the

purpose of bioremediation begun (Lange et al.,1998). All reported members of

Deinococcus can in fact grow at this dose rate and because of their individual properties,

they are foreseen as an important contributors to this technology. Recently it was

documented that expression system developed for D. radiodurans, works at the

temperature range of 50°C which is the optimal temperature for Deinococcus

geothermalis growth (Ferreira et al., 1997). So there is a possibility of easy transference of

genetic technology being developed for D. radiodurans, into D.geothermalis(Brim et al.,

2000). At the start, Deinococcus growth was doubtful, during high level prolonged

irradiation exposure, as it had been documented that upon DNA damage, DNA replication

ceases in D. radiodurans (Minton et al., 1996). However their growth in 137Cs (cesium

137) demonstrated that these bacteria are capable of simultaneous semi-conservative

DNA replication and homologous recombination (Daly and Minton 1995). These

engineered strains are being used in complex bioremediation system designing (Daly,

2000).

2.11.2. Remediation of Metals

Microorganism’s ability to toxic effects resistance the of metals is commonly

linked with their ability of transformation of those metals into a reduced amount of toxic

chemical states (Diels et al., 1995; Lovely, 1995). Diversity of metal resistance /reduction

genes are being tested in D. radiodurans .They are being examined to determine,

whether they possess ability to resist common metallic waste components along with

their capacity to transform those metals. Usually at lower oxidation states, the solubility

of metals is reduced. The enzymes with the ability to catalyze metal reducing functions

are becoming important elements of metal immobilization approach (Stephen et al.,

1999). MR-1strain of Shewanella oneidensis (formerly S.putrefaciens) is highly potent at

reducing Cr (VI), U (VI) and Tc (VII) to insoluble Cr (III), U (IV), and Tc (IV) precipitates

respectively. This has been subjected to whole-scale genomic sequencing. This might

contribute to pattern of genes targeted for expression in D. radiodurans (Daly, 2000).

Expanding on any natural metal remediating capability that D. radiodurans has, is

an alternative path to designing D. radiodurans for metal remediation. Surprisingly, in the

presence of humic acid, U (VI) and Tc (VII) can be reduced by an anaerobe D.

radiodurans). However, Cr (VI) can be decreased in the absence of humic acid. The

modification and enhancement of these functions, might be possible through genetic

engineering by using its genomic sequence as a lead to manipulation.

In DOE wastes, lead, chromium and mercury are the most ubiquitious

contaminants of heavy metals. A series of genetic vectors being analyze in D. radiodurans

that encode resistance to these metals i.e. Escherichia coli (Summers 1986), highly

characterized mer A locus has been cloned into D. radiodurans. Mercuric ion reductase

(Mer A) is encoded by mer A. Highly toxic thiol reactive mercuric ion Hg (II) is reduced by

this enzyme, to much less nearly inert elemental and volatile Hg (0). To regulate mer A

expression, by changing its cellular gene dosage, four different D. radiodurans expression

systems were developed (Brim et al., 2000). Briefly, the Mer A expressing strains of D.

radiodurans shows resistant to the bactericidal effects of ionic Hg (II), at concentrations

(30-50µM), well above the highest concentration recorded for mercury contaminated

DOE wastes sites (10µM). The strains have capability to reduce toxic substances like, Hg

(II) to Hg (0) proficiently. Other metal reducing/resistance activity that are specific to

remediate metal ions, have been cloned into D. radiodurans are being examined include

genes from Desulfovibrio vulgaris (cytc3), U (VI), Ralstonia eutrophus CH34 (czc) Cd(II), Co

(II), ), Zn (II), and Bacillus thuringiensis Cr (VI) (Daly, 2000).

2.12. Conclusion

The intention of this article has been to explore the radio-resistant microbes,

effect of UV on their genome with repair mechanisms. The correlation of different metal

ions in correlation to their survival in presence of high UV and gamma radiation doses.

The complex physiology and other biology based approaches i.e. genomics proteomic and

metabolomics can make these microbes potential candidates for development of

effective therapeutic agents. Obstacles must be overcome to utilize UVR resistant

microbes for the removal of toxic heavy metals left from the cold war. These

extremophiles can also be utilized for uranium bioremediation as they possess the ability

to reduce soluble radioisotopes of uranium into insoluble species. Various extremolytes

and other active enzymes are thought to be produced due to high UV stress that can be

used as biopharmaceutical products i.e anti-cancerous, anticholestrolic and antidiabetic

drugs and other for biodegradation of toxic radioactive compounds in different nuclear

wastes. There are various limiting factors for commercialization of these extremolytes

and extremozymes from UVR microbes. Increase research efforts need to be made via

utilizing Response Surface Methodology (RSM) approach of optimization and screening,

the bioactive compounds from these extremophiles, metabolic engineering could allow

the potent mass production.

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Chapter 3: Isolation and Characterization of Radio-resistant Bacteria

Paper 1

Title: Isolation and Characterization of Ultra Violet Rays (UVR) Resistant Bacteria from Desert

Soil Samples of Pakistan

3.1. Abstract

Living beings or dwellers of xeric environments are able to withstand adverse life

circumstances, desiccation, and extreme temperatures, sturdy thermal gaps and nutrient

deficit. The phylogenetic diversity of ultra-violet (UV) resistant bacteria from desert soil, was

investigated by culture and molecular based analysis. The bacterial strains were observed for

their tolerance to UV doses, salt concentration, and heavy metals. The effect of UV radiation

on cellular protein and lipids was also investigated. A total of 09 UV resistant bacteria were

isolated and identified through biochemical tests and 16S rRNA sequencing. Based on the

results obtained, bacterial strains were assigned to four phyla: Firmicutes, Proteobacteria,

Deinococcus-Thermus and Actinobacteria. High UV survivability was observed in case of

genus Deinococcus followed by Firmicutes. The bacteria were found to grow at wide

temperature and pH range, resistant to high salt concentration as well as various metal ions.

The microbes exhibited minor damages to protein and lipids as a result of exposure to UV

radiation as compared to Escherichia coli (ATCC 10536). The results indicated that these

microbes harbor a sophisticated phenotypic character and molecular repair mechanisms that

can prolong their survival in extreme radiations.

Key words: Radio-resistant, Phylogenetic analysis, Deinococcus-Thermus, Protein

carbonylation, Lipid peroxidation

3.2. Introduction

Radio-resistant is the term referred to the group of organisms that live under

radiation conditions. These organisms surprisingly can endure both ionizing and nonionizing

radiations, which could be lethal to others (Singh and Gabani, 2011). Diverse environments

which encompass dried food, irradiated meat and fish, high level nuclear wastes at Savannah

River in South Carolina, hot and dry desert, and warm fresh water, geothermal spring and at

Hanford in Washington have been investigated for isolation of ionizing-radiation resistant

microbes. Numerious members of the domains Archaea and Bacteria have demonstrated

extreme ionizing radiation resistance (Fredrickson et al., 2004) which appears to be an

attribute of the organisms belonging to genera, Deinococcus and Rubrobacter followed by

Kineococcus, and Kocuria (Phillips et al., 2002).

Bacteria in the extreme Ultraviolet radiation (UVR) environment is consider on of the

crucial exogenous stress factor The interest of several environmental photobiology studies is

concerned with the effects of UVB on bacteria. Exposure to solar UVR is responsible for ROS

induced oxidative stress in aquatic bacteria synthesized via photo-dynamic reactions which

are continuously involved in intracellular or extracellular photo-sensitization (Pattison et al.,

2006). The most important cellular target for ROS is proteins and lipids. These attacks the

polyunsaturated fatty acids in cell membranes and initiate lipid peroxidation, which is

accompanied by a decrease in membrane permeability and interference of transmembrane

ion gradients that eventually leads to cell death. The deleterious effects of ROS are

significantly influenced by metal ion homeostasis (Halliwell et al., 2015). In bacterial cells

which are exposed to UV-B radiations, the potential synergistic effect of some transition

metals like Cu+2, Mn+2 and Zn+2 are also been reported (Santos et al., 2013a). Intercellular

Cu+2 acquisition, presence of high intracellular Mn+2 and Zn+2 uptake by UV resistant

microbes are the adaptive response to peroxides stress by blocking the Fenton and Haber–

Weiss reactions (Bagwell et al., 2008; Daly et al., 2010). Production of different compatible

solutes trehalose and ectoine in high salt concentration play a significant role in ionization-

radiation protection. Ionizingradiation resistance in Halobacterium salinarum,

demonstrating that “metabolic route” with a combination of tightly coordinated

physiological processes contributes to irradiation resistance (Robinson et al., 2011). These

organisms also tend to produce metabolites during their defense against radiations. Recent

biotechnological development helps to identify microbial approach of self-engineering to

tolerate extreme ultra-violet radiations. Therefore, in order to explore the biological

mechanisms involved in the survival under UV radiations, it is necessary to indicate the

diversity of ultra-violet resistant microbes.

The current study focuses on determination of phylogenetic diversity of UV resistant

bacteria isolated from sand samples of Lakki Marwat and Bahawalpur deserts, Pakistan. The

effect of UVB radiations on the intracellular lipids and proteins of UV resistant bacterial

isolates was also investigated by use of standard oxidation assays.

3.3. Materials and Methods

3.3.1. Sampling

Strictly adhering to microbiological standard procedures, soil samples to a depth of

15cm were accumulated aseptically from Lakki Marwat and Bahawalpur deserts, Pakistan,

placed in sterilized polyethylene zipper bags and were carefully taken back to the lab of

Department of Microbiology, Quaid-I-Azam University, Islamabad, and stored at 4°C for

further processing.

3.3.2. Metal Analysis of Desert Soil

3.3.2.1. Sample Preparation

The soil samples were dried at room temperature for 5 days and sieved (2 mm

sieve). 1g of the soil was acidified with 05 ml of concentrated Nitric acid and 10 ml of per-

chloric acid (70% HClO4). The mixture was heated till white, dense fumes of HClO4 appeared

(RAURET 1998). The digested samples were cooled to room temperature, filtered through

Whatman # 41 and boiled to remove oxides of nitrogen and chlorine. Finally, the soil

samples were subjected to Cu+2, Ni+2, Zn+2, Mn+2, Cr+2,

Fe+2, Pb+2, Cd+2, Ca+2, Mg+2 and Na+2 analysis by Atomic Absorption Spectrophotometer (AAS)

on a Perkin-Elmer 460 Spectrophotometer.

3.3.3. Isolation of Radio-resistant Microorganisms

The soil samples were serially diluted and plated on TGY (tryptone glucose yeast

extract) agar by spread plate method. The plates were exposed to UV radiation in 119x69×52

cm UV chamber supplied with 20W and 280nm UV light source

(germicidal lamp) for a specific time (30-300 seconds). The UV fluence rate

(energy/area/time) to the test sample was measured with He=Ee×t in units of J/m2 (SAJJAD

et al. 2017). The total UV dose was determined by time of exposure UV fluence rate. All UV

irradiation procedures were performed under red light to prevent photo-reactivation.

Radiant exposure (He) = the energy that reaches a surface area due to irradiance (Ee)

maintained for a time duration (t).

3.3.3.2. Radiant exposure calculation

Exposure of each TGY plates to UV radiation for 5 min was accomplished in a UV chamber

(119x55×52 cm) prior to incubation, which conferred a 20W, 280 nm UV light placed at the

top.

=0.52 mm

= 0.55 mm

I=Ee

Now radiant exposure He=Ee×t

That is the radiant exposure (He) which can be define as irradiance (Ee) of the plate in time (t)

Where watt

If the exposure of the respective sample on the plate is 30 sec then,

So the Radiant exposure

3.3.4. UV Radiation Tolerance

The UVR resistance among bacterial isolates was determined by the method as

described by Mattimore and Battista, (1996) with some modifications in order to find out

survival curve. The UV-resistant bacteria isolated during preliminary screening were grown in

TGY broth to an OD600 0.5, and then spread on TGY agar. The plates were exposed to UV-B

(280 nm) for the variable doses (30-180 sec) and subsequently incubated at 37ºC. The

surviving fraction was calculated after 24hrs by determining the titer of culture after

irradiation divided by un-irradiated control.

3.3.5. Identification of UV Resistant Microbes

3.3.5. Biochemical and Physiological

Characteristics

Phase contrast microscopy was utilized to determine the cellular morphology of

living cells (usually contained in culture) (Labomed Lx400). The bacteria were grown on TGY

agar plates at wide temperature (15-45°C), pH (4.0-9.0) and NaCl (016%) for 3 days to

determine optimum growth conditions. Moreover, the strains were also assessed for

different biochemical tests such as catalase, cytochrome oxidase as well as starch hydrolysis,

casein, and gelatin according to the procedures outlined by Murray et al., (1981).

3.3.5.2. Sequence Alignment and Phylogenetic Analysis All bacterial strains were subjected to basic steps of genomic DNA extraction facilitated by

DNA extraction kit (QIAGEN). Following amplification of conserved

16S rRNA gene sequences, using universal primer (F27:AGAGTTTGATCMTGGCTCAG, R-

1492:TACGGYTACCTTGTTACGACTT) via PCR, amplicons were sequenced at Macrogen Service

Center (Geunchun-gu, Seoul, South Korea). The obtained sequences were computed for

closest relatives using BLAST tool at the NCBI database and homologs were analyzed for

phylogeny using Molecular Evolutionary Genetic Analysis (MEGA) version 6 (Tamura et al.,

2013). A neighbor-joining tree was constructed based upon distance matrix, for identification

and diversity among UVR resistant extremophiles with naturally occurring microorganisms.

3.3.6. Metal Resistance

Stock solutions (1000ppm) of various transition metals (Co+2, Cu+2, Fe+2, Mn+2, Cd+2, Hg+2, Ar+2,

Cr+2 and Zn+2) were prepared in deionized, filter-sterilized water from the corresponding

metallic salts. Effect of metal ions on bacterial strains was determined by inoculating them

on TGY agar supplemented with different metals at variable concentrations (20-400 ppm)

and incubated at 30°C for 48hrs.

3.3.7. Effect of UVB on Proteins and Lipids of UV Resistant Bacteria

Overnight grown culture in TGY broth was harvested by centrifugation at 10,000 rmp

for 10 mins. The pallets of respective bacterial strain (106 cells/ml) was irradiated with UVB.

The UV dose (2000 J/m2) was calculated by method described previously (Sajjad et al. 2017).

An aliquot of cell suspension was collected before and after irradiation, washed with

ultrapure water, and immediately used for lipid and protein extraction.

3.3.7.1. Lipid Extraction The entire steps of total lipid extraction were performed according to standard

protocols (Baligh et al., 1959). Lipid extracts from irradiated and un-irradiated bacterial cells

were centrifuged (13000×g for 10min, 4°C) and collected and washed with with pure sterile

desalted water. The mixture was vortexed for 5 minutes and dissolved in chloroform (125

μL), methanol (250 μL). This step was repeated 3 times to ensure the extraction procedure.

Finally 6 M HCl (8.4 μL) and chloroform (125 μL) was added and the mixture containing cell

pellets was vortexed adding pure desalted water (125 μL). Upon centrifugation (20 min at

3000×g, 4°C) the total lipid extract was collected in the lower phase and aimed to prompt

quantification by Malondialdehyde assay (MDA). All reagents used for analysis were

procured from Sigma (St. Louis, MO).

3.3.7.2. Protein Extraction Cell suspensions comprised of both Irradiated and un-irradiated, were centrifuged

and the pellets were redissolved in 10 Mm Tris-HCl (pH 8.0). the reaction mixture was

sonicated on ice bath, four times for 5 s (Branson 450, Danbury, CT) and centrifuged again

(15000×g, 10 minutes, at 4 °C). Finally, adding sarcosyl (1.5%, v/v) and 10 mM Tris-HCl (pH

8.0). The mixture was incubated at room temperature (20 min), and collecting the protein

extract by centrifugation (15000×g, 90 min, 4°C). The cell proteins extracted were suspended

in ultrapure water for quantification of degraded products facilitated by 2,4-

Dinitrophenylhydrazine (DNPH) assay. All reagents were procured from Sigma (St. Louis,

MO).

3.3.8. Lipid Peroxidation Assay

Lipid peroxidation results in formation of malondialdehyde (MDA), a lipid

peroxidation marker. The TBA (Thiobarbituric acid) assay was performed by the method

elucidated by Perez et al., (2007) with some modifications, to assess the MDA

concentration.Following standard guidelines encouraged the total lipid extraction

(Bligh et al., 1959). 250 μL of lipid samples from irradiated and un-irradiated cultures was

mixed with 125μL of 20% trichloro-acetic acid. Supernatant was collected, mixed with 0.5 mL

FeSO4 (0.07 M) and incubated at 37°C for 1hr. 300 μL of this solution was mixed with 0.8%

TBA reagent (200 μL), 8% SDS (200 μL) and incubated at 100°C for 60min. The absorbance of

chromophore developed was measured at 535 nm using UV sensitive E. coli (ATCC 10536) as

a control. The MDA concentration is presented as mM of MDA produced per mg of lipids

using a molar extinction coefficient of 1.56×105 M-1cm-1.

