Diversity of phylogenetic and lipolytic genes from soil...

Diversity of phylogenetic and lipolytic genes from

soil metagenome of Drass (Ladakh)

THESIS

Submitted to the University of Jammu

For the Award of the Degree of

DOCTOR OF PHILOSOPHY

IN

BIOTECHNOLOGY

BY

PUJA GUPTA

UNDER THE SUPERVISION OF

DR. JYOTI VAKHLU

SCHOOL OF BIOTECHNOLOGY

UNIVERSITY OF JAMMU

JAMMU – 180 006

2015

CERTIFICATE

This is to certify that:

1. the thesis entitled “Diversity of phylogenetic and lipolytic genes from soil

metagenome of Drass (Ladakh)” embodies the work done by Ms. Puja

Gupta under my supervision for the period required under statutes;

2. the candidate has put in the attendance in the School of Biotechnology for the

period required;

3. the thesis being submitted for the Degree of Doctor of Philosophy in

Biotechnology by Ms. Puja Gupta has not been submitted for any other

degree and is worthy of consideration for the award of Ph.D degree of the

University of Jammu;

4. the conduct of research scholar remained good during the period of research,

and

5. the candidate has fulfilled the statutory condition as laid down in the Ph.D

statutes.

(Dr. Jyoti Vakhlu) (Prof. M.K. Dhar)

Supervisor Director

Associate Professor School of Biotechnology

School of Biotechnology University of Jammu

University of Jammu

Acknowledgements

I take this opportunity to sincerely thank all the people involved either directly or indirectly in

my research work.

It is my pleasure and privilege to put on record my profound gratitude to my esteemed

teacher, Dr. Jyoti Vakhlu, for initiating my interest in the field of metagenomics and for her

guidance, valuable suggestions, constructive criticism, remarkable patience and

understanding throughout the course of this work.

It is my pleasure to express deep sense of gratitude and thankfulness to Prof. Manoj Kumar

Dhar, Director, School of Biotechnology, University of Jammu, for his support and providing

all the necessary facilities for the research work.

I express my heartiest gratitude to all faculty member of the department, Dr. B.K. Bajaj, Dr.

Sanjana Kaul, Dr. Madhulika Bhagat, Dr. Ritu Mahajan and Dr. Nisha Kapoor for their

invaluable help.

I would like to thanks all the research scholars of school and my lab mates, Dr. Avneet, Ms.

Deepika Trakroo, Mr. Puneet Gupta, Ms Indu, Ms. Sheetal Ambardar, Ms. Simmi Grewal,

Ms. Ranjeet Kour, Ms. Rikita Gupta, Ms. Sakshi Sharma, Sneha Ganjoo, Shanu Magotra for

their intense support throughout my course of study.

I also want to acknowledge my friends Mr Abhimanyu Jha, Ms Meenu Sharma, Ms Preeti

Choudary, Mr. Sahil Gupta, Ms Shaina Rajput, Ms Shilpi Gupta for their continual support

and affection.

I acknowledge, with utmost humility my indebtness to my parents, parents- in –law, my

beloved husband, kids and brothers for their support and encouragement.

I also would like to convey my sincere thanks to all non-teaching staff members for their

cooperation and timely help.

(Puja Gupta)

ABBREVIATIONS

U Units

TAE Tris, Glacial acid, EDTA

Sm/m Siemens per meter

SDS Sodium Dodecyl Sulphate

RNA Ribose nucleic acid

Ppm Parts per million

Pm Picomoles

PCR Polymerase Chain Reaction

PAGE Poly acryl amide gel electrophoresis

Nm Nanometer

mM Millimolar

mm Millimeter

ml Millilitre

MHz Mega Hertz

mg Milligram

kDa Kilo Dalton

Kb Kilobase

gm Gram

EDTA Ethylene diamine tetra acetate

DNA Deoxyribose nucleic acid

CFU Colony Forming Unit

bp Base pair

µl Microlitre

µg Microgram

Contents

Chapter Section Page No.

Chapter 1 Introduction 1-5

Chapter 2 Review of Literature 6-16

Chapter 3 Material and Methods 17-33

Chapter 4

Chapter 5

Results and Discussion

4.1 Sampling Site

4.2 Physicochemical analysis of soil

4.3 Microbial Diversity

4.3.1 Cultivation dependent approach

4.3.1.1 Cultivable bacterial microflora

4.3.2 Cultivation independent (metagenomic) approach

4.3.2.1 Assessment of bacterial diversity

by 454 pyrosequencing

4.3.2.2 Assessment of fungal diversity

by 454 pyrosequencing

4.4 Diversity of Lipolytic genes

4.4.1 Cultivation dependent approach

4.4.1.1 Screening cultivable bacteria for production

of lipolytic enzymes


4.4.1.2 Diversity of lipolytic genes by cultivation

independent approach.

Summary and Conclusion

Bibliography

Appendix

apter

34-52

34

34-35

35-47

35-37

37-47

37-42

43-47

47-52

47-48

48-52

53-56

57-88

Chapter – 1

Introduction

Chapter 1 Introduction

1

1. INTRODUCTION

Psychrophilic and psychrotolerant microbes inhabiting ice covered regions of

cold desert have received increasing attention during the past decade, as these play a

major role in food chains and biogeochemical cycles of these environments (Margesin

and Miteva 2011; Gesheva et al. 2012). Psychrotolerant microbes are interesting, as

besides being able to grow at temperatures close to or below freezing, they have

withheld their ability to withstand mild temperatures and are more ubiquitous and

numerous (both quantitatively and in terms of number of species) in permanently cold

environments (Ahmad et al. 2010). Various cold environments like Antarctic (Tytgat

et al. 2014), Arctic (Frank-Fahle et al. 2014), Siberian tundra (Schnecker et al. 2014),

Finland (Heino et al. 2014) have been explored for diversity of such cold adapted

microbes. The Himalayan cold deserts are characterized by a fragile ecosystem and a

complex climate due to dramatic seasonal shifts in physical and biochemical

properties. The hostile climatic conditions prevailing in these Himalayan cold deserts,

makes it an interesting habitat to study phylogenetic and functional diversity that can

be explored by the cultivation dependent and independent approach. In cultivation

dependent approach, though isolation of microbe is possible, that can be subsequently

exploited, but these methods have biases manifested by the media used and growth

conditions selected (Vartoukian et al. 2010; Delmont et al. 2011). Due to this a vast

majority of microbes resists cultivation using conventional methods (Delmont et al.

2011; Rastogi and Sani 2011) and very limited phylogenetic and functional diversity

has been catalogued so far using these traditional cultivation based methods

(Schleinitz 2011; Narihiro et al. 2014). Thus, a cultivation based cataloguing alone is

not enough for assessing the phylogenetic and functional diversity, but it needs to be

complimented with cultivation independent metagenomic techniques. Cultivation

independent metagenomic approach is based on direct isolation and analysis of

nucleic acids from environmental samples followed either by cloning & sequencing or

direct sequencing (Shivaji et al. 2011; Srinivas et al. 2011a; Serkebaeva et al. 2013;

Kim et al. 2014).

Cloning dependent metagenomic approach was the widely used cultivation

independent metagenomic method till 2011, where in the PCR products were


2

amplified from an environmental sample, cloned and sequenced (DeSantis et al.

2007; Liu et al. 2009; Rajendhran and Gunasekaran 2011; Shivaji et al. 2011). The

sequences retrieved were compared with the reference sequences in a database such

as GenBank (www.ncbi.nlm.nih.gov/genbank), Greengenes for bacterial

identification (DeSantis et al. 2006); Unite for fungal identification (Koljalg et al.

2005; Abarenkov et al. 2010; Op De Beeck et al. 2014). Ribosomal Database Project

(RDP) database used for both bacterial and fungal identification (Cole et al. 2009).

Cloning based culture-independent metagenomic approach overcomes the limitations

of cultivation based methods (Vaz-Moreira et al. 2011) and gives a larger view of the

total microbiome than cultivation based methods. This approach revealed an

abundant array of previously unknown and uncultured microbes, including entirely

new bacterial divisions with no cultured representatives (Liu et al. 2009; Shivaji et

al. 2011; Taib et al. 2013). However owing to the cloning bias, limitation of number

of clones selected and less sequencing depth, it was realized that complete

phylogenetic diversity cannot be explored by cloning based cultivation independent

metagenomic method (Shokralla et al. 2012; Fakruddin et al. 2013; McCormack et

al. 2013). Moreover, sequencing clones after metagenomic library preparation

captures only the dominant components of microbial communities that mask the

detection of low abundance microorganisms. These low-abundance microorganisms

constitute a highly diverse “rare biosphere” in almost every environmental sample

including soil (Lauber et al. 2009; van den Bogert et al. 2011; Delmont et al. 2011;

Delmont et al. 2014). These hidden rare biosphere microbial populations offer a

potentially inexhaustible genetic reservoir and could be explored only by using high

throughput direct sequencing techniques (Cloning independent). Cloning

independent direct sequencing using next generation sequencencing technology, has

not only reduced the cost of sequencing but the time consumed is also comparatively

less. Unlike Sanger sequencing that requires in vivo amplification of DNA fragments

to be sequenced (achieved by cloning into bacterial hosts first) next-generation

sequencing technology circumvents the cloning requirement by taking advantage of

a highly efficient in vitro clonal DNA amplification.

http://www.sciencedirect.com/science/article/pii/S0944501310000261http://www.sciencedirect.com/science/article/pii/S0944501310000261


3

In addition to look into what kind of microbes are present in the cold habitates,

cold active enzymes including α-amylases, cellulase, lipases and proteases are another

attraction to study these habitats. Cold active enzymes are valuable for

biotechnological applications and industrial processes (Aygan and Arikan 2008;

Pulicherla et al. 2011; Zheng et al 2011; Joshi and Satyanarayana 2013; Sarvanan et

al. 2013; Maiangwa et al. 2014). Cold adapted enzymes have high catalytic efficiency

and specificity at low and moderate temperatures, offering economic benefits through

energy savings (Gerday et al. 1997). Running processes at low temperatures reduces

the risk of contamination by mesophiles and saves energy. In addition,

thermosensitive biocatalysts can be easily inactivated by mild heat treatment.

More emphasis has been put on cold adapted lipases in the the present study

as these were predominant in soil of Drass among other hydrolases. The commercial

use of lipases of cold origin is a billion dollar business. These are widely used for

bioremediation and degrading hydrocarbons present in contaminated soil (Aislabie et

al. 2000; Paniker et al. 2006). Psychrophilic lipases have attracted attention for

synthesis of organic substances and conversion of biomass into useful products due

to their inherent flexibility in contrast to mesophilic and thermophilic enzymes with

excess rigidity (Ramani et al. 2010; Joseph et al. 2011; Nagarajan 2012; de Abreu et

al. 2014). Most of the industrially important lipases have microbial origin (Gurung et

al. 2013; Veerapagu et al. 2013) wherein the microbes are cultivated on suitable

media, purified and then further used for the production of industrially important

compounds. Several psychrophlic and psychotrophic bacteria have been exploited

for the production of a variety of coldactive lipases across different cold habitats.

The most common ones are bacterial lipases produced from the genera Aeromonas

sp. LPB4 (Lee et al. 2003), Burkholderi (Wang et al. 2009a), Pseudomonas (Yang et

al. 2009; Madan and Mishra 2010); Photobacterium sp. (Ryu et al. 2006),

Psychrobacter sp. (Xuezheng et al. 2010), Micrococcus roseus (Joseph et al. 2011),

Rhodococcus and Serratia (Joseph et al. 2007). A novel cold-active and organic

solvent-tolerant lipase displaying remarkable stability have been reported from

Stenotrophomonas maltophilia CGMCC 4254 isolated from oil-contaminated soil

samples (Li et al. 2013). Soils from Alaskan cold habitat and other cold regions have

http://scialert.net/fulltext/?doi=jm.2011.1.24#567041_jahttp://scialert.net/fulltext/?doi=jm.2011.1.24#567100_jahttp://scialert.net/fulltext/?doi=jm.2011.1.24#567100_jahttp://scialert.net/fulltext/?doi=jm.2011.1.24#566819_ja


4

been exploited as potential sources of novel cold-active lipase (Leonov 2010). Apart

from culture based screening for the isolation of lipase producers, culture-

independent approach of screening lipase/ lipolytic genes is sequence-based on any

of these methods such as PCR microarrays or next-generation sequencing platforms.

In PCR-based, approach and microarrays, oligonucleotide primers are designed on

the basis of conserved amino acid sequence motifs and these identify target lipolytc

genes, either directly or via polymerase chain reaction (PCR)/nucleic acid

hybridization. Gene-specific PCR yields a partial gene fragment, requiring additional

steps to obtain the up- and down-stream flanking regions. The partial gene fragment

can also be used as a probe to identify possible full-length genes in a metagenomic

library or enzyme restricted metagenomic DNA that can be excised and cloned. Bell

et al. (2002) designed degenerate primers using conserved amino acid regions within

lipase genes. Using these degenerate primers they obtain partial lipase gene

fragments from a metagenomic soil sample, and obtained the full-length lipase genes

using genome-walking PCR. High throughput random shotgun sequencing combined

with advanced assembly and primer walking strategies may identify particular genes

of interest. This method does not require heterologous gene expression and is

relatively less expensive and less laborious relative to PCR and microarray. This

approach generates vast amounts of sequence information that require advanced

computational approaches for assembly and gene function assignment (Piel 2011).