3.3.9. Intracellular Protein Carbonylation

Generated ROS promoted protein oxidation i.e “protein carbonylation” which was

detected by using a chemical compound DNPH (2,4-dinitrophenyl hydrazine) (Misra et al., ,

2004). Centrifugation of both irradiated and un-irradiated cell suspensions were done and

the pellets were re-dissolved in 10 mM Tris-HCl (pH 8.0), sarkosyl (1.5% v/v) and incubated at

room temperature for 20 min. The total protein contents were quantified standard protocol

procedure of Lowry et al., (1951). 2mg/ml of the protein extract was incubated with 50mM

PBS (pH 7.4) and 400 µL of 10mM DNPH in 2 M HCl for 2 hrs in dark. The precipitated

proteins in the reaction mixture was re-suspended in guanidine hydrochloride (6 M). The

unbound proteins were washed with a mixture of ethyl acetate and ethanol 50%. The light

transmittance of the supernatant was analyzed using spectrophotometer at a range of 370

nm. A protein control was run in parallel where DNPH was replaced with 2 M HCl. The

protein carbonyl content was expressed in standard units “mM/mg” of protein.

3.3.10. Statistical Analysis

Appropriate statistical examination of results was evaluated by Student’s ttest,and P

value or calculated probability <0.05 was considered as significant. Bacterial cell sensitivities

were studied by plotting the data between the %survivability and their respective UV doses

(Jm-2). Single factor and two-way ANOVA applied for analysis between and within groups.

3.4. Results.

3.4.1. Metal Analysis of Soil Samples

High solar radiation and large temperature oscillations between day and night were

attributes of the sampling areas that were representatives of Lakki Marwat and Bahawalpur

deserts. Figure 3.1 presents physiochemical analysis of soil samples harvested from two

different deserts. Both the samples presented a higher concentration of Mn+2 followed by

Mg+2, Fe+2 and Pb+2 while lower concentrations of Cd+2, Cu+2 and Cr+2 ion.

3.4.2. Isolation of Ultraviolet Rays (UVR) Resistant Microbes

Following exposure of the soil samples to different doses of UV radiation from 30 sec to 300

second with an energy dose ranges from 300-3300 J/m2. After incubation at 30°C for a week,

nine representative colonies were selected, morphological characteristics were noted and

purified on the same medium. Ultraviolet rays (UVR) resistant isolates were processed for

UV, metal, salt and pH resistance and were also identified morphologically biochemically and

molecular characterization based on 16S rRNA sequencing.

3.4.3. Resistance to UVB in Correlation with % Survivability

Selected isolates were analyzed for resistance to ionizing radiation. Radiotolerance

among the isolates was ascertained by exposing each isolate to the UV-B radiation dosage at

which they were isolated (300-3300 J/m2). Following prolonged period of UV-B exposure to

soil samples, it was found that samples exposed to UVR possessed a significantly lower

number of bacterial colony-forming units when compared to samples not exposed to UV-B

radiation over the whole study period (P< 0.01) (Fig. 3.2). The initial dose of UV-B LD50

(2.0×103 J/m2) was found to be lethal for most of the isolates and determines the bulk of

dose indispensable to kill 50 percent of a tested population. It was noted that with an

increase of UV dose the CFU count decreases and these ultraviolet rays (UVR) resistant

microbes can withstand to UV dose up to a certain extent, after that dose when gain there is

a rapid decline observed in individual microbe. Out of all the 9 isolates WMA-LM19 was most

sensitive and can withstand up to 1.30x103 J/m2energy. In case of WMA-LM9, WMA-LM30

and WMA-BD1 the percent survivability was noted as 79%, 68%, and 45%, even after

receiving high doses of radiation and were considered as most potent UV resistant microbes.

3.4.4. Identification of UV Resistant Bacterial Strains

3.4.4.1. Morphology A highly diverse kind of bacteria was observed on un-irradiated TGY plates as

indicated by their colony morphology, in comparison to the irradiated TGY plates usually

comprised of colonies of different colors such as yellow, orange, pink, or red. There was

Inverse relationship established between UV dose and CFU, demonstrated that Increase in

doses of UV radiation led to the decrease in the number of CFU/g retrieved from the soil

samples. After UV irradiation the UVR resistant isolates were examined morphologically and

microscopically Table 1 shows the cellular morphology and Gram’s reaction of all the UV

resistant bacteria from deserts sample.

3.4.4.2. Biochemical and Physiological Characteristics of UV Resistant Microbes Biochemical, physiological and other characteristics, i.e. temperature, pH range and salt

tolerance of ultraviolet rays (UVR) resistant microbes were also investigated. Table 2 shows

biochemical and other physiological characteristics of these isolates. Results revealed that all

these isolates have a good potential to grow at high salt concentration ranges from 2% to 16

with a broad pH (6-10), temperature (20-45) range and capability of producing different

hydrolytic enzymes like amylase, protease, gelatinase, DNase and others shown in the table

2. 79% of the UVR isolates showed activity of different hydrolytic enzymes.

3.4.4.3. Molecular Characterization and Phylogenetic Analysis of UVR Bacteria Partial sequencing of 16S rRNA gene of 9 isolates, representing dominant

morphotype cultured on plates, led to the assessment of phylogenetic heterogenity. The

tree with the highest log likelihood (-2463.3793) is shown. The analysis involved 54

nucleotide sequences; 462 positions while all missing data and gaps already eliminated.

Evolutionary analysis was conducted in software MEGA 6.0. Assigned accession numbers of

the associated sequences are presented in parentheses after the strain designation.

Numbers at nodes are percentage bootstrap values based on 1,000 replications. The tree is

drawn to scale, with branch lengths measured in the number of substitutions per site (Fig.

3.3).

The sequences derived are associated to four bacterial phyla: Actinobacteria (1

isolates), Proteobacteria (3 isolates), Firmicutes (3 isolates) and DienococccusThermus group

(2 isolates). Among these isolates, majority (8 out of 9) manifested close relationships (99%

of similarity) with well-identified species that are widely disseminated in soil (see Table 1).

Proeteobacteria are dominated by 3 species shown distant relationships to the genera

Stenotrophomonas, (93–99% of similarity). Two isolates were clustered near phylum

Deinococcus-Thermus and showed 99% similarity to Deinococcus sp. which is considered to

be among the most UV resistant organisms up till now. While three Firmicutes isolates

clustered near the genera Bacillus (2) and Staphylococcus (1) with 99% similarity (Fig. 3.3).

Finally,

Actinobacteria comprised of only one isolate that showed minute relatedness to genus

Kocoria (99% similarity), as shown in table 3. It is seeking attention that the 16S rRNA gene

sequences of the 9 isolates and database sequences, obtained from different

environments(arid, semi-arid environments and polluted soils) have close relatedness. (Table

2.1)

3.4.4.4. Nucleotide Sequence and Excision Number Pure culture -based driven 16S rRNA sequences were submitted in the GenBank

database with following accession numbers: KT008382 (Stenotrophomonas maltophilia

WMA-LM10); KT008383 (Stenotrophomonas sp. WMA-LM19);, KT008384 (Deinococcus sp.

WMA-LM30);, KT008385, (Bacillus licheniformis

WMA-BD2); KT008386 (Staphyphylococcus lugdunensis WMA-BD4); KT008387

(Kocuria turfanensis WMA-BD1); KT008388 (Bacillus pumilus WMA-LM4);

KT008389 (Deinococcus radiopugnans WMA-LM9); KT008390 (Bacillus subtilis WMA-LM15).

3.4.5. Minimum Inhibitory Concentration of UVR Microbes to Different Metals

Preliminary experiments were conducted with different metal concentrations. The

majority of the UV resistant strains were sensitive to Hg+2, WMA-LM10 is considered to be

more resistant to Hg+2 (40ppm). All isolates were more resistant to Mn+2, Co+2, Cr+2, and Ni+2,

which can be directly correlated to UVB resistance. In the presence of Mn+2, an interesting

change in cultural characteristics of strain WMALM9, WMA-LM30 and WMA-LM19 was

observed, that is an increase in size of the colony and brighter coloration. Some strains, for

instance, WMA-BD1, WMA-LM15, WMA-LM9, WMA-LM30 (240-280 ppm) had

demonstrated greater potential for intracellular Cu ion sequestration that displayed a

protective barrier against the detrimental effects of ionizing radiation. Mn+2 ions also

safeguard against oxidative damage induced by several exogenous stresses, including UV-B,

gamma-irradiation, wet and dry heat and H2O2.

3.4.6. Effect of UVB on Whole Cell Proteins

Effect of UVB on the whole cell protein was measured, UV resistant isolates provide

a strong protection to cellular protein as compared to E. coli (ATCC 10536) that is a UV

sensitive strain run as a control. Strain WMA-LM9, WMA-LM30, WMA-BD1 and WMA-BD2

shown in the figure have a strong potential to survive in high UV dose and protected their

protein. An increase in total intracellular protein was observed in strain WMA-LM4 (Fig. 5).

Table 3.1: Microscopic characteristics with cultural morphology of UV resistant bacteria

isolated from desert soil samples.

Culture code Sampling site Morphology Microscopy

WMA-BD1 Bahawalpur

desert

Small to medium sized light pink colored mucoid

and circular, raised colonies with entire margins

G (+) cocci

WMA-BD2 Bahawalpur

desert

Large light off-white oval shaped colonies dry

surface, forming crystal like structure when

aggregates

G (+) rods

WMA-BD4 Bahawalpur

desert

Medium to large yellow colored circular raised

colonies with entire margins

G (+) rods

WMA-LM4 Lakki Marwat

desert

Small to medium sized off white smooth and

circular, flat colonies with entire margins

G (+) rods


desert

Medium brick red colored colonies mucoid circular

with entire margins

G (+) cocci


desert

Large flat off white in color with dry surface

colonies with irregular margins

G (˗) rods


desert

Large flat off white in color oval shape dry colonies

with entire margins

G (˗) rods


desert

Large off white in color, flat colonies with shiny

surface, circular with entire margins

G (˗) rods


desert

Medium brick red colored colonies with dry

surface, circular with entire margins occur singly

or tetrads

G (˗) cocci

Figure: 3.1: Metal analysis (in ppm) of soil samples collected from deserts. No significant

difference was observed among the groups as p>0.05

Figure 3.2: Survivability of total UVR resistant isolates from desert soil at varying UV-B

exposure. % survivability was measured using the formula N1/N0*100. N1 is the number of

colonies after UV irradiation while N0 number of colonies after UV irradiation. Statistically it

is proved that there is a significant effect of dose level (Jm2) on UVR isolates and significant

effect of UV on various strains at different time exposure (p<0.05).

Table 3.2: Biochemical and physiological characteristics of UV Resistant isolates from Lakki

Marwat and Bahawalpur desert soil.

Characteristics Bacterial Strains

WMA-

LM9

WMA-

LM4

WMA-

LM15

WMA-

LM19

WMA-

LM30

WMA-

BD1

WMA-

LM10

WMA-

BD2

WMA-

BD4

Temperature 10-30 25-37 25-45 20-45 10-30 20-35 20-45 25-40 20-37

pH 7-8 7-9 6-9 6-10 7-8 7-9 6-10 7-10 7-9

Salt tolerance 2% 14% 16% 12% 6% 12% 10% 10% 10%

Catalase + + + + + + + + +

Oxidase - - + + - - + - -

Amylase + - + + - + + + +

Protease + + + + + - + + +

Gelatinase + - + + + + + + -

DNase + - + + - + + - -

Table 3.3: 16S rRNA sequence homologues, closest related species, % survivability, gene

bank accession number and query coverage of Ultraviolet radiation (UV subtype –B)

resistant isolates from desert samples.

Isolates GenBank

Accession

Number

Closest related

Species

Query

Coverage

%

similarity

Score* % UVR

resistance

J/m2

Survival

Rate

(%)

WMA-BD1 KT008387 Kocuria

turfanensis 100 99 3.3×103 45.45

WMA-BD2 KT008385 Bacillus

licheniformis 100 99 2.0×103 43.18

WMA-BD4 KT008386 Staphylococcus

lugdunensis 100 99 2.0×103 48.27

WMA-LM4 KT008388 Bacillus pumilus 99 99 2.60×103 45.28

WMA-LM9 KT008389 Deinococcus

radiopugnans

100 99 3.30×103 79.47

WMA-LM10 KT008382 Stenotrophomonas

maltophilia 100 99 1.30×103 46.15

WMA-LM15 KT008390 Bacillus subtilis 100 99 3.30×103 38.72

WMA-LM19 KT008383 Stenotrophomonas sp. 99 93 1.30×103 51.69

WMA-LM30 KT008384 Deinococcus sp. 100 100 3.30×103 68.03

Figure 3.3: Phylogenetic analysis of UV resistant bacterial strains by maximum likelihood

method based on Tamura-Nei model (1993). Bar=0.05 sequence variation.

3.4.7. Protein and Lipid Oxidation of UVR Isolates

UVB can strongly affect the cell lipids and proteins and can cause a serious damage

to cells. The effect of UV on cellular protein oxidation and lipid per-oxidation was measured

using E. coli (10536) UV sensitive strain. E. coli (10536) displayed a significant damage to its

cellular lipids and protein upon UV treatment with lipid oxidation of 12 µM and protein

oxidation 189 mM/mg. Figure 2.5 revealed that strain WMA-LM9, WMA-LM30 and WMA-

BD1 having a strong scavenging system to

detoxify the different superoxide’s that can damage the cell protein and lipids. A lower lipid

oxidation (6.3µM) and protein oxidation (128mM/mg) was measured in WMA-LM9 followed

by WMA-LM30 (8 µM, 138 mM/mg) and WMA-BD1 (10 µM, 135 mM/mg) respectively.

Figure 3.4: Effect of UVB on total cell protein content in mg/ml of radio-resistant bacteria from

desert soil samples.

Table 3.4: Effect of the metal ions (in ppm) on growth of UVR resistant bacteria from desert

samples on TGY agar plates. Values are shown in ppm.

Strain code Cd+2 Zn

+2 Cr+2 Fe

+2 Cu+2 Ni

+2 Hg+2 Ar

+2 Mn+2

WMA-BD1 380 160 360 360 280 280 10 320 300

WMA-BD2 200 200 320 360 200 200 10 300 260

WMA-BD4 240 240 300 360 200 280 20 300 240

WMA-LM4 360 280 360 300 240 280 0 280 200

WMA-LM9 200 80 380 280 200 280 0 200 220

WMA-LM10 240 200 300 360 200 280 40 300 300

WMA-LM15 220 280 360 360 240 360 0 320 340

WMA-LM19 280 120 280 360 200 200 10 300 280

WMA-LM30 280 200 360 200 280 380 0 280 340

Figure 3.5: Protein oxidation to quantify carbonylated protein and lipid peroxidation assay for

TBARS in UV treated isolates from desert soil.

3.5. Discussion

The soil samples were collected in June-July from Lakki Marwart and Bahawalpur deserts

in Pakistan. Few environmental studies have been investigated by scientists on the bacterial

community inhabiting the extreme environment like high hypersaline environment and high UV

radiations, no screening programs have been designed to explore these UV resistant microbes for

different hydrolytic enzymes and other therapeutic agents of biotechnological use. These

microbes can not only be a potential source for enzyme industries, but also their secondary

metabolic products by which these microbes prevent themselves from high UV dose could be a

potential source of drugs for aging and cancer. The high correlation of UV, salt and metal's

resistance investigated in this study will make the readers more attractive to investigate the

molecular mechanisms of this synergistic effect in the microbes for UV resistant. The

physiochemical analysis of the soil samples collected from these deserts indicated that Mn+2,

Mg+2, Fe+2 and Pb+2 were the most abundant ion followed by Ca+2, Ni+2, Na+2 and Zn+2. Studies

associated with scrutinization of several biological systems have validated the significant role

exhibited by transition metal ions in protecting against the damaging effect of radiations, wet and

dry heat and H2O2 (Daly et al., 2004; Bagwell et al., 2008; Ghosh et al., 2011). Desert varnishing

contributes to the existence of high levels of Mn+2 in sand formation. During the process of sand

formation in the desert, the occurrence of manganese oxide in rock varnish has an efficacious role

in obstructing the transmission of ultraviolet radiation. Perhaps the microbes which dwell in the

rock have attributes of synthesizing their own manganese-formula sunscreen. The bulk of varnish

together with manganese oxides confers the dark color to desert soil (Fleisher et al., 1999).