Whole-metagenome sequencing of mangroves areas using the 454 GS-FLX titanium

technology revealed profile of α/β-hydrolase fold proteins in mangrove soil

metagenomes from oil-contaminated sites (Jiménez et al. 2014). Cloning

independent direct pyrosequencing catalogue the diversity and access the novel

enzyme genes from previously uncharacterized soils (Wang et al. 2010; Mhuantong

et al. 2015). The information generated by metagenomics can also be used to develop

cultivation techniques to isolate novel microbes, hence novel gene products (Gong et

al. 2013; Narihiro et al. 2014).

The current study aimed to explore the diversity of phylogenetic and lipolytic

genes from composite soil sample of Drass by cultivation dependent and cultivation

independent metagenomic approaches. This place is unique in many respects for its


5

geoclimatic conditions, such as high altitude, extremely cold and dry weather

characterized by hostile climatic conditions. Drass, the second coldest place in world

located at 34.428152°N, 75.75118°E is situated 60 km west of Kargil on the road to

Srinagar with an average elevation of 3,280 metres (10,764 feet) (Fig.1) It starts

from the base of the Zojila pass (the Himalayan gateway to Ladakh), a trans-

Himalayan region that separates the western Himalayan peaks from the Tibetan

plateau. The place is snowbound and inaccessible for half of the year, from October

to April giving it a cool, temperate climate. Summers are warm up to 30°C with cool

nights, while winter is long and cold with temperatures often dropping to −45°C.

Annual precipitation is almost entirely concentrated in the months from December to

May with 360 mm (14 inches) of snow. Considering the wide range of temperature

of the site and other geoclimatic conditions, it was expected that the soil sample from

this site would contain novel microbial communities encoding enzyme able to work

at wide range of temperature.

With this background, following objectives were set forth for the present study

1. Assessment of microbial diversity (both cultivable and yet to be cultivated) in

Drass soil

2. Screening and identification of lipolytic psychrozyme producing microflora

3. Isolation of lipolytic genes from the metagenomic library of Drass soil

4. Characterization of isolated lipolytic genes

http://tools.wmflabs.org/geohack/geohack.php?pagename=Dras&params=34.428152_N_75.75118_E_

Chapter – 2

Review

of

Literature

Chapter 2 Review of Literature

6

2. REVIEW OF LITERATURE

Microbes have been around for billions of years and are present everywhere.

They inhabit almost all the environment, from cold (below 0°C) to hot (above 100°C),

acidic to alkaline, and saline environments etc. that might not look optimum for life to

human beings. In the last century lot of microbial secrets have been unraveled, but

there are still many unanswered questions e.g How many microbes are there? Who are

they? And what exactly are they doing? Therefore the microbial diversity associated

with various niches needs to be explored to answer these questions.

Cold habitats dominate the biosphere. About 85% of the biosphere are

permanently exposed to temperatures below 5°C. Oceans representing 70% of the

earth’s surface and 90% by volume are at or below 5°C (Margesin and Miteva 2011).

According to Morita and Moyer (2001), cold environments can be divided into two

categories: psychrophilic (permanently cold) and psychrotrophic (seasonally cold or

where temperature fluxes into mesophilic range) environments. Psychrophiles usually

grow at or below zero (0°C) and have an optimum growth temperature ≤15°C with an

upper limit of ≤20°C. In contrast, psychrotrophs can grow close to zero but have

optima temperature limits above 30°C; hence these could be considered as being cold-

tolerant mesophiles (Russell 2006).

Psychrophiles and Psychrotrophs belong to diverse genera ranging from Gram

negative bacteria (e.g. Acinetobacter, Serratia, Pantoa and Pseudomonas) to Gram-

positive bacteria (e.g. Exiguobacterium, Arthrobacter, Bacillus and Micrococcus) and

Fungi (Penicillium and Hypocrea). These organisms play a key function in food

chains, biogeochemical cycles and mineralization of pollutants (Margesin and Miteva

2011; Gesheva et al. 2012). Psychrotrophs are more interesting than psychrophiles,

besides being able to grow at temperatures close to or below freezing, they have

withheld their ability to withstand mild temperatures and are more ubiquitous and

numerous (both quantitatively and in term of number of species) even in permanently

cold environments (Kanbakan et al. 2004; Ahmad et al. 2010). Various cold

environments like Antarctic (Tytgat et al. 2014), Arctic (Frank-Fahle et al. 2014),


7

Siberian tundra (Schnecker et al. 2014), Finland (Heino et al. 2014) has been explored

for microbial phylogenetic and functional diversity.

Functional diversity is the direct response of the soil microbial community to

it’s metabolic requirements and available nutrients. Knowledge of functional diversity

is equally important as taxonomic diversity, as it reflects the capacity of microbial

communities to flourish through disturbance, stress or succession that could

ultimately be more important to ecosystem productivity and their stability. Although

microbes produce various hydrolytic enzymes, the present review is focused only on

the functional diversity of cold adapted lipases owing to their wide biotechnological

and industrial applications (mentioned in detail in Table 4).

Diversity of phylogenetic and lipolytic (lipolytic) genes can be assessed by

cultivation dependent as well as independent methods. The cultivation dependent

approach has an advantage of isolation of microbe that can be subsequently

manipulated for various processes. Very limited phylogenetic and functional diversity

has been catalogued so far using traditional cultivation based methods (Schleinitz

2011; Narihiro et al. 2014). Thus, culture based cataloguing alone is not the answer

for assessing the phylogenetic and functional diversity but it needs to be

complimented with cultivation independent metagenomic techniques.

Based on the above mentioned facts the layout of the review as follows:

2.1) Microbial diversity

2.1.1) Cultivation dependent approach

2.1.2) Cultivation independent approach

2.2) Functional diversity


2.2.2) Cultivation independent approach


8

2.1) Microbial diversity


Microbial diversity of various polar environments such as the Arctic and

Antarctic (Tropeano et al. 2012; Moller et al. 2013; Shivaji et al. 2013) and non-polar

alpine environments such as Italian Alps and Himalayan regions (Lipson et al. 2004;

Gangwar et al. 2009; Shivaji et al. 2011; Franzetti et al. 2013) have been assessed by

cultivation dependent methods. Diversity in polar regions differ from several high-

altitude regions such as the Himalayan ranges due to seasonal variations in

temperature that results in different physical and biochemical properties. Moreover,

polar regions are permanently frozen unlike alpine regions.

Numerous selective and nonselective media have been used for enumerating

and isolating micro-organisms. Selective media are mostly used when targeting

particular genus or species or biopropecting for specific enzymes. Nonselective

medium is preferred if the aim is to harvest diversity. Various non-selective media,

e.g Beef extract Agar medium, and YM Agar medium, PYGV agar, TSA, R2A agar,

Nutrient agar (Miteva et al. 2004; Bai et al. 2006; Zhang et al. 2008b; Sahay et al.

2013) are used for isolating microbes. The relative abundance of various taxonomic

groups of microorganisms changes with the type of the medium (Sorheim et al. 1989).

Different media giving comparable plate counts, may select for different bacterial

types thus leading to different estimates of diversity for the same soil. The choice of

the growing medium markedly affects the growth of microbe (Sorheim et al. 1989).

Small bacteria cells (dwarf cells or ultramicrobacteria) are not cultivable as they

cannot form colonies in agar. Bakken (1997) hypothesized that the culturable larger

bacteria cells have an ecological significance in soil more important than that which

appears from their small numbers as larger cells account for about 80% of the

bacterial volume.

A comprehensive work done in the last 11 years on the bacterial diversity of

cold habitats by cultivation dependent approach has been tabulated in Table-1


9

As cultivation dependent approach can assess limited phylogenetic diversity,

so further needs to be complemented with cultivation independent metagenomic

approach.

2.1.2) Cultivation independent metagenomic approach

Cultivation independent approach (metagenomics) involves the study of

collective genomes of microorganisms simultaneously. The term metagenomics was

coined by Jo Handelsman in 1998. The discipline of metagenomics was born when

Torsvik and Goksoyr (1978) and Pace et al. (1986) independently gave the idea that

the genomes of micro-organisms can be assessed without cultivating them. It

combines the power of Genomics, Bioinformatics and System biology.

The initial step in metagenomics is the extraction of total DNA to be used as a

template. There are two ways of extracting DNA (1) direct (in situ) extraction where

the cells are lysed within the matrix/ sample and then the DNA is recovered (Robe et

al. 2003) and (2) indirect extraction where the cells are first recovered from the

sample and then lysed for DNA recovery (Brady et al. 2007; Inceoglu O et al. 2010).

In both the techniques, the common step is to break open the cell either by shearing it

mechanically or chemically. Both methods have their own advantages and limitations.

Chemical and enzymatic treatment of the sample is gentle method and results in the

recovery of high molecular weight DNA but, can select certain species only by

exploiting biochemical properties of their cell wall. Mechanical shearing, on the other

hand, does not show such bias and is known to recover nucleic acid from more

diverse cells. However, the quality of DNA is not so good. Microbial community

(metagenomic) DNA isolation is a compromise between vigorous extractions required

for the representation of all microbial genomes and minimization of the DNA

shearing. Extracted metagenomic DNA can be used to explore both microbial

diversity and functional diversity.

Cultivation independent approach to study microbial diversity involves

analyses of selected genes such as SSU ribosomal and housekeeping genes from total

DNA extracted from an environmental sample followed by PCR amplification. The

most critical step in this approach is the primer selection used for amplification. PCR


10

amplification of conserved genes such as 16S rRNA from an environmental sample

has been used extensively in bacterial ecology primarily because these genes, i) are

ubiquitous, i.e. present in all prokaryotes ii) are structurally and functionally

conserved and iii) contain variable and highly conserved regions (Rastogi and Saini

2011; Rajendhran et al. 2012). In addition, the suitable gene size (~1,500 bp) and a

growing number of 16S rRNA sequences available for comparison, in sequence

databases make it a “gold standard” in microbial ecology. 16S rRNA gene used for

bacterial identification comprises nine hypervariable regions, V1-V9, that exhibit

considerable sequence diversity among species (Elgaml et al. 2013). However, V3

hypervariable region is the target that has been the most extensively used (Kumar et

al. 2011). These hypervariable regions are generally flanked by conserved sequences

that can serve as anchors for universal or specific primer pairs (Jany and Barbier

2008). They are therefore used for species identification and allow the evaluation of

community diversity. No single region can differentiate among all bacteria therefore

different regions are amplified depending on the aim of the study (Chakravorty et al.

2007). Genomic copy number of the 16S gene varies greatly from 1 in many species

to up to 15 in some bacteria (Steven et al. 2012). Other primers used for bacterial

phylogeny are designed as per conserved genes such as RNA polymerase beta subunit

(rpoB), gyrase beta subunit (gyrB), recombinase A (recA), and heat shock protein

(hsp60) have also been used in microbial investigations (Das et al. 2014).

In fungus, internal transcribed spacers (ITS) provide a greater taxonomic

resolution than ssu rRNA genes and are generally used for fungal community surveys

in different environments (Porras-Alfaro et al. 2014). The ITS is a region located

between the 18S rRNA and 28S rRNA genes, including the 5.8S rRNA gene that

splits the ITS into two parts: ITS1 and ITS2. The ITS region undergoes a faster rate of

mutation than rRNA genes but, its sequence remains homogenous within a species.

Indeed, both ITS1 and ITS2 fulfil significant functions during rRNA maturation and

are under selective pressure (Jany and Barbier 2008).

The cultivation independent (metagenomic) community analysis can be

i) Cloning dependent

ii) Cloning independent (direct analysis)


11

i) Cloning dependent method:

Till 2011, Cloning dependent was the widely used cultivation independent

method. In this method, the PCR products were amplified from an environmental

sample and then cloned and sequence the individual gene fragments (DeSantis et al.

2007). The obtained sequences are then compared to known sequences in a database

such as GenBank, Ribosomal Database Project (RDP), and Greengenes for bacterial

and UNITE fungal identification. The phylogenetic relatedness is estimated by

comparing sequence of amplicon to known microorganisms based on the homology of

16S rRNA/18S/ITS sequences and the closest affiliation of a new isolate (Taib et al.

2013). Cloning dependent culture-independent approach has revealed an abundant

array of previously unknown and uncultured microbes, including entirely new

bacterial divisions with no cultured representatives (Liu et al. 2009; Shivaji et al.

2011; Taib et al. 2013). Microbial diversity catalogued by Cloning dependent

approach has been tabulated in Table 2

Cloning dependent approach is limited in several aspects regarding

microbiome characterization and is very labor intensive. Each individual colony

represents one 16S rRNA gene, limiting most studies to relatively few sequences per

sample (sometimes in the tens or hundreds). Although it gives a larger view of the

total microbiome than cultivation based methods but complete bacterial diversity

cannot be explored, especially for rare biosphere bacteria (Carlos et al. 2012). In

addition to limited representation, restricted by the number of clones analysed, this

approach is also subjected to the bais induced by cloning.

ii) Cloning independent (direct analysis)

The alternative cloning independent metagenomic based on massively high-

throughput pyrosequencing has enabled acquisition of millions of sequences from

days worth of lab work, the same amount of data that would take years to obtain using

cloning methods, and at a fraction of the cost. It involves direct sequencing of the

phylogenitic/ functional gene amplicon without cloning them. Unlike Sanger

sequencing that requires in vivo amplification of DNA fragments to be sequenced

(achieved by cloning into bacterial hosts first) next-generation sequencing technology


12

circumvents the cloning requirement by taking advantage of a highly efficient in vitro

DNA amplification. Some of the examples where microbial diversity has been

catalogued by Cloning independent (cultivation independent) approach has been

tabulated in Table 3

In addition to look into the diversity of microbial communities present in the

cold habitates, cold active hydrolytic enzymes including α-amylases, cellulase, lipases

and proteases produced by such microbes are another attraction to study these

habitats.