When 16S rRNA gene sequences were compared, most of the UVB resistant bacteria were

gram positive and assigned to four different clusters. 47% of the ultraviolet resistant isolates were

from Fermicutes phylum followed by Deinocuccos the most UV resistant genera, Proteobacteria

and Actinobacteria Deinococcus thermus and Actinobacteria showed 99% similarity to

Deinococcus sp. and Kocuria turfanensis Gram positive pink colored bacteria on TGY agar. Our

findings were accordance to (Flores et al., 2009; Moreno et al., 2012). Confinement of four

radiotolerant species of the Bacillus class, elucidates its well documented radio-tolerance as they

are resistant spore formers (Nicholson et al., 2000). The hydrolytic bacteria in our studies

predominant and were from four distinct clusters or phyla with high survival rates at high dose of

radiations. Other studies showed only -Proteobacteria that dominates in hydrolytic enzymes

production in extreme environ (Rohban et al., 2009; Baati et al., 2010) where only a few of the

Firmicutes were found. Isolation of Proteobacteria- related species in the waste contaminated

with radio-isotopes of radionuclide were studied for its survival that exhibited resistance to 2.5

kGy of gamma radiation, with 0.0017% survival was given by Fredrickson and colleagues (2004).

The stimulation of bacterial growth with UVB exposure has already been reported on

culturable bacteria and in total community analysis as well (Dib et al., 2009). The number of cells

increases rapidly after exposure to UV and this increase in population and diversity under UVB

stress need deeper studies in order to explain the mechanism triggered by radiations that

enhances cellular survival and replication (McGlynn and Lloyd 2002). Most of the UVR isolates

showed colored compounds when grown on TGY agar plates after the exposure to UV that may

absorb radiation in order to protect the cells from damage. The production of UV absorbing

compounds might be induced as a result of exposure to radiation stress. We have reported a UV

resistant bacterium that has 91% similarity to Stenotrophomnas sp. representing an interesting

phenomenon of enlargement in colony size and light pink to red coloration upon radiation

exposure in presence of Mn+2. These secondary metabolite or extremolytes production during

UVR exposure can be of high significance in pharmaceutical industry. Deinococcus genus is well

known for their extreme resistance to UV radiation. Previously a number of studies have been

carried out to isolate radio-resistant bacteria from desert soil, radiation resistant property is the

result of evolution that protect cells from desiccation (Rainey et al., 2005). Recently it has been

proposed that Deinococcus possesses a remarkable ability to cope with adverse conditions such

as UV radiation and desiccation. This resistance is due to binding of the S-layer protein DR-2577 to

deinoxanthin that could suggest its protective role against these two stresses (Farci et al., 2016).

The ability of these UV resistant microbes to survive in several extreme conditions is suggested to

be as a result of three combine mechanisms like prevention, tolerance and repair (White et al., ,

1999).

The 09 UV resistant microbes were found to produce hydrolytic enzymes like amylase,

DNase, protease, and were capable of growth at high salt concentration ranges from 2-16%. The

results demonstrated a strong linkage between salt, metal and UV radiation tolerance. Metals like

Mn+2, Cu+2, Zn+2 and Co+2 enhance the survivability of UVR microbes up to great extent These

metals block the Fenton reactions and play an indirect role to prevent the formation of several

toxic oxides and byproducts, which can alter the different cell membranes (Imlay 2008; Baati et

al., 2010; Santos 2013). Responses to heavy metals tested in this study were very homogenous

and all the strains isolated from both deserts showed high resistance to different metal ions with

exception of Hg+2. This suggests that these UV resistant microbes express a very homogeneous

behavior in connection with their individual natural resistance to different metal ions tested. The

majority of the isolates showed resistant to Mn+2, Cr+2, Co+2, Ni+2 and Zn+2 which play an important

role in the cell resistance to UV. The possible protective role of several metal ions against UV

radiation has been discussed recently. Kineococcus radiotolerans (Actinobacteria) showed a high

ability for intracellular copper ion sequestration that provided protection against the deleterious

effects of ionizing radiation (Asgarani et al., 2012; Paulino-Lima et al., 2016). Manganese (Mn2+)

ions also prevent oxidative damage instigated by several stresses encompassing UV-B radiation,

gamma-irradiation, wet and dry heat and H2O2 (Daly et al., 2007; Barnese et al., 2008; McEwan,

2009; Daly, 2010; McNaughton, 2010; Slade, 2011). Adaptive response is mediated by the uptake

of Zinc (Zn2+) which confers resistance against peroxide stress (Gaballa et al., 2002), thereby act as

a barrier for copper-treated Escherichia coli against superoxide killing (Korbashi et al., 1989) and

opposing the effects of oxidative stress in Lactococcus lactis (Scott et al., 2000). Sensitivity to Hg+2

by all isolates might be due to the fact that it has not been found to be essential for the UVR

biological activities, furthermore the strong effect of Hg to cells relates to their strong affinity for

thiol groups in proteins (Robinson et al 1994; Velasco et al., 1999). The ability of these microbes to

grow in high UV radiation and high metal concentration make these more attractive for in-situ

bioremediation of radioactive wastes.

Meticulous comprehension of the molecular effects of UVR on bacteria may pave the way

for an indulgent of environmental repercussion of intense UV levels affiliated with global climate

changes and will be followed by the optimization of UV-based disinfection strategies. Bacterial cell

features such as small size,unavailability of effective UV-protective pigmentation and short

generation time confers susceptibility to the effects of UV radiation, (Garcia-Pichel, 1994). The

biological effects of UV radiation on all the resistant isolates were assayed that showed different

survival rate and protein carbonylation and lipid peroxidation. The lipid and protein damages

were analyzed by standard assays in UVB oxidative damages which are characterized by

thiobarbituric acid reactive substances (TBARS) accumulation and loss of main metabolic activity.

These UV damages also resulting in alteration in cell protein and lipids that ultimately hinder the

growth or kill the cell. Deinococcus genra was considered the most resistant with less protein and

lipid damages as compared to other isolates and E. coli (10536) ATCC used a control. The UV

sensitive strain E. coli (10536) displayed significantly higher number of protein carbonyls than

resistant isolates. The consequences associated with UVR exposure were determined by gas

chromatography (Santos, 2013). An increase in the methyl groups was observed in the lipids upon

its oxidation to UV. It is also investigated that change in the lipid chains and its composition occurs

to the stress for survival.

In addition to DNA damages by high UV exposure changes in the lipid membranes and

protein tertiary structure also have a crucial role in bacterial inactivation. The targets (e.g. nucleic

acids, proteins, lipids) for UV radiation inactivation may differ among the genus, species and

strains and thus all these factors contribute to the prolong cell survival in high UV radiations It has

also been suggested that UV-induced DNA damage in Gram-positive bacteria is lower than that in

Gram-negative bacteria because of a shielding effect by the cell wall (Jagger, 1985). Presence

Mn/Fe ratio is another factor that can contribute to cell resistant in high radiations. Presence of

high concentration of Fe+2 in Shewanella oneidensis MR1 makes it sensitive to UV radiations.

These intracellular Fe promotes the formation of ROS via Fenton type reactions (Qiu et al., 2005).

The efficiency of the defence and highly sophisticated molecular repair mechanisms, that may

also differ among bacteria play an important role in cellular resistance in extreme environments

(Arrieta et al., 2000; Matallana-Surget et al., 2009; Santos et al., 2011).

3.6. Conclusion

Analyzing and characterizing the culturable UV resistant bacteria from deserts, this study

comes up with implications such as the discovery of a unique environment for UV resistant

microbes in the context of Pakistan, production of UV absorbing compounds and investigation for

heavy metal tolerant microorganisms. These results open the exiting standpoints on investigating

bacterial lenience to desiccation, radiation and survey in the deserts. The implication of the

outcomes is conferred from an environmental and industrial perspective and with admiration to

potential expansions in UV-based disinfection technologies.

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Chapter 4: Extremolytes Extraction and Bioassays

Paper 2

Title: In-Vitro Cytotoxic and Antioxidant Activities of Extremolytes from RadioResistant

Bacteria

4.1. Abstract Historically the study of microbial driven natural product as drug has largely ignored the

radio-resistant extremophiles. From the limited courtesy received, many compounds of

extremophilic origin have been described that displayed anticancer drugs, cholesterol-lowering

agents, antibacterial activities and tools for research across a range of potencies. The secondary

intracellular bio-active compounds isolated from radio resistant microbes were assayed for

cytotoxic, antioxidant and antibacterial activities to evaluate their potential to be considered as

therapeutic drugs. The methanolic extracts of these radio-resistant isolates were also scanned for

it excitation wavelength using photo luminescence spectroscopy. 2,2-diphenyl-1picrylhydrazyl

(DPPH) reducing assay, 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay

using HeLa cell line and the disc diffusion test was carried out in order to evaluate the partially

purified crude extracts. A pronounced cytotoxic effect on HeLa cell lines and anti-oxidant activity

was shown by Deinococcus WMA-LM9 (50 µg). Similarly, the high anti-bacterial activity was

detected in case of extract from Stenotrophomonas sp. WMA-LM19 against N. gonorrhoea MS11

and Rhodococcus sp. The current research work concluded that isolated strains from extreme

environment have great potential to produce potent metabolites with a wide range of anti-

oxidant and cytotoxic activities. Exploration of these microbes and metabolites are basic need for

new drug discovery.

Keywords: Radio-resistant, Deinococcus WMA-LM9, Stenotrophomonas sp. WMALM19, DPPH

assay, MTT assay, Anti-bacterial assay

4.2. Introduction Ionizing radiation, distinctly, the low linear energy transfer (LET), happens to destroy the

vital macromolecules like protein structures, lipid membranes, DNA and RNA of the target cell by

generating free radicals. Cellular targets, i.e. macromolecules, suspended in the biological

aqueous environment react with high energy free radicals and become ionized or convert into

toxic free radicals (Halliwell et al., 2015). These types of chemical alterations lead to transformed

cell metabolism, structural and functional damage and cell death by disrupting molecular

organization (Goodson et al., 2015). Radiation-induced disruptions in DNA includes damaged

pyrimidine and purine bases, cross-linking of DNA with adjacent protein molecules, removal of

bases and single and double strand break (Rastogi et al., 2010). The most adverse effect of

irradiation in general is on lipid as lipid peroxidation in all biological membranes while

mitochondrial membrane in particular. Lipid peroxidation leads to the formation of short chain

fatty acyl derivatives, which interfere in lipid-lipid as well as lipid-protein cross-linkages formation

(Pizzimenti et al., 2013). This type of interference encourages drastic deformity in biological

functions also protein denaturation and cleavages of disulphide bond in protein and oxidation of

approachable amino acids leads to oxidative stress. All these changing leads to membrane

permeability and fluidity which trigger the release of effective physiological mediators of

apoptosis (Upadhyay et al., 2005). These stresses induced protein and lipid modifications leads to

premature aging cancer.

The radio-resistant microbes are supposed to have a high efficiency system for proteome

protection as compared to genome (Santos et al., 2013). Proteome is believed to ensure recovery

of cell from adverse radiation effects by molecular repair, followed by repair of disintegrated DNA,

that’s the reason why death correspond with protein damage instead of DNA by irradiation. The

proteome conserves and maintain life while genome makes sure the uniformity of life by

renewing proteins. The process depends on the preceding proteome that restore, replicate and

expressed genome. Moreover, other small metabolites cofactors for protein interaction and

catalysis are of equivalent importance of proteome functionality however the role of protein

damage on conservation of life is underated in biology and medicine (Krisko et al., 2013).

However, considering above fact, many therapeutic agents, having ability to rescue organisms

from radiation injury has been discovered (Ghose, 1983; Nair et al., 2004). Many health benefits

are associated with biological activities of carotenoids and other bio-active compounds like

antioxidants and precursors for vitamin A biosynthesis (Grune et al., 2010). The carotenoid rich

dietary intake is associated with enhancement of the immunity and devaluation of liability for

degenerative diseases such as cancer, cardiovascular defects, macular degeneration, and cataract

(Krinsky et al., 2000; Fiedor et al., 2014). These benefits diversified the novel functionalities of

carotenoids in foods, cosmetics, and pharmaceutical applications. A monumental documentation

available about the expression of secondary metabolite genes under stress conditions. The

transformation of a single variable or factor and provoking a stress response in growth conditions

of microbes has already been investigated in one strain many compounds (OSMAC) approaches

for the exploration the possibility of secondary metabolites through various strains of

cyanobacteria (Edwards and Ericsson, 1999). A group of oxigenases (hemoprotein) like

Cytochrome P450s CYPs are ubiquitous present in all domain of life (Nebert et al., 1989). Modern

biotechnological approaches could be used to activate the induction of the biosynthesis of

radiation responsive pigments/bio-active compounds and cellular metabolites which can be

utilized by other organisms to provide protection to live in a radiation rich environment (Singh,

2011). In UV-light, UV-resistant microorganisms can be grown in these conditions leads to

induction of genes that produce metabolites which defensive against harmful radiations. It has

been expected that useful drugs exclusively anticancer and antibiotics and further agricultural

products of commercial implications can be obtained these compounds i.e extremolytes (Kumar

et al., 2010). The Deinococcus radiodurans is well-documented radio-resistant microorganism, its

survival lies in the fact that it is able to induce certain genes, proteins and DNA repair mechanism

enzyme (Hockberger, 2002).

Development has been made to search extremophiles having ability to produce

extremolytes with an indication as productive as well as therapeutic agents. The modern

biotechnological techniques and biosynthesis of radiation responsive pigments or bio-active

compounds could be enhanced to furnish a chance for other organisms to sustain radiation rich

surroundings (Singh and Gabani, 2011). Numerous extracts (i.e. extremolytes) are known to yield

vital drugs, specifically antibiotics and anticancer drugs (Kumar et al., 2010). These extremolytes

are of prime importance for industrial use as it has been investigated, however, therapeutic

intimations are remade to investigate. The challenges for further research include the

implementation of these UVR resistant antimicrobial, anti-oxidant is to prevent aging, DNA

mutation and cancer related protein modifications (Slade and Radman, 2011).

A comprehensive review has been made on bioactive compound derivatives from ultra

violet radiations (UVR) resistant bacteria. Their remarkable biological properties as antimicrobial,

cytotoxic activities using HeLa cell lines and detailed antioxidant system in terms of hydroxyl

radicals scavenging for toxic superoxide’s was studied.


4.3. Materials and methods The radio-resistant bacteria were isolated from Lakki Marwat and Bahawalpur desert soil of

Pakistan. Isolates were identified on the basis of method described in

Bergey’s Manual of Systematic Bacteriology (Buchanan and Gibbons, 1974; Wilson,

1987) and phylogenetic analysis was done on the basis of 16S rRNA sequence (Marchesi et al.

1998).

4.3.1. Growth Medium

The isolates were grown in tryptone glucose yeast (TGY) medium (5% Bactotryptone, 1%

glucose, 3% Bacto yeast extract) and incubated in shaking incubator at 30°C with 150 rpm for 72

hrs.

4.3.2. Collection of Intracellular Crude Extracts

500 mL of each UVR resistant bacterial isolates were grown with steady shaking under

aerobic conditions. Cells were collected after 40hrs of incubation by centrifugation at 5000xg for

10 min. The cells in sediment were used to collect intracellular extract. Once washed with

sterilized water, the cells were re-suspended in 5 mL chloroform:methanol (7:3), probe sonicated

and then washed with 100% dichloromethane to ensure extraction process, finally the mixture is

centrifuged. The supernatant with solvent mixture was evaporated and the dried intracellular

extract was collected on freeze drying. The dried crude extract was then again put through solid

phase extraction using 500 mg of C18 cartridge with methanol/water (50:50 v/v)

acetone:methanol (7:2 v⁄v) and dichloromethane and methanol (2:1).

4.3.3. Excitation vs Emission Spectra by using Photoluminescence Spectroscopy

Photoluminescence spectroscopy is a non-destructive method that is concerned with

transitions of photons from the excited state to the ground state. Fluorescence spectra of

methanolic extract were taken on an LS-50B fluorescence spectrometer (Perkin-Elmer Corp.,

Forster City, CA, USA) with an external 980 nm laser as excitation source. The spectrum range

used (300–900 nm) with a double beam Fluorescent using a 5-cm path quartz cuvette against

methanol as a reference.

4.3.4. Sample Preparation for Biological Assays

The intracellular crude extract was dispersed in dimethyl sulphoxide (10mg/mL DMSO),

protected from light and stored at room temperature. For the cytotoxic assay, the extract volume

was set in such a way that the final concentration of DMSO does not exceed 0.1%.

4.3.5. DPPH Radical Scavenging Activity

The antioxidant ability of the extracts was evaluated by a standard procedure method

described by Rao et al., (2006), using 2,2-diphenyl-1-picrylhydrazyl (DPPH). Different

concentrations of the partially purified extract (ranges from 5-20µg) were added to ethanolic

solution of DPPH (0.2mM), only DMSO was mixed in case of control. The reaction mixture was

then vortexed and incubated for 30 min at room temperature under dark condition. The

absorbance of the reaction mixture was measured at 517 nm using spectrophotometer. The

scavenging capacity of the extracts was measured by a decrease in the absorbance of DPPH (a

negative control). Ascobic acid was run as positive control.

4.3.6. Cytotoxic Evaluation of Crude Extracts

In vitro tests were performed on HeLa cells to check cytotoxic effect of extract using a rapid

colorimetric assay with MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5Diphenyltetrazolium Bromide) and

untreated controls were also present to compare results. The HeLa cells were purchased from

ATCC by Mark Leid's lab (Department of Pharmaceutical Sciences, Oregon State University)

cultured in RPMI-1640 (Roswell Park Memorial Institute) (Gibco, Scotland) in 75 cm2 tissue flask

(Nunc, Denmark), cultured in the T75 flask with MEM (minimal essential medium) 10% FBS (fetal

bovine serum) and 1% pen/step (penicillin/streptomycin). The assay is based on the reduction

potential of the soluble MTT of tumor cells by the mitochondrial enzyme action (2000 and 10,000

cells/well), into an insoluble product called formazan. Which can be measured

spectrophotometrically after dissolving in DMSO (Denizot and Lang 1986). The cytotoxic effect of

the extract was indicated as relative viability (% control) and is calculated. Percentage of the cell

survival in negative control was presumed as 100% (Ngeny et al., 2013).

4.3.7. Evaluation of Antimicrobial Activity

Analysis of antimicrobial activity of extracts was assayed by the agar diffusion test against

clinically and ATCC strains on agar plates. Neisseria gonorrhoea MS11 (Meyer et al., 1982) strains

were cultured on gonococcal base (GCB) solid medium

(Difco) augmented with Kellogg’s supplements I and II (Kellogg et al 1982; Spence et al., 2008) at

37°C, in the presence of 5% atmospheric CO2 for 20 hours. Nonpalliated variants of bacterial

colonies were sub cultured in fresh GCB medium for additional 20 hours. Vibrio cholera N16961

(laboratory collection) was cultured on LB agar (Difco) plates medium at 37°C. Rhodococcus

fascians ATCC 12975, S. aureus ATCC 6538, E. coli ATCC 11303 and Pseudomonas aeruginosa

ATCC 10145 were cultured on Muller Hinton agar (MHA) plates. The antibacterial activity was

analyzed by following standard guidelines of Clinical and Laboratory Standard Institute (CLSI) by

using disk diffusion method (Schwalbe et al., 2007; Mbaveng et al., 2008). Antimicrobial activity

was determined by measuring zones of inhibition in mm and the results recorded in triplicate. The

extract fractions with zone of inhibition more than 10 mm were considered to be active.

4.3.8. Statistical analysis

Mean, standard deviation (SD) and coefficient of variation (CV) were determined, for

concentrations of each drug. Significance was presumed at p<0.05.

4.4. Results

4.4.1. Solid Phase Extraction of intracellular metabolites Once the intracellular metabolites extracted in a solvent mixture, dried and further fractionated

to 3 fractions in water: methanol, acetone/methanol and dichloromethane vacuum evaporated

and dissolved in DMSO for further studies.

4.4.2. Solid Phase Extraction of Intracellular Metabolites

Once the intracellular metabolites extracted in a solvent mixture, dried and further

fractionated to 3 fractions in water:methanol, acetone:methanol and dichloromethane vacuum

evaporated and dissolved in DMSO for further studies.

4.4.3. Excitation of Methanolic Extract Photo-excitation aids electrons within a material to move into legitimate excited states.

Extracts of Deinococcus sp. WMA-LM9 showed a greater excitation state followed by Kocuria sp.

WMA-BD1 and Deinococcus sp. WMA-LM30. The excitation is due to the colored part or

chromophore of these compounds. All the compounds extracted from these UV resistant

microbes showed a peak of photoluminescence at wavelength ranges from 400-600 nm as shown

in figure 4.1.

.

Figure 4.1: The fluorescent quenching spectrum of methanolic extracts 1mg/ml from UVR

bacteria with an excitation wavelength of 316 nm. The data was plotted by using Origin 8.6

software.

4.4.3. Cytotoxic activity of the crude extracts MTT staining method revealed about the cytotoxic effect of different metabolites extracted from

all isolates. Our results demonstrated that the extracts of 5 isolates had strong effects on HeLa

cells mostly in a time-dependent manner (Fig. 4.2). The most remarkable activity against HeLa cell

lines was shown by methanolic fraction of Stenotrophomonas sp. WMA-LM19, Deinococcus sp.

WMA-LM9 and Kocuria sp.

WMA-BD1 and showed 90% killing of HeLa cells when used in concentrations of 50 µg.

4.4.4. Comparative IC50 Values of Extracts by Anti-oxidant Assay

The DPPH radical scavenging activity of different extracts was measured as shown in

figure 4.3. The scavenging activity was found to be concentration dependent. Deinococcus sp.

WMA-LM9 (F2; methanolic fraction) and Deinococcus sp. WMA-LM30 (dichloromethane extract)

was the most potent one with having IC50 of 10 µg. IC50 was acquired by linear regression analysis

of dose response curve that has been plotted between % inhibition and carotenoid concentration

that led to 50% inhibition of free radical activity of DPPH.

4.4.5. Anti-microbial Activity of Fractionated Extracts Significant anti-microbial activity against N. gonorrhoea MS11, E. coli ATCC 11303,

P. aeruginosa ATCC 10145, S. aureus ATCC 6538 and R. fascians ATCC 12975 by UV resistant

microbial organic and aqueous extracts. The highest activity was achieved by WMA-LM19-F1 and

F2 against N. gonorrhoea MS11 and R. fascians. ATCC 12975 (25mm) followed by WMA-LM9-F2

(22 mm and 18 mm) respectively

(table 4.1 and figure 4.4).

Figure 4.2: The cytotoxic effect of partially purified fractionated extracts on HeLa cell line by

using MTT assay. The various concentrations of extracts used were 50 µg/mL. The data represent

the percentage (%) inhibition. Values are expressed as mean ± SD (n=3). F1= Water:methanolic

fraction, F2= acetone:methanolic extract and F3= Dichloromethane fraction.

Figure 4.3: Comparative analysis of IC50 values of different fractionated extracts of UV resistant

microbes. IC50 of microbial extracts used for all the activities are measured in µg/ml. Data is

expressed as mean ± SD (n=3). *p< 0.0001 vs 0 µg/ml.

Figure 4.4: Anti-microbial assay of partially purified extracts from radio resistant bacteria. Each

extracts were used at the concentration of 10µg and zone of inhibitions were measured in mm

after 18 hours of incubation at 37 °C. The values are mean ± standard deviation (n=3). (tet =

tetracycline, car= carbencillin, pen= penicillin, amp= ampicillin).

Table. 4.2: Anti-microbial activity of partially purified extracts from UV resistant microbes.

Diameter of the inhibitory zones recorded in mm. n = 3, Values mean ± SEM.

S.no Crude extracts S.

aureus

6538

E.

coli

11303

P.

aeurogenosa

10145

N.

gonorrhoeae

MS11

R.

fascians

12975

1 WMA-BD2-F1 11 NZ NZ NZ 11

2 WMA-LM4-F1 8 14 NZ NZ NZ

3 WMA-LM9-F2 12 NZ NZ 22 18

4 WMA-LM19-F1 12 10 12 15 25

5 WMA-LM19-F2 NZ NZ NZ 25 NZ

6 WMA-LM30-F2 NZ NZ NZ 8 14

7 WMA-BD1-F1 NZ NZ NZ 22 NZ

8 PC 28 20 20 20 8

F1=Water:Methanol, F2=Methanolic and F3=Dichloromethane fraction. NZ= No zone, PC= Positive

control

4.5. Discussion:

The radiations induced oxidative damage is a versatile phenomenon which affects the

regulation of key cellular processes such as proliferation, repair and recovery (Szumiel et al.,

2015). Reactive nitrogen/oxygen species (RNS/ROS) lead to DNA fragmentation, membrane

damages and lesions. In current study, partially purified extracts from UVR resistant bacterial

strains that have been isolated from two different deserts of Pakistan were assessed for their

cytotoxic, antioxidant and antibacterial activities. Several biochemical methods like free radical

scavenging properties by using DPPH assay, cytotoxic assay by using HeLa cell lines, antibacterial

assay by disc diffusion method were performed.

The photoluminescence spectra of methanolic extracts were recorded showing that all

the compounds seem to show a high peak of excitation at wavelengths ranging from 400-700. It

indicates that all extracts from radio-resistant isolates have UV absorbing potential. The

fluorescence of these UV absorbing compounds is the result of photons being emitted (vibrational

energy), as molecules come to ground state from excited state’s Comparatively fastest

mechanism is followed as molecule returns to their ground state, fluorescence can only be

observed when there are more systematic ways of relaxation (Hodak and Valeur, 2008). Photo-

resistance can be achieved by quenching the energy in an excited state from UV absorbing

compounds and coupling it with photosensitive cells or tissues. Photoluminescence is associated

with the energy level differences between two electron states that are being in transition

between excited and the equilibrium state (Valeur et al., 2012). In most of the photo luminescent

systems, the chromophore aggregation mostly quenches light emission via aggregation caused

quenching (Yuan et al., 2010).

High production of various super oxide and nitric oxide radicals confers to the

pathogenesis of some inflammatory diseases and other pathological conditions (Guo et al., 1999).

Current research shows that the different metabolic extracts from radioresistant bacteria can

effectively quench DPPH, a free radical containing reactive oxygen/nitrogen species (ROS/RNS) by

using DPPH assays. In in-vitro system extracts that were obtained from UV resistant bacteria are

used to inhibit oxide radical pathways and remarkable inhibition is reported. Compounds from

these UVR resistant bacteria may be contemplated with their same antioxidant activity under

radiation-induced pathological processes. The reaction between bioactive compounds having free

radicals at their conjugated double bonds following additional reaction, leads to the formation of

stable products that keep the cell tissues from significant damage (Krinsky et al., 2005). Our

investigations showed the scavenging abilities of extremolytes against non-site-specific hydroxyl

ion from the UVR resistant microbes.

The ability of the extracts from UV resistant bacteria to exhibit cytotoxic activity against

HeLa cells and antimicrobial activity against some of the bacteria suggested the presence of

hydrophobic and hydrophilic bioactive compounds. Previous studies on preliminary screening of

extracts from bacteria and fungi revealed the presence of saponins, triterpenoids and the

flavonoids but no alkaloids (Ellithey et al., 2013). The results obtained from this study indicate that

most of the microbial extracts from UV resistant microbes have displayed significant antibacterial

and cytotoxic activity. MTT assays to predict the cytotoxic effects that targets the activity of

succinate dehydrogenase in mitochondria reducing tetrazolium salt into formazan crystals (Duh et

al., 1998). The color intensity of formazan dye corresponds to the sum of potent cells. The MTT

assay evaluates the activity cell on the bases of generation of reducing equivalents in

metabolically active cells (Cartuche et al., 2015).

Not much research has been done on the anticancer activities of Deinococcus UV

resistant genera. We reported 5 potent and high priorities hits as an anti-cancerous C18 partially

purified compounds. The extracts from Kocuria, Deinococcus and Stenoptrophomonas species

were found to have high cytotoxic activity against cell lines of cancer and perhaps the potential

candidates for the isolation of bioactive molecules. As far as we know, no report is available

concerning the isolation of bioactive molecules that have cytotoxic activity, from

Stenoptrophomonass, Deinococcus and Kocuria sp. These bioactive compounds with significant

cytotoxic activity with cancer cell lines might be very useful as antitumor, anti-proliferative and

other bioactive agents. The present study showed that different fractions of the cellular extract

were active against E. coli ATCC11303, S. aureus ATCC 6538, R. fascians ATCC 12975 (a plant

pathogen) and most importantly drug resistant N. gonorrhoea MS11. Di-terpenoid and phenolic

compounds are hydrophobic in nature, thus targets the cytoplasmic membrane and preferentially

create partition into the lipid bilayer (Kyrikou et al., 2005). The results of our antibacterial study

showed that the extract from Stenotrophomons sp. WMA-LM19 and Deinococcus sp. WMA-LM9

showed better inhibitory activity against N. gonorrhoea MS11 and other gram positive and

negative bacteria in comparison to other antibiotics.

4.6. Conclusion Some of the extracts showed good antioxidant, cytotoxic and antibacterial activities. Our

results suggested that these UVR resistant microbes can be a potential source of new therapeutic

drugs and metabolite isolation in the field of biotechnology. To our knowledge this is the first

report of anti- N. gonorrhoea and R. fascians12975 (plant pathogen) activities of bio active

compounds isolated from a UV resistant Stenoptrophomonas sp. and other isolates. In this study,

we also investigated that strain Stenotrophomonas sp. WMA-LM19 and Deinococcussp. WMA-LM9

not only able to survive in high UV doses, H2O2 and Mitomycin C but can also produce highly

active and UV absorbing compounds. These two strains were selected for further studies.

Acknowledgment

This work was supported by grants from Higher Education Commission of Pakistan under

international research support initiative program (IRSIP). We also highly acknowledge Oregon

State University for providing the opportunity to work in collaboration.

Conflict of interest

No conflict of interest is associated with this work.

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Chapter 5: Radio-protective and Antioxidative Activities of Astaxanthin

Paper 3

Title: Radio-protective and Antioxidative Activities of Astaxanthin from Newly

Isolated Radio-resistant Bacterium Deinococcus sp. Strain WMA-LM9

Published in Annals of Microbiology

5.1. Abstract A radio-resistant bacterium, designated as strain WMA-LM9, was isolated from desert

soil. 16S rRNA gene sequencing indicated that the bacterium belongs to genus Deinococcus with

maximum similarity to Deinococcus radiopugnans. Deinococcus sp. strain WMA-LM9 was found

to be resistant to an ultraviolet (UV) dose of 5 × 103 J/m2, hydrogen peroxide (50 mM) and

mitomycin C (10 μg/ml). A carotenoid pigment was extracted using chloroform/methanol/acetone

(7:5:3) and purified by high-performance liquid chromatography on a C18 analytical column. The

compound was characterized as mono-esterified astaxanthin by 1H, 13C nuclear magnetic

resonance and mass spectrometry. It was tested for antioxidant activity, total flavonoids and

phenolic content, radio-protective potential in correlation to the prevention of protein oxidation

and DNA strand breaks in vitro. The carotenoid pigment showed a very potent antioxidant activity

and significantly stronger scavenging ability against superoxides, with an IC50 (concentration

causing 50% inhibition of the desired activity) of 41.6 μg/ml. The total phenolic and flavonoid

contents were 12.1 and 7.4 μg in terms of gallic acid and quercetin equivalents per milligram of

dried mass, respectively. Astaxanthin also showed a higher inhibitory action against oxidative

damage to collagen, elastin and bovine serum albumin than did β-carotene. The carotenoid also

inhibited breaks to DNA strands, as indicated by the results of the DNA damage prevention assay.

We conclude that astaxanthin from Deinococcus sp. strain WMA-LM9 has protective effects

against radiation-mediated cell damage, and it also protects cellular protein and DNA against

oxidative stress and other anti-oxidant activities.

Keywords: Deinococcus sp., radio-resistance, astaxanthin, HPLC, NMR, MS, antioxidant, protein

oxidation,

5.2. Introduction Extremophiles are all those organisms that can survive and optimally grow in extreme

conditions e.g. volcanic areas, deep seas, hot springs, as well as in environments with oxygen

shortage (Kumar et al., 2010). Among these extremophiles, radio-resistant are very important

because they can survive under high radiations (both ionizing & non-ionizing). These radiations

have severe effects like oxidative damage to biomolecules such as nucleic acid and proteins.

Extreme energy radiations tolerance has been detected in various members of archaea and

bacteria domains. Most important genera having ionizing-radiation-resistance includes

Rubrobacter and Deinococcus that exhibits a high level of resistance (Fredrickson et al., 2004).

Some other extremely UVC radiations resistant bacteria have been reported from the Atacama

Desert Chile, Sonoran Desert Arizona and from manganese mine in northern Argentina that

includes Hymenobacter sp. and Geodermatophilus sp. The isolates comprised 28 genera grouped

within six phyla, which we ranked according to their resistance to UVC. Hymenobacter sp. showed

a higher survival profile than D. radiodurans a bacterium, generally considered the most

radiation-resistant organism (Rothschild et al., 2016). Many radiation-resistant organisms with

desiccation resistance have been reported from dry areas in Xinjiang, Taklimakan Desert and

displayed diverse metabolic properties. A total of 52 Ɣ-radiation-resistant bacteria were isolated

from the Taklimakan desert sample that were clustered into five group’s based on the 16S rRNA

sequencing (Yu et al., 2015). These extremophiles (radio resistance) protects themselves by

synthesizing certain antioxidants to hostage oxygen radicals or else they have a well-developed

DNA repair mechanisms (Betlem et al., 2012). Further down penetrating light, bacteria crease

excess light and quench what is not required, thus escaping undesirable photochemical

impairment. Deinococcus radiodurans is one of the most studied radio-resistant, red pigmented

and nonphotosynthetic bacteria. The red pigment is assumed for the microbial resistance against

highly energetic radiation (Battista and Cox, 2005).