2) Functional diversity

Cold active enzymes have huge industrial and biotechnological significance.

The total market for industrial enzymes reached $3.3 billion in 2010 and it is

estimated to reach a value of 4.4 billion by 2015 (Sanchez et al. 2011; BBC Research

Report 2011; Adrio and Demain 2014). Among industrial enzymes, Lipases/esterases

represent a major product segment in the global industrial enzymes market with high

growth potential (Lopez-Lopez et al. 2014). The commercial use of lipases of cold

origin is a billion dollar business as in addition to other applications they are gaining

importance for the production of biodiesel (Takaya et al. 2011; Yan et al. 2014).

Global market of biodiesel is increasing rapidly (CAGR >20%). A new report

from Pike research predicts the global biofuels market shall double over the next

decade, from $82.7 billion in 2011 to $185.3 billion in 2021. Jatropha oil containing

20% saturated and 80% unsaturated fatty acids, represents a potential source for

biodiesel producton (Kumar et al. 2011b). Since feedstocks costs are about more than

85% of the total cost of biodiesel production (Fan and Burton 2009), lipase

transesterification has attracted much attention for biodiesel production as it allows

use of lower-cost feedstocks including waste cooking oil (WCO), grease, soapstocks.

It also produces high purity product, enables easy separation from the byproduct,

glycerol (Takaya et al. 2011). Other applications of cold active lipases have been

tabulated Table 4 for easy comprehension.

Many microbial lipases have been commercialized in the world by various

manufacturers like Novazyme (Denmark), Amano Enzyme Inc (Japan, Biocatalysts

http://www.pikeresearch.com/


13

(UK), Unilever (Netherlands) and Greenock (USA). Bacterial lipases produced from

the genera Burkholderia and Pseudomonas are commercially available. Lipase PS

isolated from Burkholderia cepacia and Lipase AK isolated from P.fluorescens are

supplied by Amano and Lipase SL and Lipase TL isolated from B.cepacia and

P.stutzeri are supplied by Meito Sangyo (Japan). Cold adapted organisms expoited for

lipase production are tabulated below in Table 5.

As a large pool of microbes and their enzymatic activities remain unexplored

by cultivation dependent approaches so complementation with the cultivation

independent metagenomic approach gives a better insight of the functional diversity

in any habitat.

Cloning dependent approach to access functional diversity includes

construction of small insert library in a standard cloning vector such as pUC (Ferrer et

al. 2009). However, for the detection of large gene clusters or operons, BAC (Kakirde

et al. 2011), Cosmid (Neufeld et al. 2011; Cheng et al. 2014) and Fosmid (Geng et al.

2012; Martínez

and Osburne 2013) may be used as vector. Metagenomic

investigations have been conducted in several environments such as soil,

phyllosphere, ocean, and acid mine drainage and have provided access to

phylogenetic and functional diversity of uncultured microorganisms (Gupta and

Vakhlu 2011). This approach (Cultivation independent) is useful in mining novel

esterases and lipolytic enzymes from environmental samples. Most of the esterases

and lipases have a size of 1-2 Kb (Nacke et al. 2011) hence the use of pUC series

vector is considered to be the best available vectors to clone these genes.

Once the metagenomic library is constructed, the most important step is

screening for the desired trait. Screening of a metagenomic library can be either

functional or/and sequence based.

Function-based screening involves assay based identification of clones that

exhibit lipolytic activity. The most popular screening method for detecting positive

clones exhibiting the desired lipolytic activity uses tributyrin agar plates, in which the

appearance of clear halos around the colonies indicates hydrolysis of the substrate.

Screenings for specifically detecting true lipases have also been used with

http://www.ncbi.nlm.nih.gov/pubmed?term=Kakirde%20KS%5BAuthor%5D&cauthor=true&cauthor_uid=21112378http://www.sciencedirect.com/science/article/pii/S016770121400027Xhttp://www.ncbi.nlm.nih.gov/pubmed?term=Mart%C3%ADnez%20A%5BAuthor%5D&cauthor=true&cauthor_uid=24060119http://www.ncbi.nlm.nih.gov/pubmed?term=Osburne%20MS%5BAuthor%5D&cauthor=true&cauthor_uid=24060119


14

metagenomic libraries, using longer substrates that are not hydrolyzed by esterases

(such as emulsified triolein, tricaprylin or olive oil) in the presence of the fluorescent

dye rhodamine B. In this case, orange fluorescent halos appear around lipase-

producing colonies when irradiated with UV at 350 nm (Kouker and Jaeger 1987).

The success of such screening relies on the compatibility of the cloned genes with the

transcription and translation machinery of the heterologous host, usually Escherichia

coli. Moreover, expression of lipases can be hampered by the requirement for specific

chaperones for the correct folding of the enzyme or by its toxicity to the host cells. It

has been reported that about 40%, are recovered by functional screening when E. coli

is used as host (Craig et al. 2010; Lopez-Lopez et al. 2014).The usefulness of a broad-

host range vectors for overcoming the barrier of host compatibility has been assessed.

One of the studies proves its effectiveness using six different Proteobacteria as hosts

for the same metagenomic cosmid library, recovering different positive clones in each

host (Craig et al. 2010). Cold adapted lipases have been screened from mountain soil

(Chow et al. 2012; Ko et al. 2012), high altitude soil Taishan, China (Wei et al. 2009),

deep-sea sediments (Jeon et al. 2009), marine sediment (Chu et al. 2008; Jeon et al.

2009) and tidal flat sediment (Wu et al. 2009).

Sequence-based screenings implement PCR-based methods, microarrays or

next-generation sequencing platforms. In PCR-based, approach and microarrays,

oligonucleotide primers are designed on the basis of conserved amino acid sequence

motifs and these identify target genes either directly or via polymerase chain reaction

(PCR)/nucleic acid hybridization. Gene-specific PCR yields a partial gene fragment,

requiring additional steps to obtain the up- and down-stream flanking regions. In such

instances, the partial gene fragment can be used as a probe to identify possible full-

length genes in a metagenomic library or enzyme restricted metagenomic DNA, that

can be excised and cloned.

Bell et al. (2002) designed degenerate primers using conserved amino acid

regions within lipase genes. Using these degenerate primers they obtain partial lipase

gene fragments from a metagenomic soil sample, and obtained the full-length lipase

genes using genome-walking PCR.