Carotenoids have no role in normal cell growth, reproduction or development of organisms, but

its absence does upset the survivability. These are effectual trackers of reactive oxygen species

(ROS), predominantly that of singlet oxygen 1O2 and peroxyl radicals (ROO) (Tatsuzawa et al.,

2000; Stahl and Sies, 2003). These metabolic assets of microbes have widely been studied for

various industrial uses (Ferrer et al., 2007; Gostincar et al., 2010). The assembly of carotenoids

from natural sources has been a region of demanding exploration (Scarpa et al., 2015). D.

radiodurans produces deinoxanthin that is a unique keto carotenoid, which contributes to D.

radiodurans resistance under oxidative stress with a potent quenching ability of ROS than -

carotene and lycopene (Saito et al., 1998; Tian et al., 2007; Peng et al, 2009). In fact, carotenoids

defend DNA strands from oxidative damage, membranes from lipid peroxidation and proteins

from carbonylation (Zhang and Omaye, 2000).

All types of ROS like hydroxyl radicals (•OH), hydrogen peroxide (H2O2), superoxide (O2−), and

singlet oxygen (1O2) and reactive nitrogen species (RNS) like 2, 2-diphenyl-1-picrylhydrazyl (DPPH)

can be hunted by carotenoids from D. radiodurans in vitro (Tian et al., 2007; Zhang et al., 2007).

These carotenoids also have the ability to protect plasmid DNA (20%) uncover to OH, and

recovers the supercoiled plasmid form, that is completely crushed (Tian et al., 2009). The

advancement in metabolomics, proteomics, and genomics, has increased the interest to study the

genes and its proteins that are helpful in regulation of microbial metabolic assets in such an

extreme environment (Ferrer et al., 2007; Hammon et al., 2009; Singh et al., 2010).

Figure 5.1: Astaxanthin synthesis from isophorone, cis-3-methyl-2-penten-4-yn-1-ol and a

symmetrical C10-dialdehyde has been discovered and is used commercially.

Figure 5.2: Structure of Deinoxanthin from Deinococcus radiodurans.

Accordingly, in current study we investigated the role of astaxanthin extracted from

Deinococcus sp. strain WMA-LM9 in a resistance to UVB, H2O2 and Mitomycin C. The carotenoid

was purified and investigated for antioxidant and cytotoxic activity. Oxidative damage in bovine

serum albumin, collagen and elastin with and without carotenoids and their possible role in

Deinococcus sp. WMA-LM9 resistance to intracellular protein carbonylation was also studied. We

also confirmed that these carotenoids can neutralize the effect of different reactive oxygen

species resulting from oxidative stress that can damage microbial DNA. To the best of our

knowledge, this is the first report of astaxanthin-producing Deinococcus with highly anti-oxidant

and protein oxidation preventing activities.

Phenolic Flavonoid’s

Figure 5.3: Structure of phenolics and flavonoids




5.3. Material and methods

5.3.1. Isolation of Radio-resistant Bacteria Soil samples were collected aseptically from the desert of District Lakki Marwat, Khyber

Pukhtoonkhwa, Pakistan, in sterile zipper plastic bags, immediately transported to the laboratory

and kept at 4°C before investigation for the radioresistant microbial community. Soil samples

were serially diluted in phosphate buffer saline (PBS) and inoculated by spread plate method on

TGY (Trypton glucose yeast extract agar) medium containing 10 g/L tryptone, 5 g/L YE, 1 g/L

glucose, was used as basic medium. TGY plates were exposed to UV radiation for 5 min prior to

incubation. Each sample was UV irradiated in UV chamber (119x55×52 cm), which was supplied

with a 20W, 280 nm UV light placed at the top. The choice of UV-B was based on the fact that UV

280 nm can cause a serious damage to cell DNA because of its shorter wavelength. Moreover UVB

is considered to be the only choice for causing both types of direct and indirect damages.

The UV fluence rate (energy per area per time) to the test sample was calculated by

using the following equation in J/m2.

Where He is the radiant exposure that is the energy reaches a surface area due to

irradiance (Ee) maintained for a time duration (t).

5.3.2. Radiant Exposure Calculation

TGY plates were exposed to UV radiation for 5 min prior to incubation. Each sample was UV

irradiated in UV chamber (119x55×52 cm), which was supplied with a 20W, 280 nm UV light

placed at the top.

Total UV dose was determined by time of exposure to the UV fluence rate. All UV

irradiation procedures were performed under red light to prevent photoreactivation. After

irradiation, the plates were incubated at 37ᵒC for 5-7 days. The isolates were subcultured from

irradiated plates and again exposed to UV radiation for further confirmation. Several fractionated

doses ranging from 300 to 3300 Jm-2 (30 to

300 sec) were used to find the survivability rate of all the UVR isolates. Strain WMALM9 was

selected on the basis of tolerance to maximum doses of UV radiation.

5.3.3. Identification of Radio-resistant Bacterium

Based on high tolerance to UV radiations, H2O2 and mitomycin C strain WMA-LM9 was

identified morphologically as well as biochemically by previously described methods (Shah et al.,

2013). Molecular identification was carried out by sequencing of 16S rRNA gene. For this purpose,

for the extraction of DNA extraction kit (qiagen) was used. Amplification of 16S rRNA gene was

carried out by 27F’ (5’-

AGAGTTTGATCCTGGCTCAG-3’) and 1492R’ (5’-

CTACGGCTACCTTGTTACGA-3’) bacterial primers.

The amplified PCR product was sequenced by Macrogen Service Center (Geunchun-gu,

Seoul, South Korea). The obtained sequence was computed for closest relatives using BLAST tool

at the NCBI and homologues were analyzed for phylogeny using Molecular Evolutionary Genetic

Analysis (MEGA) version 6. The neighbour-joining phylogenetic (Tamura and Nei, 1993) tree was

constructed for the identification of isolated bacterial strain and diversity among UVR resistant

extremophiles was studied (Tamura et al., 2013). Afterwards, the sequence was submitted to

NCBI GeneBank in order to assign an accession number (KT008384).

5.3.4. Bacterial Survival Curves and Oxidative Stress Strain WMA-LM9 was inquested for radiation resistance and survival curve was plotted

(Mattimore and Battista, 1996). Cell culture of strain WMA-LM9 was serially diluted (1:1000) by

phosphate buffer saline (PBS) and spread on TYG agar plates, then exposed to different doses of

UV radiation at 280 nm. For the determination of survival rate UV-irradiated plates were divided

by colonies from unirradiated plates. For determination of oxidative stress and mitomycin C

tolerance, an overnight grown culture of strain WMA-LM9 in TGY broth was diluted in sterile

normal saline up to an OD600 of 0.5. The cells suspension was treated with different molar

concentrations of hydrogen per-oxide (5-40 mM) for 30 min and mitomycin C (2-10 µg/ml) for 20

min, cell were cultured on TGY agar plates. Former to persisting colonies counting, plates were

incubated at 30°C for 3 days. The survival rate was expressed as the difference in number of

colonies between treated and untreated samples. All the experiments for survival curve were run

in triplicates.

5.3.5. Measurement of Intracellular Protein Carbonylation

Protein carbonylation was measured using the DNPH (2,4-dinitrophenyl hydrazine)

method (Cao and Cutler, 1995; Misra et al., 2004). To obtain cell free extract for protein

carbonylation assay cells were lysed by sonication. For the estimation of total protein

concentration method by Lowry et al., (1951) was used. The cell-free extract (2 mg/ml of protein)

in 50 mM PBS (pH 7.4) was incubated with 400 µl of 10 mM 2,4-dinitrophenyl hydrazine (DNPH) in

2M HCl for 2 hrs. Proteins were precipitated, and then re-suspended in 6M guanidine

hydrochloride. After centrifugation of solution supernatant was analysed spectrophotometrically

at 370 nm. As protein control DNPH was replaced with 2M HCI, was run in parallel. The protein

carbonyl content was expressed in mM/mg protein.

5.3.6. Preparation of the Carotenoid Extract

500ml of the Deinococcus sp. WMA-LM9 culture under continuous shaking and aerobic

conditions was harvested after 48 h by centrifugation for 10 min at 5000xg.Cell pellet was

extracted with [acetone:methanol:chloroform (7:5:3)], after washing with sterilized water by

probe sonication (150 watts of power at 40 kHz). The cell suspension was again centrifuged at

10000 × g for 10 min and a clear red colour supernatant was recovered. It was allowed to dry and

then dissolved in methanol for further study.

5.3.7. Reversed-Phase High-Performance Liquid Chromatography (R-P HPLC)

The crude extracts were inspected by HPLC and flash chromatography using a Waters

2690 Alliance system. A Hypersil ODS-C18 column (5 µm pore size, 4.6 × 250 mm) protected by a

guard column. All solvents of HPLC grade were degassed (Merck, Millipore Corporation, Merck

KGaA, Darmstadt, Germany) and filtered through a 0.2 µm filter prior to HPLC analysis. Using

standard column thermostat, constant temperature of 25°C was maintained in column. An

isocratic technique eluted with a mixture of isopropanol, methanol and acetonitrile (10:50:40,

v/v) at a flow rate of 0.8 ml/min was used as mobile phase (Saito et al., 1998). The fractions eluted

were monitored with a Waters 996 photodiode array detector.

5.3.8. Liquid Chromatography–tandem Mass Spectrometry Analysis

The carotenoid extracts from Deinococcus sp. WMA-LM9 was subjected to LC-MS/MS

an ABX3200 Q-TRAP mass spectrometer equipped with a

TurbolonSpray ESI source, and connected to a Shimadzu HPLC system with dual LC20 pumps, a

SPD-M20A UV/Vis photodiode array (PDA) detector and auto sampler. 10 μL of the sample

dissolved in LC-MS grade methanol were injected onto a column

(C18; 5 μm, 250 × 4mm, Bischoff, Germany). The mobile phase comprised of Methanol (solvent A)

and Acetonitrile (solvent B) with 0.1% (v/v) formic acid, used in a gradient mode for B: 0.0/30;

25/100; 35/100; 45/30 (min/%); with a flow rate of 0.8ml/min. A computer equipped with

Thermos Scientific Xcalibur 2.2 (Thermo Fisher Scientific, USA) was used to analyze data in control

manner. The system was controlled and data were analyzed on a computer equipped with

Thermo Scientific Xcalibur 2.2 (Thermo Fisher Scientific, USA). The MS was used in positive ion

mode to detect m/z transitions [M+H]+.

5.3.9. 1H and 13C NMR Studies

A nuclear magnetic resonance spectrum gives the prime evidence about the structure of a

compound. NMR spectroscopic data were recorded at room temperature on Bruker Avance 400

MHz NMR spectrometer in CDCl3 referenced to residual

77.0 ppm) with tetramethylsilane (TMS) as an

internal standard.

The purified sample was placed in an inert solvent [deuteron-chloroform (CDCl3), and the

solution was positioned between the poles of a powerful magnet. In respond to their molecular

environs the diverse chemical shifts of proton and carbon within molecule were measured in the

NMR apparatus relative to a standard, usually tetramethylsilane (TMS). The strength of the signals

was combined to reveal the number of carbons and protons resonating at any one frequency.

Each chemical shift value corresponds to a set of protons and carbons in a particular environment.

The strength of each signal signifies the number of protons and carbon of each type.

5.3.10. Anti-Oxidant Activity and Determination of Total Phenolic/Flavonoid Contents

To determine the anti-oxidant activity of carotenoid Commonly used Diphenyl-1-

Picrylhydrazyl (DPPH) assay was used (Xu et al., 2005). Various concentrations of carotenoid (5-20

µg) were taken in 96 well microtiter plate, volume was raised uniformly up to 200 µl using DPPH

and incubated for 30min at 37°C. Ascorbic acid was taken as a standard in same concentration to

the test samples. Methanol was used as a blank and absorbance was measured at 517 nm on UV-

H C

visible spectrometer. The IC50 values were calculated for carotenoid along with standard. The

following formula was used for calculation of free radical scavenging activity.

% Scavenging = (Control-Sample/Control) x 100

Total phenolic and flavonoid phenolic contents were analyzed in the extracted carotenoid

from Deinococcus sp. strain WMA-LM9. Singleton et al (1999) method was used for the

determination of total phenolic content (TPC) using gallic acid as standard. While total flavonoid

contents were calculated by aluminium chloride colorimetric assay (Zhishen et al., 1999) by

plotting quercetin standard curve.

Phenolic and flavonoid contents were measured in µg of gallic acid and quercetin equivalents per

milligram (GAE/mg and QE/mg) of dried extract.

5.3.11. Protein Oxidation Inhibition Assay

Elastin, collagen and bovine serum albumin as standard proteins were used to analyze

inhibitory effect of carotenoid on protein oxidation. Two hundred micro-litter of targeted proteins

(1mg/ml) were incubated with hundred micro-litter of carotenoid dissolved in tetrahydrofuran

(THF) and treated with hundred micro-litter of (1 mmol/L) FeSO4 and 100 µl of 80 mmol/L of H2O2

at 37oC for 1 hr. 15 U of catalase was added to stop the reaction. The mixture was then incubated

with 600 µl of 10 mmol/L DNPH for 1 hr. 10% TCA was added to precipitate out the unbound

protein afterwards. The mixture was dissolved in 6M guanidine hydrochloride and quantified

spectrometrically at 370 nm. Percent inhibition of protein oxidation by carotenoids was calculated

using H2O to replace FeSO4 and H2O2 as a blank.

(%) Inhibition of protein oxidation= (Control-sample/control) x 100

5.3.12. DNA Damage Prevention Assay

The DNA damage preventing capability of carotenoid pigment was determined by

incubating plasmid pUC18 in a reaction mixture that contains: plasmid 2 µl, carotenoid solution 3

µl (6 µg) and 6 µl (12 µg), 2mM FeSO4 3 µl, 1M sodium nitroprusside 4 µl and 30% H2O2 4 µl, for

1hr at room temperature. Hydrogen per oxide and sodium nitroprusside usually produces single

strand breaks in DNA. The pattern of bands of treated samples, as well as positive and negative

control, was examined using gel electrophoresis technique.

5.3.13. Cytotoxic Assay

Brine shrimp assay was performed to analyze cytotoxicity of the carotenoid (Maridass,

2008). In distilled water 34 gm of sea salt per litre was added for the prepration of artificial

seawater and taken in vials with 15-20mg eggs of brine shrimp (Artemia salina). Carotenoid

dissolved in DMSO, was added to vials in different volume (100µl, 50µ, 25µl) and incubated for

24-48h at 30°C. Cytotoxicity of carotenoid was determined by counting the number of live

shrimps.

5.3.14. Statistical Analysis

To assess the significance between the results, Student’s t-test was used and P < 0.05 was

considered as significant. Bacterial sensitivities to UV, H2O2 and mitomycin C and scavenging %

activity of carotenoid were studied by regression analysis between the percentage of

survival/inhibition and their respective concentrations, single factor and two-way ANOVA applied

for analysis of protein carbonylation and in vitro % protein oxidation inhibition assay between

groups and within single group.

5.4. Results

5.4.1. UVB Selection and Isolation of Radio-resistant Bacteria

The shorter wavelength UVB (280 nm) radiations carry 3.94–4.43 eV energy per photon

and can damage cellular DNA.most common types of DNA damage are pyrimidine-pyrimidone (6-

4) photoproducts (6-4PPs) and cyclobutane pyrimidine dimers (CPDs) that can be caused by UVB

and lead to CC-TT or C-T transitions.

5.4.2. Identification of Strain WMA-LM9

Strain WMA-LM9 was found Gram-positive diplococcus, tetrad in arrangement, with

round, red, raised mucoid and opaque colonies. 16S rRNA sequence of strain WMA-LM9 indicated

that strain belongs to genus Deinococcus having 99% similarity with Deinococcus radiopugnans.

The strain was submitted to NCBI under accession number KT008384.

5.4.3. Resistance to UV radiation, Oxidative Stress and Mitomycin C

Strain WMA-LM9 was exposed to different energy doses of UV radiation in order to

determine its radiation resistant potential. Strain WMA-LM9 maintained nearly 50% viability at 5K

Jm-2 energy dose of UV radiation, whereas the E. coli (10536) couldn't survive at such high energy

UV radiation (Fig. 5.4A). A gradual decrease in survival of strain WMA-LM9 was observed with

increase in concentration of H2O2 and maintained up to 49% viability at 50mM H2O2 for 60min

(Fig. 5.4B). The bacterium was quite resistant to mitomycin C and more than 50% of survival rate

was observed up to 8µg/ml whereas the E. coli couldn't even survive at this concentration (Fig.

5.4C). Results are expressed as means ± SD and are compared using the Student’s unpaired t-test.

Moreover, the percentage values had an exponential distribution. Error bars represent standard

deviation for triplicate experiments. P value < 0.05 is considered significant.

5.4.4. Measurement of Intracellular Protein Carbonylation Level

The total protein oxidation in Deinococcus sp. WMA-LM9 and E. coli (10536) was

measured as 0.128091± 0.00585 and 0.197378 ± 0.0191 µM/mg respectively that indicates lower

protein oxidation in the radio-resistant WMA-LM9 sp. as compared to E. coli (10536). The results

shown in figure 3 indicated that lack of ability to produce carotenoids in E. coli (10536) becomes

more sensitive to oxidative damages like UV stress and H2O2 (Fig. 5.5). Results are highly

significant and expressed as means ± SD with p<0.05.

Figure 5.4: Survivability of strain WMA-LM9 from desert soil at varying UV-B exposure. (A) UV

radiation resistant potential of WMA-LM9 (B) Resistance to different concentrations of hydrogen

peroxide (mM) (C) Resistance to different concentrations of Mitomycin C (µg/ml). % viability value

is calculated as N1/N0x100 where Ni is the value after exposure to irradiation, H2O2 and Mitomycin

C, while N0 is the value at time 0, for each condition tested. Results are highly significant different

in among group (Fig. 5.4) as p value is less than significant level 0.05. Values are mean ± SD.

5.4.5. Carotenoid extraction and purification

The carotenoids were extracted from the cells of Deinococcus sp. WM-LM9 and analyzed

by HPLC. The fraction 7 of flash chromatography (Fig 5.6) showing highest scavenging activity was

subjected to RP-HPLC. Two distinct peaks were observed with a retention time of 2.3 and 4.9

collected separately marked as LM9F1 LM9F2 as shown in Fig. 5.7. 15 mg of pure astaxanthin was

extracted from Deinococcus sp. WM-LM9 with percent purity of more than 90.

5.4.6. LCMS/MS

The fraction LM9F1 that exhibited the highest antioxidant activity was subsequently

subjected to LC–MS/MS for mass identification for C40H52O3 by elemental analysis and electron

ionization liquid chromatography (EIMS). The positive ESI-MS spectrum of carotenoid extract

exhibits the signals at m/z 597 [M+H] + (LM9F1) and 610 [M+H] + (LM9F2) (Fig. 5.7). The major

component, m/z 596 accounted for more than 70% of the total carotenoids. The UV spectrum of

the compound showed λmax at 475 nm.

5.4.7. 1H and 13C NMR

The purified extract once dissolved in an inert solvent [deuteron-chloroform (CDCl3) was

analysed for carbon and proton spectra to investigate the chemical structure properties. The

peaks obtained by NMR spectroscopy were found to contain astaxanthin. All the peaks were

compared with available spectra of astaxanthin and confirmed.

double bonds. The formation of cis bond results in characteristic shift differences compared to all-

trans compounds. The proton NMR data shows that signals between 6.0 and 7.8 ppm represent

the 14 (-CH) methine protons on the ASTX backbone and a near bilateral symmetry around the

central double bond. The conjugation system described imparts carotenoids with excellent light

absorbing properties in the bluegreen (450–550 nm) range of the visible spectrum. The signals in

range of 0.99 to 2.1158 ppm represents 30 protons from methyl group and two OH protons show

peak at 3.627ppm. In addition, the presence of unsaturated fatty acids is detectable by the

appearance of multiplets between 5.2–5.4 ppm attributed to methine protons (Fig. 5.8).

In the 13C NMR spectra, the signals attributed to the carbon atoms found in the carbonyl moieties

are good indicators of structure of astaxanthin. The C-1 is a quaternary carbon and it gives peak at

29.69ppm. In the spectrum C-4 (C=O) signal is downfield at 200.14 ppm. The 13C NMR spectra of

compound reveals the presence of C=C carbon-3 position with 162.27 ppm on both side of ring

showing the presence ester carbonyl. It also confirms the monoester linkage in the compound. C-

5 an alpha carbon to carbonyl and olefinic region shows peak at 126.76-136 ppm. The 8 methyl

groups at these olefinic carbons show peaks at 14.00 and 26.13 ppm (Fig. 5.9). The signals

corresponds to the carbon atoms of the carotenoid were found to be the same as those of

already reported astaxanthin (Fig. 5.10) in literature.

1 H NMR spectroscopy of the pure extract showed diastereomeric arrangement in the

olefinic region that gives a significant evidence for the presence of carbohydrate backbone

in carotenoids. NMR spectra showed hydrophobic nature of the pure compound arranged in

isoprene residues with long conjugated chain of double bonds. Therefore, our attention was

directed to the chemical shift area for

Figure 5.5: Comparison of intracellular protein carbonylation level between radioresistant

Deinococcus sp. strain WMA-LM9 and E.coli (10536) following UVR and H2O2 treatment. Results

are highly significant different with p value less than 0.05.

Values are mean ± SD.

Figure 5.6A: Flash chromatography of the extract using different solvent system of Hexane

Dichloromethane water and methanol.

Figure 5.6B: Different fractions collected upon flash chromatography for bioassay guided

fractionation.

Figure 5.7: HPLC chromatogram/positive ESI-MS spectrum of carotenoid extract exhibits signals

at m/z 597 [M+H]+, and 609 [M+H]+ LM9F1 and F2 respectively.

Figure 5.8: 1H NMR spectra of purified compound LM9F1. The purified compound was identified

as ―Astaxanthin‖

Figure 5.9: 13C NMR spectra of purified compound LM9F1. The purified compound was identified

as ―Astaxanthin‖

Figure 5.10: Astaxanthin chemical structure from NMR peaks using ChemDraw software.

5.4.8. Anti-oxidant Activity of Carotenoid

Methanol dissolved carotenoid extract DPPH· radical scavenging activity was measured as

shown in figure 5.11. Concentration dependent scavenging activity was found to and almost 50%

(IC50) of the DPPH· radicals were scavenged at 41.6 µg/ml carotenoid that was greater that the

activity of -carotene (20%). However, the carotenoid extract scavenged less DPPH radical (80 ±

1.7%) than ascorbic acid positive control. IC50 was obtained by linear regression analysis of dose

response curve plotting between % inhibition and carotenoid concentration that led to 50%

inhibition of free radical activity of DPPH. Results are expressed as means ± SD or SE and are

compared using the Student’s unpaired t-test p<0.05.

The phenolic and flavonoid contents in the carotenoid extract (astaxanthin) were

quantified by using standard calibration curve equation. Various concentrations (5-20 µg) of gallic

acid and quercetin were used as standards to plot calibration curve and the results were

expressed as µg of standard equivalents. Total phenolic and flavonoid contents were 12.1 ± 1.3

and 7.4 ± 1.0 µg/mg of the respective standards equivalence (µg of GAE/mg and µg of QE/mg)

from the calibration curve equation.

5.4.9. Protein Oxidation Inhibition Assay

The inhibitory effect of astaxanthin from strain WMA-LM9 against oxidative damage of

three standard proteins i.e., bovine serum albumin, collagen and elastin were studied, using -

carotene as standard. Astaxanthin is able to inhibit protein oxidation by 40-45% ± 10.3, which is a

greater inhibition than that achieved with carotene (15-20 ± 3.3%). 10 µg of this carotenoid

inhibited protein oxidation better than -carotene, with significant results as p<0.05 using t-test

as shown in Fig. 5.12.

5.4.10. Inhibition of Protein Oxidation and DNA Damage Prevention

The effect of astaxanthin on DNA damage prevention was examined using a hydroxyl

radical-induced DNA breaks system in vitro. Specifically, plasmid pUC18 was incubated with H2O2

and sodium nitroprusside in the presence and absence of carotenoid. The plasmid DNA was

broken down by the attack of the •OH generated from the Fenton reaction, as indicated by smear

formation in the negative control in Fig. 5.13. The DNA was completely protected from oxidative

damage by H2O2 and sodium nitroprusside by the presence of carotenoid in test samples T1 and

T2 (6 and

12 μg) in the reaction mixture, showing promising results in DNA prevention (Fig

5.13).

5.4.11. Cytotoxic Activity of Carotenoid

Cytotoxic activity of astaxanthin was determined by brine shrimp assay. For the

evaluation of cytotoxic effect assay was carried out at four different concentrations of carotenoid.

At lowest concentration of carotenoid no toxic effect was noticed that is 25-100 µg, while at

higher concentrationts only 30% cytotoxicity was observed with an IC50 of 1567.62 µg.

Figure 5.11: Anti-oxidant activity of carotenoid extracted from strain WMA-LM9. DPPH radical

50% (IC50) was obtained by linear regression analysis of dose response curve plotting between %

inhibition or % activity (y-axis) and carotenoid concentration (41.6 µg/ml).

Figure 5.12: Inhibitory effect of carotenoid from strain WMA-LM9 of different protein oxidation

in-vitro. Commercially available β-carotene (Sigma Aldrich) was used as standard. Values are

mean ± SD.

Figure 5.13: Role of carotenoids in prevention of oxidative damage to pUC18 plasmid DNA after

exposure to oxidative agents. Lane PC: Positive control (only plasmid DNA); Lane NC: Negative

control (plasmid DNA treated with hydrogen peroxide and sodium nitroprusside). Lane T1 and T2:

Test samples (plasmid DNA, hydrogen peroxide, sodium nitroprusside and carotenoid in different

concentration).

5.5. Discussion Hot and dry desert can be considered as paradigm of extreme environment for all forms

of life because of several limiting factors i.e. nutrient availability, extreme dryness, and high

temperature. Continuous exposure to high sunrays and dryness, it may receive an ample amount

of radiations. In current years, mechanism underlining ROS-induced oxidative stress mechanisms

and the exploration for appropriate strategies to fight oxidative stress has become one the

foremost goal of medical research efforts (Vílchez et al., 2011). In the present study surface soil,

collected from the desert of District Lakki Marwat, was screened for the isolation of radio-

resistant bacteria. Carotenoid extract from this bacterium was purified and evaluated for DPPH

radical, protein and DNA oxidation inhibition activity.

A radio-resistant bacterium, Deinococcus sp. strain WMA-LM9 was isolated from desert soil.

Strain WMA-LM9 showed prolonged resistance (50% survivability) to different energy doses of

UVB radiation and also was found viable after incubating with mitomycin C (10 µg/ml) for 20 min,

50mM hydrogen peroxide for 60 min suggested that this strain has a strong CAT (catalase) and

SOD (super oxide dismutase) antioxidant system that protects the cell from oxidative damages

(Prazdnova et al., 2014). It has already been reported by several researchers that resistance to

ionizing radiation is directly linked with resistance to hydrogen peroxide, mitomycin C and

desiccation (Daly et al., 2007; Fredrickson et al., 2008; Daly 2009).

Deinococcus strain’s resistance to high concentration of mitomycin C for 10 min generates 100 to

200 cross-links per genome without a loss of viability (Kitayama,i 1982). However, the detailed

antioxidant mechanisms of this bacterium are still unknown.

The carotenoid compound in LM9F1 was analysed for astaxanthin (C40H54O4) by elemental

analysis and EIMS giving molecular ion at 597 m/z. Astaxanthin protected cellular proteins from

oxidative damage in case of strain WMA-LM9 as compared to E. coli (10536). The carotenoids are

effective scavengers of different toxic oxides, therefore, block the formation of all superoxide’s

and Fenton reaction pathways that can contribute to protein oxidation (Imlay, 2003; Hua et al.,

2009). The ability of this genus to survive in several extreme conditions is suggested to be as a

result of three combined mechanisms like prevention, tolerance and repair (White et al., 1999).

The mono-esterified astaxanthin from WMA-LM9 showed two-fold stronger quenching abilities

of super oxides than already reported deinoxanthin and carotenes, that might be attributed to

the extra keto-group substitution and length of their conjugated double bond system as

compared with the other carotenoids. Antioxidant potential of carotenoids from radio-resistant

microbes has been documented as the contributory factor to radioprotection presented by any

compound (Albrecht et al., 2000). A carotenoid extract from D. radiodurans was clearly able to

scavenge superoxide anions using the DPPH assay (Zhang et al., 2007).

Free radicals formed during DPPH assay showed that astaxanthin have capability of

donating electrons to neutralize free radicals and can scavenge free radicals and therefore has

potential as chemotherapeutic drugs to eradicate pathological diseases related to free radical

from a system. The photo-protection against toxic super-oxides offered by astaxanthin is based on

electron exchange energy transfer quenching (Galano et al., 2010). Protein rather than DNA was

proposed to be a possible target for UVB radiations and free radicals. The carotenoid isolated

from this bacterium was more effective to prevent the oxidation of different standard proteins

like bovine serum albumin, collagen and elastin. The protective effect of purified astaxanthin on

proteins in Deinococcus sp. is the result of free radical quenching ability. DNA repair proteins and

many other important cell enzymes and proteins involve in cell recovery are protected by these

naturally occurring carotenoids in the cells.

Carotenoid form stable product upon reaction with free radicals and additional reactions

at their conjugation double bonds, which further contribute to the inhibition of protein and lipid

damages from oxidative products formed during stress (Krinsky et al, 2005; Hua et al., 2014). We

also investigated that this newly reported monoesterified astaxanthin from WMA-LM9

neutralized the effect of superoxide’s, hydrogen peroxide and sodium nitroprusside and

prevented pUC18 plasmid DNA from oxidative damage. It also restricted protein oxidation by

inhibiting protein carbonylation that led to prevention of DNA indirect damage. The carotenoid

might block the formation of 8-oxo-2-deoxyguanosine during oxidation of DNA in stress. Many

important proteins including DNA repair proteins and other enzymatic antioxidant are protected

by carotenoids in order to prolong cells survivability in extreme conditions. Antioxidant-rich

metabolites from radio-resistant extremophiles significantly reduce the risk of DNA damages

(Singh and Gabani, 2011).

The carotenoid was found either non-toxic or less toxic even with higher concentration

that makes it more effective to use in drugs and other therapeutic applications. Little is known

about the total phenolic and flavonoid contents in carotenoid of Deinococcus radiopugnans,

which may contribute to the radioprotective ability and anti-oxidant activity of these compounds.

As the presence of aromatic hydro carbons, double bonding system and different keto groups

indicate the high resonance structure of the extracted carotenoid from Deinococcus sp.WMALM9.

So we investigate the phenolic and flavonoid groups that can contribute to antioxidant and DNA

damaging preventive abilities for such carotenoids. Mostly flavonoids imparts a characteristic

colors (orange, violet, crimson, scarlet, and mauve, blue) with beneficial health effects ( ili et al.,

2012). The OCH3 groups shifts the color toward more red (Grotewold 2006). The presence of oxo

groups at position 4 and nine or more double bonds in the carotenoids enhances singlet oxygen

and super oxides quenching activities (Terao, 1989). Contribution of the carotenoids and phenolic

contents to the radical scavenging activity was described by several researches previously

(Fernandez et al., 2007; Wang et al., 2010). Our results confirm that both carotenoids and

phenolic are contributing to the radical scavenging property of the extract. Carotenoid with high

phenolic and flavonoid spectrum determines its medicinal importance. Deinococcus sp. strain

WMA-LM9 has higher levels of CAT, hence more resistant to UV, mitomycin C, and hydrogen

peroxide than Escherichia coli.

The results concluded that astaxanthin from the newly isolated Deinococcus sp. WMA-

LM9 play a key role in protection against UV-photo-oxidation, protect proteins and DNA from

oxidative damage and contribute to cell resistance. Furthermore investigations are required to

find its biosynthetic pathway in order to produce highly active carotenoid via metabolic

engineering.