15

Another approach for gene isolation is microarrays technology primarily based

on DNA-DNA hybridization. It is used to monitor differential gene expression to

quantify the bacterial diversity of the environment and catalogue genes involved in

key processes (Sebat et al. 2009). Microarray technology can be used before the

shotgun sequencing for the pre-selection of genes from metagenomic libraries,

thereby reducing the sequencing burden and reducing the proportion of sequences

unassigned by the database sequence similarity searches (Sebat et al. 2009). An

advantage of such method of screening is the direct identification of gene containing

clones without expression requirements while the possible disadvantage is the high

risk of false positive clones and the detection of partial genes (Booijink et al. 2007).

High throughput random shotgun sequencing combined with advanced

assembly and primer walking strategies may identify particular genes of interest. This

method does not require heterologous gene expression and when combined with next

generation sequencing technologies may be relatively less expensive and less

laborious relative to PCR and microarray. This approach generates vast amounts of

sequence information that require advanced computational approaches for assembly

and gene function assignment (Juncker 2009). Shot gun sequencing of metagenomic

libraries of human gut microbiomes revealed clear differences between adult and

infant microbiome pool (Qin et al. 2010) while this method also revealed a higher

similarity between family relatives as compared with unrelated individuals

(Turnbaugh 2009). This approach requires that the targeted gene has sufficiently

conserved regions of appropriate distance for emulsion PCR, which is required for

pyrosequencing, so that primers or sets of primers will sufficiently cover a gene

family. This approach is likely to be most useful for genes directly responsible for

important ecosystem functions or ecological processes, such as biogeochemical

cycles, biodegradation, pathogenesis, antibiotic resistances and cell signaling.

Allgaier 2010 applied high throughput sequencing (454-titanium

pyrosequencing) for the discovery of glycoside hydrolases from a Switchgrass-

adapted Compost Community and identified 800 genes encoding glycoside hydrolase

domains that were biased toward depolymerizing grass cell wall components. Of

these, 10% were putative cellulases belonging to families GH5 and GH9. Wang and


16

coworkers (2014) explored the genetic diversity of glycoside hydrolase (GH) family

10 and GH11 xylanases in Lake Dabusu, a soda lake with alkaline pH and high

salinity (10.1%). A total of 671 xylanase gene fragments were obtained, representing

78 distinct GH10 and 28 GH11 gene fragments respectively, with most of them

having low homology with known sequences. Phylogenetic analysis revealed that the

GH10 xylanase sequences mainly belonged to Bacteroidetes, Proteobacteria,

Actinobacteria, Firmicutes and Verrucomicrobia, while the GH11 sequences mainly

consisted of Actinobacteria, Firmicutes and Fungi.

High throughput sequencing has been also applied to reveal the extensive

diversity of aromatic dioxygenase genes in the environment among human and animal

fecal microbiota (Iwai et al. 2010) analysis of glucan-branching enzyme gene profiles

among human and animal fecal microbiota (Lee et al. 2014). Whole-metagenome

sequencing of mangroves areas using the 454 GS-FLX titanium technology revealed

profile of α/β-hydrolase fold proteins in mangrove soil metagenomes from oil-

contaminated sites (Jimenez et al. 2014). High throughput sequencing has not been

yet used to study the diversity of lipase genes in any cold environment.

Chapter – 3

Material &

Methods

Chapter 3 Materials and Methods

17

3. MATERIALS AND METHODS

3.1. Sampling site

The soil samples were collected from different regions of Drass mountains at

34.450N, 75.77

0E located in Ladakh (J&K) during May 2010 and pooled into one

composite sample. The soil was collected 20 cm cm deep into the earth by digging

and collected in aseptic plastic bags. Hands, trowels were treated with 70% ethanol

immediately before use. The samples were transported to the laboratory in ice and

stored at -20 °C (Foght et al. 2004).

3.2. Physiochemical analysis of soil

The soil samples were sent for analysis of pH, electrical conductivity, water

holding capacity, metal ion concentration, QC research lab IIIM Jammu, India. The

pH value was measured in a 1:5 (w/w) soil water suspension using electric digital pH

meter and the salinity of a soil was determined by measuring the electrical

conductance (EC) of soil water saturation extract with the help of a conductivity

meter (Das et al. 2012) where as chemical profiling of the soil for different metallic

ion was done by following the method developed by (Raman and Sathiyanarayanan

2009). The organic carbon and organic matter were determined by rapid titration

method (Walkley and Black 1934). Sodium and Potassium were determined by flame

photometerically. Total Nitrogen and Total phosphorous was estimated by using

Kjeldahl method (Bremner and Mulvaney 1982).

3.3 Soil phylogenetic Diversity

3.3.1 Cultivation dependent approach (Bacteria)

3.3.1.1 Media used

All the chemicals used in the study were purchased from Himedia Pvt. Ltd,

India and biochemicals and enzymes were procured from Bangalore Genei India Ltd,

New England Biolabs and Fermentas (MBI).


18

3.3.1.2 Comparison of cultivable bacterial load

1gm of soil was added to 10 ml normal saline and serially diluted. The samples

were diluted upto10-3

and 10-4

dilution level and spread on respective agar plates (Table

6) and incubated at 4 °C, 15 °C, 20 °C, 30

°C for 1 week. Colony forming units

(CFU)/gm soil/sample were calculated for each of the sample using a given formula

(Devi et al. 2012).

CFU/gm = Number of colonies x dilution factor/ volume spread

3.3.1.3 Polyphasic characterization

Bacteria were characterized on the basis of colony morphology, microscopy,

pigment color, growth pattern and biochemical analysis (Hamid et al. 2003). Microscopy

of bacterial isolates was done using Gram’s staining kit (Sigma). Isolates were screened

for biochemical property using biochemical test strips (Himedia). Pure cultures were

cryopreserved in 50% glycerol at -80 °C (New Brunswick, Effendorf).

3.3.1.4 Molecular identification of bacterial isolates

Selected bacterial cultures were further identified at molecular level.

3.3.1.4 (a) Genomic DNA isolation

Genomic DNA was isolated from all the bacteria using the GES protocol

(Pitcher et al. 1989)

Reagent preparation:

GES:

Guanidium thiocyanate (5 mol/l)

EDTA (100mmol/l)

Sarkosyl (0.5% v/v)

Guanidiumthiocyanate (60 g), 0.5 mol/l EDTA at pH 8 (20 ml) and deionized

water (20 ml) were heated at 65 °C with mixing until dissolved.After cooling, 5ml of

10% v/v sarkosyl were added; the solution was made up to 100 ml with deionized water

and stored at room temperature.