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Chapter 6: Ectoine a Compatible Solute in Radio-halophilic Bacteria

Paper 4

Title: Ectoine: a Compatible Solute in Radio-halophilic Stenotrophomonas sp. WMALM19 Strain to

Prevent Ultraviolet-Induced Protein Damage

In review Journal of Applied Microbiology

6.1. Abstract

Ectoines are amorphous water-binding molecules that form large hydrate envelopes to stabilize

the natural structure of biopolymers and other vital biomolecules. The purpose of this study was

to assess the antioxidant and radioprotective properties of a compatible solute, ectoine, extracted

and purified from radio-halophilic bacterium. Nine different radio-resistant bacteria were isolated

from desert soil, where strain WMA-LM19 was selected on the basis of its high tolerance for

ultraviolet radiation among all these isolates. 16S rRNA gene sequencing indicated the bacterium

was closely related to Stenotrophomonas sp. (KT008383). A bacterial milking strategy was applied

for extraction of intracellular compatible solutes in 70% ethanol, purified by high performance

liquid chromatography (HPLC) using a C18 analytical column. The antioxidant and radio-protective

properties of the pure compound were evaluated by hydroxyl scavenging, DPPH reducing, lipid

peroxidation inhibition assays and protein radio-protection assays with SDS-PAGE (sodium

dodecyl sulfate polyacrylamide gel electrophoresis) analysis. The compound was characterized as

ectoine by 1H and 13C NMR (Nuclear Magnetic Resonance), and mass spectrometry (MS). Ectoine

exhibited strong Fe2+ chelation in comparison to EDTA (38.58± 0.846%). The OH- radical

scavenging efficiency of ectoine (53.68 ± 0.48863%) was estimated in terms of % inhibition of

deoxy D-ribose degradation in a non-site-specific assay using a concentration of 10.0 μg/mL.

Maximum reduction in DPPH (~60.45 ± 1.185%) was observed at 10 μg/mL ectoine concentration.

Ectoine effectively inhibited oxidative damage to proteins and lipids in comparison to the

standard ascorbic acid. Furthermore, a high level of ectoine-mediated protection of bovine serum

albumin against ionizing radiation (1500-2000 Jm-2) was observed, as indicated by SDS-PAGE

analysis. The results indicated that ectoine can be used as a potential mitigator and radio-

protective agent to overcome radiation- and salinitymediated oxidative damage in extreme

environments.

Keywords: Extremophiles, Ectoine, Antioxidant, Sun screen, Nuclear Magnetic

Resonance

6.2. Introduction

A capability of microorganisms to regulate osmotic pressure is a critical consideration when

evaluating their ability to successfully compete and grow in a given habitat. This unique feature of

extremophiles facilitates extensive applications in biotechnology, ranging from bioremediation of

contaminated composites to the assembly of medicinal important drugs (Gabani et al., 2013).

Most living cells have to adapt to fluctuations in the osmotic strength of their environment within

certain limits. The proliferation of extremophiles in a given habitat, is strictly dependent upon

their ability to maintain an internal osmotic pressure. In order to maintain an osmotic equilibrium,

these microbes in extreme environment (bacteria, archea and eukarya) can equilibrate to saline

environments by accumulating compatible solutes (Csonka et al., 1991). The diverse chemical

structures of compatible solutes range from sugars, polyols to amino acid derivatives, and are not

only considered as osmoregulatory solutes, but also valuable to bacterial cells in protecting

proteins from the damaging effects of drying, freezing, high temperatures and ultraviolet

radiations (Kunte et al., 2014; Ventosa and Nieto, 1995).

a) Structure of Betaine b) Glycine zwitterion c) Structure of glycine

Figure 6.1: Structure of a) betaine b) glycine zwitterion c) glycine, compatible solutes from

extremophiles.

The compatible solute ectoine (1,4,5,6-tetrahydro-2-methyl-4-

pyrimidinecarboxylic acid) is a cyclic amino acid derivative of aspartate, which has achieved

extensive interest as a protective agent, and appears to be a universal organic solute in bacteria. It

was originally discovered in the phototrophic, halophilic bacterium Ectothiorhodospira

halochloris (Galinski et al., 1985). Ectoine is considered one of the most effective protectants of

nucleic acid, proteins, cell membrane and even whole cells against freezing, heating, drying or

other chemical agents (Louis et al., 1997; Severin et al., 1992). Action biosynthesis has been

documented in a great variety of halotolerant and halophilic species, particularly in those with

simple growth demands. Ectoine biosynthesis is far more widespread in the bacterial domain than

betaine and glycine (Oren, 2008). Ectoine is currently produced and has extensive applications in

cosmetic additives, biological processes and enzyme preparations, pharmaceutical industries and

other fields for commercial usage (Kanapathipillai et al., 2008; Lentzen and Schwarz, 2006; Zhang

et al., 2006). There is hence great interest in the technology that leads to the efficient production

of ectoine. Abundantly halophilic and halotolerant microorganism were investigated that could

produce ectoine in salt stress environments (Pastor et al., 2010). The bacterial process for

production of ectoine has been modified in the past two decades to meet increasing market

demands. The yield of ectoine can be maximized by a technical bioprocess called “bacterial

milking” using the halophilic eubacterium H. elongata (Sauer et al., 1998), which involves a cyclic

upturn and diminution of the salt concentration for ectoine synthesis and secretion,

correspondingly. Ectoines and their derivatives, like hydroxy ectoine, are now being mainly

produced biologically widely. These compatible solutes are used in cosmetics as anti-aging agents,

for oral care and as adjuvants for vaccines (http://www.bitop.de; http://www.merck.de). In

addition, these solutes are not only limited to use as stress protectants, but can serve as an

important source of carbon and nitrogen for energy production (Salar-García et al., 2017)

intracellularly, and upon release to the surrounding medium, can induce cell death or hypo-

osmotic shock in other micro-organisms.

Ectoine’s protective properties of proteins can be explained by its strong osmotropic interaction

with water and subsequent exclusion from protein surfaces, the strengthening of intra-molecular

hydrogen bonding (secondary structures) and the decrease of solubility of the peptide backbone

(Kunte et al., 2014). Ectoine as a novel active component in health care products and cosmetics

has attracted industry because of its stabilizing and UV protective properties. The halophilic

bacterium Halomonaselongata has been used as a producer strain in joint efforts of industry and

research to develop large-scale fermentation procedures (Hahn et al., 2015). Bacterial milking

procedure and the development and application of ectoine excreting mutants

https://en.wikipedia.org/wiki/glycine

https://en.wikipedia.org/wiki/zwitterion



http://www.merck.de/

http://www.merck.de/

(“leaky mutants”) are the two key technologies that allow the annual production of ectoine on a

scale of tons. Despite of being more successful method bacterial milking also has some

drawbacks. This method is inexorable with the decline in cell growth rate, corrosion of equipment,

and the difficulty of downstream handling due to the discontinuous production pattern and high

salt mediation (Schubert et al., 2007).

To overcome these shortages, Stenotrophomonas sp. strain WMA-LM19, a UV resistant strain

isolated from desert soil, a halophilic bacterium that can nurture and excrete ectoine in high

manganese and salt concentration, was chosen for ectoine production using monosodium

glutamate as a carbon and nitrogen source in 0.5M NaCl. Ectoine was purified and their possible

protective role as an antioxidant to protect the cellular protein in stress was also evaluated.

Protective effect offered by ectoine to BSA and red bold cells membrane was also evaluated using

SDS-PAGE and membrane damage assay.


6.3.1. Bacterial Strain and Growth Conditions Soil samples were collected aseptically in sterile zipper plastic bags from the dessert of District

Lakki Marwat, Khyber Pukhtoonkhwa, Pakistan, and kept at 4°C until further investigation for

radio-resistant microbial community. Serially diluted soil sample were inoculated by spread plate

method on Spizizen's minimal medium (SMM) medium containing: (NH4)2SO4 (2 g/L); Na-

citrate.2H2O (1 g/L); K2HPO4 (14 g/L); KH2PO4 (6 g/L), MgSO4.7H2O (0.2 g/L), MnCl2.4H2O (0.01g/L),

and NaCl (30 g/L), with 0.5% glucose as the carbon source supplemented with phenylalanine (18

mg/L), and tryptophan (20 mg/L). SMM plates were exposed to UV radiation for 5 min in UV

chamber (119x69×52 cm) supplied with a 20W, 280 nm UV light, prior to incubation. The UV

fluency rate was calculated as He = Ee×t in units of J/m2. The isolates were subcultured from

irradiated plates after incubation at 37°C for 5-7 days.

6.3.2. Bacterial Survival Curves at UVB and Oxidative Stress Strain WMA-LM19 was tested for radiation resistance and survival curve was plotted (Mattimore

and Battista, 1996). Cells were serially diluted with phosphate buffer saline and spread on SMM

agar plates, then exposed to different doses of UV radiation at 280 nm. The survival rate was

determined by dividing the number of colonies appeared on UV-irradiated plates with colonies

from un-irradiated culture.

6.3.3. Identification of Radioresistant Bacterium Strain WMA-LM19 was identified morphologically as well as biochemically by the method as

previously described (Murray et al., 1981). 16S rRNA gene was sequenced for molecular

identification of strain WMA-LM19. DNA was extracted by DNA extraction kit (QIAGEN) and 16S

rRNA gene sequence was amplified using

27F’ (5’-AGAGTTTGATCCTGGCTCAG-3’) and 1492R’ (5’-

CTACGGCTACCTTGTTACGA-3’) bacterial primers. The amplified PCR product was sequenced by

Macrogen Service Center (Geunchun-gu, Seoul, South Korea). The sequence obtained was

computed for nearest relatives in the NCBI database using

BLAST tool and homologues were analyzed for phylogeny using Molecular Evolutionary Genetic

Analysis (MEGA) version 6. The neighbour-joining phylogenetic tree was constructed for the

identification of isolated bacterium and diversity among UV resistant extremophiles was studied.

The sequence was submitted to NCBI GeneBank in order to assign an accession number.

6.3.4. Extraction and Determination of Ectoine by NMR Spectroscopy Extraction of intracellular compatible solutes was carried out by the method as previously

described with slight modification (Wang et al., 2006). Strain WMALM19 was grown in 200 ml

SMM at a high salt concentration [8 % (w/v) NaCl] to induce the accumulation of ectoine. When

the optical density (OD600) was reached up to 1 (1 OD/mL = 0.31 g DCW/L), cells were collected by

centrifugation at 1000 x g for 10 min at 4°C and washed with 50mM potassium phosphate buffer

(pH 7.2) containing isotonic NaCl concertation. The cells were transferred to a deionized water of

low salinity [hypo-osmotic shock from 8% to 3% (w/v) NaCl], the accumulated solutes within the

cells were rapidly released to achieve the osmotic equilibrium. These solutes were extracted by

re-suspending the pellets in 80% ethanol for 10 hr and then centrifuged. After the centrifugation

supernatant was collected, dried and dissolved in 0.8 ml methanol-d4 (CD3OD) for proton (1H) and

carbon (13C) NMR. NMR spectroscopic data were recorded at ambient temperature on Bruker

AMX-400 MHz in CDCl3 with tetramethylsilane (TMS) as an internal standard (temperature of 20-

22°C).

For HPLC the extract was further purified by using whatman filter paper, applying a second

crossflow filtration in a mixture of water and ethanol.

6.3.5. High Performance Liquid Chromatography (HPLC) Analysis of Ectoine

The cellular extract was filtered using a small pore size membrane filter (0.22 m) and subjected

to isocratic HPLC (Agilent 1260 series, Hewlett-Packard) C18 (2.6 × 250 mm, 5 um). The extract

was eluted with a mixture of methanol/water (70:30) as mobile phase for 10 minutes. The flow

rate was adjusted to 0.8 ml/min using UV detection at 210 nm to measure ectoine and confirmed

by LC-MS/MS.

6.3.6. Liquid Chromatography–Mass Spectrometry LC-MS Analysis of Ectoine The m/z (mass to charge ratio) was determined by using triple quardrupole mass spectrometer

with Electro-Spray Ionization (ESI) source in positive mode ionization (Agilent, USA). The

compound was eluted with a mixture of water and methanol (5/95%) containing formic acid

(0.1%) with an isocratic conditions at a flow rate of 0.8 mL/min and mass scan ranges from m/z

80-1500. The capillary voltage and atomizing gas pressure were 4.0 kV and 35 psi, respectively.

The flow rate of drying gas (nitrogen as collision gas) was 12 mL/min and the temperature of

solvent removal was 350°C.

6.3.7. Evaluation of Antioxidants and Radio-Protective Properties of Ectoine 6.3.7.1.

Hydroxyl radical scavenging activity estimation

The hydroxyl scavenging activity of ectoine was carried out by fenton method described by

Halliwell and Gutteridge (1987). 0.1mM of ferric chloride solution was mixed with different

concentrations of ectoine (2-10 g) previously reacted with 3.6 mM 2-deoxy-D-ribose, 0.1 mM L-

ascorbic acid along with 1 mM H2O2 and 0.104 mM EDTA-Na in potassium phosphate buffer (pH

7.4) in a total assay volume of 1 ml followed by incubation at room temperature for 1 hr. The

reaction mixture was then treated with 10% TCA and 0.5% thiobarbituric acid (in 0.025M NaOH),

incubated for 15 min at 55ºC. The resulted pink chromogen generated from degradation of

deoxyribose by OH was measured at 532 nm and scavenging potential of ectoine was calculated

by following formula.

% Inhibition=[(Abscontrol – Abstest)/Abscontrol]x100

6.3.7.2. Radical Neutralizing Activity by DPPH Assay

The free radical scavenging ability of ectoine was based on decay of DPPH (2,2-Diphenyl-1-

Picrylhydrazyl) (Xu et al., 2005). Different concentration of ectoine ranges from 2-10 g was

added to 1.0 ml of DPPH solution (100 μM). The mixture was incubated at room temperature for

30 min. using ascorbic acid as positive control (Rao et al., 2006). The absorbance was taken at 517

nm and percent scavenging activity of ectoine was calculated according to the formula

%Inhibition = (Abscontrol – Abstest)/Abscontrol x 100

6.3.7.3. Lipid peroxidation inhibition assay

Thiobarbituric acid reactive substances assay was carried out for lipid peroxidation by using lipid

rich medium (mice liver homogenate) (Ohkawa et al., 1979). 1 gm of liver tissue was homogenized

in tris HCL buffer of pH 7.4 and centrifuged at 3000 ×g for 5 minutes. 1 mL of the homogenized

mixture was mixed with different concentration ranges from 2-10 g of ectoine. 10 M FeSO4 was

added and the volume was made up to 0.3 ml. 0.2 ml of SDS (8.1%), 0.5ml acetic acid (pH 3.4) and

an equal volume of thiobarbituric acid (0.6% v/v) were added and incubated for 1 hr at 100oC. The

absorbance of colored mixture was measured at 532 nm and the absorbance was compared with

that of a standard curve using malondialdehyde (MDA) using the blank without adding FeSO4.

6.3.7.4. Protein Oxidation Inhibition Assay

The inhibitory effect of ectoine on protein oxidation in-vitro was quantified using bovine serum

albumin as standard proteins (Tian et al., 2009). Different concentration of ectoine (2-10 g) was

incubated with 200 l of BSA (target protein).

The mixture was then treated with 100 l of oxidant FeSO4 (1 mm), 100 l H2O2 (80mm) and

incubated for 1hr at 37oC. The reaction was stopped by adding 15 U of catalase. 10mmol/l of

DNPH was added to the mixture and incubated for 1hr, the unbound proteins to DNPH were

precipitated by adding 10% TCA. The supernatant was dissolved in guanidine hydrochloride (6M)

and protein-oxidation was quantified spectrometrically at 370 nm using H2O to replace FeSO4 and

H2O2 as blank. Percent inhibition offered by ectoine was calculated using following formula.

(%) Inhibition of protein oxidation= (Control-sample/control) x 100

6.3.7.5. Assay of Reducing Power

The reducing potential of ectoine was determined by the method described by Oyaizu (1986).

Ectoine in different concentrations ranges from (0-10.0μg/mL) was mixed with 1 ml of

ferricyanide [1% K3Fe(CN)6] and 0.1mM phosphate buffer (pH 7.0), incubated at 50oC for 20min.

10% TCA was added to mixture to stop the reaction. The supernatant was removed and mixed

with FeCl3 (0.5 mL, 0.1% w/v). % reducing potential was calculated my measuring the absorbance

at 700 nm using the formula (A0-A1)/A0 × 100.

6.3.7.6. Iron (Fe+2) Chelating Activity

The chelation potential of ectoine ranges in different concentration (010.0μg/mL) was determined

by the method as described previously (Dinis et al., 1994). 50 μl of the sample was mixed with a

solution of FeCl2 (2mM). The rection was initiated by adding 50μl of ferrozine (5mM). The mixture

was incubated at room temperature for 10 minutes. The optical density was measured at 562 nm

once the reaction had reached equilibrium. %chelating activity of ectoine was calculated using the

formula.

Chelating activity (%) = (A0- A1)/A0 × 100. EDTA was used as positive control.