Ammoniumacetate (7.5 mol/l)


19

Lysozyme(50 mg/ml)

Chloroformand isoamyl alcohol mixture (24:1 v/v)

TE buffer: TrisHcl (10mM), EDTA (1mM)

Protocol:

3ml of broth cultures were harvested at the end of the exponential growth phase by

centrifugation at l000 g for 15 min and a small (rice grain-sized) cell pellet was obtained.

The cells of Gram-positive species were resuspended in 100 µl of fresh lysozyme in TE

buffer and the suspensions were incubated at 37 °C for 30 min.

The Gram-negative species were resuspended in 100 µlof TE buffer without enzymic

treatment.

Cells were lysed with 0.5 ml of GES reagent and cell suspensions were vortexed briefly

and checked for lysis (5-10 min).

The lysates were cooled on ice and 0.25 ml of cold ammonium-acetate (7.5 mol/l) was

added with mixing gently.

It was held on ice for further 10 min and then 0.5 ml chloroform and isoamylalcohol

mixture (24:1) was added.

The phases were mixed thoroughly, transferred to a 1.5 ml eppendorf tube and

centrifuged (12000 g) for 10 min.

Supernatant fluids were transferred to eppendorf tubes and 0.54 volumes of cold 2-

propanol was added.

The tubes were inverted for 1 min to mix the solutions and the fibrous DNA precipitate

was deposited by centrifugation at 6500g for 20 s.


20

Pellet of DNA was washed in 70% ethanol and air dried; and dissolved in 50µl of milliQ

3.3.1.4 (b) Purification of genomic DNA: Genomic DNA was purified by gel elution,

phenolation and purification through column or the combination of two methods.

Gel elution: DNA was eluted from 0.7 % Low melting agarose gel by elution kit

(Macherey – Nagel, Nucleospin Extract II kit) using the manufacturer’s protocol as

under

100 mg of the gel slice mixed with 200µl binding buffer (NT) , incubated at 5-10 min

(50°C)

The contents transferred to a column, centrifuged 1 min (11000g) and flow through was

discarded

700µl of wash buffer (NT3) was added to column and centrifuged as above followed by

dry spin at for 2 minutes (11000g)

Column was kept in a fresh eppendorf tube (collection tube) and 20µl milliQ was added

to the column (incubated for 10 minutes (25°C) and centrifuged as above to collect the

DNA.

Phenolation

Crude DNA was diluted with milliQ water and an equal volume of P:C:I

(Phenol: chloroform: isoamylalcohol; 25:24:1) was added and centrifuged at 13000g for

10 minutes.

The upper aqueous layer was carefully collected in a fresh eppendorf and again

an equal volume of C: I (24:1) was added followed by centrifugation.

The upper aqueous layer was carefully collected in a fresh eppendorf and DNA

was precipitated by adding 5M NaCl(1/10 volume) and ethanol (double the volume) and

incubated at -20°C for 2 h.


21

The precipitated DNA was centrifuged at 13000g for 30 minutes and the pellet

was washed with 70% ethanol, air dried and suspended in milliQ

Purification through column

200µl of Isopropanol was added per 100µl of crude DNA

Centrifugation was done at 11000g for 1 minute and flowthrough was discarded

700µl of 70% ethanol was added and centrifuged as above

Dry spin was done for 2 minutes and flowthrough was discarded

Column was kept in a fresh eppendorf tube (collection tube) and 20µl milliQ was added

to the column (incubated at room temperature for 10 minutes) and centrifuged as above

to collect the DNA

3.3.1.4(c) PCR amplification of 16S rRNA gene from bacterial culture and

ARDRA (Amplified Ribosomal DNA RestrictionAnalysis)

Universal bacterial primers, namely Bac8f (AGTTTGATCCTGGCTCAG) &

Univ529r (ACCGCGGCKGCTGGC) based on Escherichia coli positions, were used to

amplify internal fragments of 16S rRNA gene that amplify ~ 500 bp (Fierer et al. 2007).

The sequences that showed less than 98% homology with the reported sequences in the

database were reamplified by bac8f (5-AGAGTTTGATCCTGGCTCAG-3) and 1492r

(CGG TTA CCT TGT TAC GAC TT ) corresponding to Escherichia coli positions 8–27

and 1492–1509 respectively to amplify ~1500 bp region (Yong et al. 2011b). PCR

products were analyzed by electrophoresis on 1.5% agarose gel, followed by staining

with ethidium bromide and visualization under UV light. The amplified PCR products

were purified with a PCR product purification kit (Himedia cat no. ≠ MB512).

The PCR (with Bac8f & Univ529r primers) was performed following the

protocol standardized by Fierer and coworkers (2007) with modifications: instead of

0.5µM, 100pM primer were used and instead of 25 cycles, 30 cycles PCR were run. The

template DNA concentration for PCR reaction was used as 10-50ng per 10µl of PCR


22

reaction volume and the PCR reaction mix is tabulated in Table 7. The PCR program

was denaturation at 950C for 5 minutes followed by 30 cycles of denaturation at 95

0C

for 60 seconds, annealing at 540C for 30 seconds followed by extension at 72

0C for 90

seconds and final extension at 720C for 10 min. The PCR (with 8f & 1492r primers)

was performed following the protocol standardized by (Yong et al. 2011b). The PCR

program was denaturation at 940C for 5 minutes followed by 30 cycles of denaturation

at 940C for 1 minute, annealing at 55

0C for 40 seconds followed by extension at 72

0C

for 90 seconds and final extension at 720C for 10 min.

The amplicons were screened for duplicacy by ARDRA using Alu and Hha I

restriction enzymes. The PCR amplified 16S rDNA were purified with a purification kit

(Himedia cat no. ≠ MB512). Aliquots of purified 16S rDNA PCR products were

digested separately with two restriction endonucleases AluI, Hha I in 25 lL reaction

volumes, using the manufacturer’s recommended buffer and temperature. The

restriction was done at 37 o

C for 1 hour. Restricted DNA was analyzed by horizontal

electrophoresis in 2.5 % agarose gels.

3.3.1.4(d) Sequencing and phylogenetic analyses:

16S rDNA were custom sequenced at SciGenom Labs Private Ltd., Cochin,

Kerala, India. The resulting nucleotide sequences were assigned bacterial taxonomic

affiliations based on the closest match to sequences available at the NCBI database

(http://www.ncbi.nlm.nih.gov/) using the BLAST (ww.ncbi.nlm.nih.gov/BLAST).

Sequences of bacteria thus obtained were deposited in the GenBank nucleotide

sequence database.

3.3.2 Cultivable independent (Cloning independent)

3.3.2(a) Metagenomic DNA isolation

Manual metagenomic DNA extraction protocols developed by Zhou et al. 1996;

Wechter et al. 2003; Brady et al. 2007; Amorim et al. 2008; Pang et al. 2008; Liles et al.