6.3.7.7. Membrane Protection Assay

Human red blood cells (erythrocytes) were isolated by centrifugation of citrated blood at 2000xg

for 15 minutes. Cells were washed with phosphate buffer saline and then re-suspended in the

same buffer to the desired hematocrit level (Yang et al., 2006). Cells were pre-treated with

Ectoine (1%) and lecithin (1%), in separate, and then kept under stress for 1 hr at 37oC in the

presence of surface damaging substances like sodium dodecyl sulfate, alkylpoly glucoside, Na-

Laurylethersulfate, benzalkoniumchloride. 200 μl of the reaction mixture was taken and

centrifuged at 3000×g for 5minute. The supernatant was collected and absorbance was

determined at 540 nm. The percentage haemolysis was calculated by following formula:

% haemolysis = (control-test/control) x100

6.3.7.8. Analysis of Radio-Protection to BSA by Ectoine

The structural damage to BSA in response to different doses of irradiation (1500-2000 Jm-2) and

protection in presence of ectoine was confirmed by SDS-PAGE analysis. The analysis was

performed by preparing 0.75 mm thick polyacrylamide gel (10%). Protein was irradiated with

different UV doses in the presence and absence of ectoine, then added to PAGE loading buffer

[0.0625M Tris/HCl, pH 6.8, 5% (v/v) glycerol, 2% (v/v) 3-mercaptoethanol and 0.01% (w/v)

Bromophenol Blue]. Sample was loaded on 10% polyacrylamide gel (0.75mm) and electrophoresis

was carried out at constant voltage (90v). The gel was stained in 0.1% Coomassie Brilliant Blue

R250 by gentle shaking at room temperature for 1 hr and detained in methanol/acetic acid/water

mixture (4:2:4), bands were observed over a clear background.

6.4. Results:

A total of 09 different radio-resistant bacterial strains were isolated, strain WMA-LM19 was

selected on the basis of high resistance to UV radiation among all these isolates, i.e., 50% survival

rate to UV dose (280 nm) was 5 ×103 Jm-2.

6.4.1. Resistance to UV radiation, oxidative stress and Mitomycin C Strain WMA-LM19 was exposed to different energy doses of UV radiation in order to determine

its radiation resistant potential. Strain WMA-LM19 maintained nearly 50% viability at 1300 Jm-2

energy dose of UV radiation, whereas the E. coli (10536) couldn't survive at such high energy UV

radiation (Fig. 6.2A). A gradual decrease in survival of strain WMA-LM19 was observed to increase

in concentration of H2O2 and maintained up to 50% viability at 6mM H2O2 for 60min (Fig. 6.2B).

Results are expressed as means ± SD and are compared using the Student’s unpaired t-test.

Moreover, the percentage values had an exponential distribution. Error bars represent standard

deviation for triplicate experiments. P value < 0.05 is considered significant.

6.4.2. Identification of Strain WMA-LM19 Strain WMA-LM19 was aerobic, gram-negative rod shaped and grown white to light pink, raised,

mucoid colonies. 16S rRNA sequence of strain WMA-LM19 was assembled by DNA baser software

and subjected to blast search in National Centre for Biotechnology Information (NCBI). The results

indicated that strain belongs to genus Stenotrophomonas with 93% similarity. The phylogenetic

tree was constructed and WMA-LM19 was clustered into Proeteobacteria group among the

sequences obtained from NCBI. The nucleotide sequence reported here can be obtained from

NCBI nucleotide sequence database under accession number KT008383.

Figure 6.2: Survivability of strain WMA-LM19 from desert soil at varying UV-B exposure. (A) UV

radiation resistant potential of WMA-LM19 (B) Resistance to different concentrations of hydrogen

peroxide (mM). % viability value is calculated as N1/N0x100 where Ni is the value after exposure to

irradiation and H2O2 while N0 is the value at time 0, for each condition tested. Results are highly

significant different in among group as p value is less than significant level 0.05. Values are mean ±

SD.

Figure 6.3: Neighbor joining phylogenetic tree based on 16S rRNA gene sequence analysis,

showing the position of isolate WMA-LM19 to other strains of Stenotrophomonas obtained from

NCBI. Accession numbers of the sequences used in this study are shown in parentheses after the

strain designation. Numbers at nodes are percentage bootstrap values based on 1,000

replications.

Figure 6.4: HPLC chromatogram/positive of the polar extract for WMA LM19, that exhibit signal

at 210 nm with a retention time of 3.0 minutes.

6.4.3. Purification and Identification of Compatible solute from strain WMA-

LM19

6.4.3.1. LCMS analysis of the extract

The ethanol extract of compatible solute from Stenotrophomonas sp. strain WMA-LM19 exhibited

a single peak on HPLC at 3.0 minutes retention time (Fig.

6. 4). For further confirmation the extract was subjected to LCMS on an ABX3200 QTRAP mass

spectrometer equipped with a TurbolonSpray ESI source, and connected to a Shimadzu HPLC

system with dual LC-20 pumps, a SPD-M20A UV/Vis photodiode array (PDA) detector and

autosampler. Mass data were collected in positive ionization mode and the mass scan width was

set to m/z 100-1700. This peak showed a mass of 143.68 by ESI/MS, and matched with the formula

C6H10N2O2 (Fig.

6.5) and was identified as ectoine by 1H and 13C NMR.

6.4.3.2. 1H and 13C NMR

NMR data were acquired on a Bruker Avance 500 NMR spectrometer in XXX solvent, referenced

to residual protonated solvent ( H 3.35 ppm, C 49.3 ppm) Representative 1H NMR spectra for the

extract from the bacterial cells (Fig 6.6) showed the presence of diastereotopic protons in the

compound, which together with the N-H singlet at H 2.2 ppm, was consistent with the structure

of ectoine. The 1H NMR data were found to be identical to those reported in the literature for

ectoine, as were the 13C NMR data (Fig 6.7). The presence of signals at C 161.671 (C-CH3) and

175.993 (C=O) in the 13C spectrum confirmed the presence of a carboxyl group, while signals at

18.7, 23.1, 38.7, and 55.0 ppm showed the 2-CH and 3-CH aliphatic carbons in ectoine. Thus, on

the basis of the NMR and LC-MS data, ectoines were accumulated in the cells.

Figure 6.5: ESI-MS spectrum of purified ectoine extract exhibits signals at m/z 143.2036 [M+H]+.

Figure 6.6: 1H NMR spectra of purified compound LM19F2.

Figure 6.7: 13C NMR spectra of purified compound LM19F2. The purified compound was

identified as “Ectoine”

Figure 6.8: Chemical structure of ectoine from NMR spectroscopy.

6.4.4. Antioxidants and Radio-Protective Properties of Ectoine

6.4.4.1. DPPH and Hydroxyl Radical Scavenging Potential DPPH is a stable radical and appears as violet colour solution. Any compound with good

antioxidant activity can reduce DPPH and resulted disappearance of the characteristic violet

colour. Concentration dependent decrease in absorbance of DPPH solution by ectoine was

noticed. Ectoine mediated DPPH reduction up to

60.45±1.1876% at 10 μg/ml concentration. This concentration was found highly comparable to

the positive control ascorbic acid in this study (10 μg/ml) as shown in figure 6.9. The scavenging

potential of hydroxyl radical with ectoine was evaluated by % deoxyribose degradation inhibition.

Maximum % inhibition in deoxyribose degradation was observed as 53.6879±1.1856% at 10

μg/mL concentration of ectoine (Fig. 6.9).

6.4.4.2. Reducing capacity and Fe2+ chelating activity

The reducing and Fe chelating potential of ectoine was increased with increase in its

concentration. Ectoine demonstrated a significant reducing and chelation activity than those of

ascorbic acid and EDTA, both of them were considered as positive control (Fig. 6.10). The reducing

capacity and Fe2+ chelating activities were

53.81% ± 0.531 and 49.49% ± 0.3068 at concentrations of 10.0 μg/mL, respectively in comparison

to ascorbic acid (30.78%) and EDTA (38.59%) (Fig. 6.10).

6.4.4.3. Lipid peroxidation and protein carbonylation inhibition

Ectoine showed a concentration dependent inhibitory effect on lipid peroxidation and protein

carbonylation/oxidation. The oxidative damage to lipid (mice liver homogenate) and proteins

(BSA) was inhibited to 66.01% and 72.09%, respectively at 10 μg/mL of ectoine that was

comparatively higher than ascorbic acid (55.45%) (P > 0.05). The study demonstrated a significant

effect on protein and lipid oxidative damages in the presence and absence of ectoine (Fig. 6.11).

6.4.4.4. Membrane protection

A marked protection was provided by ectoine against all kind of surface membrane-damaging

active substances used in the membrane protection assay. Ectoine demonstrated more efficient

preventive activity (54.80%) to erythrocytes membrane in the presence of Benzalkoniumchloride

in comparison to 1% lecithin (28.915%) that serves as a positive control (Fig. 6.12).

6.4.4.5. Protective efficacy of ectoine against radiation induced damage

An aqueous solution of bovine serum albumin (BSA) was irradiated with different doses of UV in

the presence of ectoine and protein was analyzed by SDSPAGE, its breakdown was observed in

terms of intense smeared band. The radiation dose dependent (1500-2000 Jm-2) degradation of

BSA was observed in the form of intense smeared bands appeared on SDS-PAGE as compared to

untreated control. However, no smearing of BSA bands was found upon irradiation (1500-2000

Jm-2) in the presence of ectoine. The results clearly demonstrate significantly high radioprotective

efficacy of ectoine towards BSA against supra lethal ionizing radiation induced damage (Fig. 6.13).

Figure 6.9: DPPH and OH Free radical scavenging assay using ascorbic acid as positive control. The

reduction of the DPPH radicals was estimated as the function of decrease absorbance as

monitored at 517 nm.

Figure 6.10: Assay of reducing power and Fe chelation of ectoine using ascorbic acid and EDTA as

positive control. Ectoine exhibited significant % inhibition.

Figure 6.11: Protein oxidation and lipid peroxidation inhibition activity offered by ectoine using

bovine serum albumin (standard protein) and mice lipids as test samples.

Figure 6.12: Membrane damage preventing assay by ectoine using human RBCs and Lecithin as

positive control to different membrane damaging substances.

Figure 6.13: Analysis of protein protection offered by ectoine on SDS-PAGE.

Observance of protein protection (BSA) offered by ectoine (25 μg/ml) at radiation doses 1500-

2000 Jm-2.

6.5. Discussion

Strain WMA-LM19 (KT008383) was found to produce ectoine intracellularly under UV radiation

and salt stress via bacterial milking strategy. This result was confirmed by the HPLC, LC–MS

analyses as well as NMR data. Furthermore the inhibitory role of ectoine against protein and lipid

oxidative damage was also determined. To the best of our knowledge, there are limited reports

on ectoine production from wild-type strain of radio-resistant halophilic bacteria.

Among total 09 bacterial strains isolated from desert soil, strain WMA-LM19 was found to grow

under high UV dosage and salt concentration (15%). The results of 16S rRNA gene sequencing

indicated that strain WMA-LM19 showed 93% sequence similarity to Stenotrophomonas genus.

Strain WMA-LM19 accumulates high concentration of ectoine intracellularly in the presence of

manganese chloride, high salinity and UV radiation. The slight change in morphology such as an

increase in colony size might be due to high concentration of Mn. Accumulation of Mn and high

ectoine might be two interesting mechanisms of survival under high UV radiation and salinity, as

Mn+2 block the Fenton reactions and quench superoxides, thus preventing the cells from oxidative

damage. On the other hand, ectoine act as an anti-oxidant and preventing cells from desiccation

and water loss. In this report, the productivity of ectoine from ectoine-non excreting type strain

Stenotrophomonas strain WMA-LM19 was 2.9 gl-1d-1, which is the highest level of ectoine

productivity. Sauer and Galinski (1998) investigated the production of ectoine 3.3 g l-1d-1 from Halomonas elongata

(an ectoine excreting strain) DSM 142 using bacterial milking strategy. While Brevibacterium

epidermis DSM 20659 an anaerobic denitrifying halophilic bacterium capable of producing ectoine

up to 2 g l-1d-1 (Onraedt et al., 2005).

HPLC analysis of the medium reveals no peak after centrifugation and filtration of the cell pellets,

demonstrating that ectoine is produced intracellularly preventing the excessive loss of water from

the cells in extreme environment of high salinity and UV radiations. Ectoine from strain

Stenotrophomonas sp. WMA-LM19 was investigated for its role in cellular protection against

radiation mediated oxidative damage. Hydroxyl (OH-) radical is the most reactive oxygen formed

during the reaction of transition metals with various peroxides. These OH-radicals ultimately

attack on cellular macromolecules such as DNA, poly unsaturated fatty-acids and proteins, thus

damaging the cells (Aruoma et al., 1999). In our findings ectoine demonstrated a significant

scavenging potential of non-site specific hydroxyl ion. The presence of ectoine makes the Fe un-

available for attacking deoxyribose (Kitts et al., 2000). Our results demonstrated that ectoine is a

strong iron chelator. Chelating agents may protect DNA from hydroxyl radicals by protecting

enzymes responsible for DNA repair. Tissues enriched with iron containing proteins readily

releases iron and copper ions in the form that are capable of catalysing such free radical

formation, lipid peroxidation and autoxidation of neurotransmitters (Spencer et al., 1994). Iron

chelation therapy leads to low cytosolic iron concentrations that facilitate resistance by protecting

proteins, more than DNA, from IR-induced oxidative damage. By three folds decrease in iron

content in some bacterial species may lead to increase in radioresistance up to 2000 times than

human lethal dose (Daly, 2009; Flora et al., 2010).

There seems to be a direct relationship between quenching ability of ectoine for hydroxyl radicals

and prevention of lipid peroxidation process. DPPH assay is the most efficient tool to evaluate the

antioxidant properties of any compound. The antioxidants react with free radicals and extract

electrons from it to get reduced. We investigated that ectoine extracted and purified from

Stenotrophomonas sp. WMALM19 offered an efficient scavenging activity of DPPH radical in a

concentration dependent manner. The two fold stronger quenching abilities of ectoine may be

the results of keto group that can quench the super-oxides and thus preventing the cell from

photo-oxidation and excessive damages in extreme environments (Buenger et al., 2004; Ventosa

et al., 1998).

As antioxidant potential has been recognized as the contributory factor to radio-protection

offered by any compound, ectoine-mediated radio-protection to BSA was evaluated by

performing native PAGE. A clear, intense smearing of BSA was observed as a result of oxidative

damage to its secondary structure due to increase in radiation dosage in the absence of ectoine.

However, no smearing was observed in the presence of ectoine that provides enough evidence of

ectoine-mediated UV protection. Ectoine treatment to BSA results into formation of relatively

more stable product, which in turns contributes to inhibition of oxidative damages in proteins and

lipids in stress condition (Cheng et al., 2014). The kosmotropic nature of ectoine decreasing the

solubility of peptide back bone and strengthens the intramolecular hydrogen bonding or

secondary structures thereby offering a significant protection to proteins. Furthermore, ectoine

and other compatible solutes effect on preventing aggregation of amyloidogenic proteins by

stabilizing the native state (Arora et al., 2004; Botta et al., 2008; Furusho et al., 2005). Ectoines

have considerable industrial and pharmacological interest due to function preservation and

stabilizing effects (Pastor et al., 2010). Ectoine and their derivatives have gained significant

commercial market value. Recent biotechnological techniques via “bacterial milking” enhanced

the annual ectoine production and reached the scale of tons (Sauer et al., 1998; Schwibbert et al.,

2011).

Concerning the interaction with other biomolecules, as for example, lipids of the cytoplasmic

membrane, research on native fluid systems (as apposed to dry stabilization of liposomes) are still

at the beginning. Ectoine also stabilizes the membranes of pre-treated cells (erythrocytes from

human RBCs) against the damaging effect of stress factors and surfactants as well that lead to

dehydration (hemoglobin release). In the same way skin cells also have a double lipid layer

configuration within targeted and linked proteins. Cell survival largely depends upon function of

this membrane that control permeability, structure of molecules and ions in the cell by special

ducts and pumps. A number of external parameters like temperature, pH, radicals and other

chemical factors can disturb this equilibrium and damage the membranes. However, it has been

shown that the cell membrane of pretreated cells are protected and stabilized by ectoine against

damaging effect of surfactants and suggesting its kosmotropic nature. Thus, ectoine features long-

term moisturizing efficacy as it is more potent moisturizer than glycerol and other membrane

protective agent like lecithin (Graf et al., 2008). Ectoine attracted industry due to its UV protective

and stabilizing properties with potential market value as active component in cosmetics and

health care products (Bünger et al., 2001).

6.6. Conclusion

Based on the current study, it can be concluded that ectoine carried strong antioxidant properties,

and thus can neutralize in vitro radiation-induced free radicals efficiently. Ectoine can be used as

potential mitigator and radio-protective agent to overcome the radiation and salinity mediated

oxidative damages in extreme environment. Further studies are required to evaluate the radio-

protective efficacy of ectoine in vivo. It will certainly explore the future applications in sunscreens

and other UV absorbing compounds to treat different skin infections due to radiation exposure.

Acknowledgment

This research was supported by grants from Higher Education Commission of Pakistan under

international research support initiative program (IRSIP). We also highly acknowledge Oregon

State University for providing the opportunity to work in collaboration.

Conflict of interest

No conflict of interest is associated with this work.

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