2009, Inceoglu et al. 2010 were applied. The protocols are mentioned in detail below

http://www.ncbi.nlm.nih.gov/)%20using


23

Zhou’s protocol:

a) Buffer I: (Working concentration)

Tris HCL 100mM (pH 8)

Na2HPO4 50mM

NaH2PO4 50mM

EDTA 100mM (pH 8

NaCl 1.5 M

CTAB 1%

b) Lysis buffer:

SDS 20% (9 ml)

GITC 5M (4.5 ml)

c) Purification buffer:

Choloroform 24 ml

Isoamylalcohol 1 ml

Isopropanol

5 gm of soil sample was weighed in centrifuge cups and passed through sterile

sieve (2mm mesh sieve), 75 ml of buffer I was added. Dry ice was crushed and

isopropanol was added to make slush. The cups were incubated in crushed dry ice to

freeze (~40 minutes) and shifted to water bath at 65°C for thawing (~40 minutes).

13.5 ml of lysis buffer was added to the cups and incubated at 65°C and mixed

throughly by inversion for several times. Further 2 hr incubation with gentle inversion

mixing was done. Centrifugation was carried out at 15,000 g at 10C for 20 minutes.

The supernatant was transferred to fresh cups and 25 ml of choloroform:

isoamylalcohol (24:1) to each cup was added and mixed for 10 minutes.

Centrifugation at 15,000 g at 10 minutes was carried out for 20 minutes. The

supernatant was transferred in fresh cups and 0.7 % of isopropanol was added, mixed

for 5 minutes and the cups were incubated at room temperature for 20 minutes.

Centrifugation of cups at 15,000g for 40 minutes was done to collect the pellet and

supernatant was discarded. The DNA pellet was dissolved in 1 or 2 ml of T.E. Equal


24

volume of Tris buffered with phenol-choloroform (pH 8.0) was added and then mixed

gently. The solution was centrifuged for 10 minutes. The supernatant was transferred

to a new centrifuge cup and isopropanol was added and again centrifuged for 20

minutes to retrieve the DNA pellet. The pellet was dissolved in 100μl of T.E and the

DNA was stored for few days at 4°C and -80

°C for long term storage.

Wechter’s protocol:

a) Extraction buffer

Phosphate buffer 0.2 M (pH 7.2)

PVPP 10% (w/v)

CaCl2 3M

b) Lysis buffer

SDS 20% (w/v)

Lysozyme 25 mg/ml

Proteinase K 20 mg/ml

c) Washing Solution

Ethanol 70%

d) TE (Tris Cl)

Tris-HCL 10mM (pH 8)

EDTA 1mM

One milliliter of sterile PPB (pH 7.2) was added to a 2ml microcentrifuge tube

containing 500mg of soil. After vortexing for1 min at high speed, the mixture was

centrifuged at 325x g for 30s in a micro centrifuge. The supernatant was transferred to

a 1.5-ml microcentrifuge tube, 400µl of a poly vinyl polypyrrolidone slurry (100 mg

PVPP/ml of PPB at pH 7.2) was added using a large-bore pipette tip, and the mixture

was vortexed at high speed for 30 second. To this, 2µl of 3M CaCl2 was added, and

the microcentrifuge tube was vortexed for 30s and centrifuged as stated above. The


25

supernatant then was transferred carefully to a clean 1.5ml microcentrifuge tube, to

which 20µl of a lysozyme solution (25mg/ml) and 10µl of a Proteinase K solution

(20mg/ml) were added. The tube was inverted several times to mix the contents and

placed in a 37°C water bath for 30min and then placed in a 55°C water bath for

30min. 30µl of 20% (w/v) SDS was added to the tube, the contents were mixed by

inverting several times, and the tube was placed in an 80°C water bath for 30min.

Immediately after removal from the water bath, 400µl of the PVPP slurry was added

to the contents of the tube which was mixed by gentle inversion, placed on ice for 15

min, and then centrifuged at 8,000x g for 30min.The supernatant was transferred to a

clean 1.5ml microcentrifuge tube followed by the addition of 0.7x volumes of 100%

isopropanol, the tube was inverted several times and centrifuged at 8,000 x g for

30min. After centrifugation, the supernatant was discarded and the remaining pellet

was washed in 500µl of 70% ethanol, centrifuged for 5min at 16,000x g, and air dried

for 5min. The pellet was resuspended in 25µl of TE.

Brady’s protocol

a) Lysis buffer

Tris-HCl 100 mM

Na EDTA 100 mM

NaCl 1.5 M

Cetyl trimethyl ammonium bromide 1% (w/v)

SDS (pH 8.0) 2% (w/v)

b) Washing solution

Ethanol 70%

c) TE (Tris Cl)


EDTA 1mM


26

Soil (5 g), 10 ml of preheated (70°C) lysis buffer were mixed and incubated at

70°C for 2h with constant inversions after every 30 min. The content of the bottles

was cooled to room temperature and supernatant was extracted twice by

centrifugation for 20 min. Supernatant was precipitated with two volume of ethanol

(25°C, 1hr). Following centrifugation for 30 min, DNA pellet was washed with 70%

ethanol, air dried and resuspended in TE.

Amorim’s protocol

a) Tween 80 0.1%

Sodium Phosphate buffer (pH 7)

b) TE (Tris Cl)


EDTA 1mM

c) Purification buffer

Choloroform 24 ml

Isoamylalcohol 1 ml

Isopropanol

Soil (5 g), 0.1% Tween 80 and 50ml Sodium Phosphate buffer (pH 7) were

mixed in a nalgene bottle (250-mL) followed by overnight incubation at room

temperature (25°C) with constant shaking. Pellet was obtained by centrifugation

(6000 g, 10 min) and washed four times with TE buffer (Tris-HCl 50 mM, EDTA 50

mM, pH 8.0) by centrifugation at 5000 g for 5 min. Cell pellet obtained was lysed

mechanically using liquid nitrogen, macerate was then transferred to a 15-mL

centrifuge tube containing 2 mL TE buffer 50/50 and an equal volume of PCI

(25:24:1). Supernatant was obtained by centrifugation at 5000g for 10min. DNA was

precipitated using 0.7 vol of chilled Isopropanol and 1/10 vol of 3M sodium acetate,

followed by overnight incubation at -20°C. DNA pellet was collected by

centrifugation at 10,000g for 15 min (4°C), washed with 70% ethanol, air dried and

resuspended in T.E


27

Liles’s 2009

a) Lysis buffer

Sarkosyl 1%

Sodium deoxycholate 1%

Lysozyme 1 mg/ml

Tris-HCl [pH 8.0] 10 mM

EDTA [pH 8.0] 0.2 M

NaCl) 50 mM

b) ESP buffer

1% Sarkosyl,

1 mg/ml Proteinase K

0.5 M EDTA [pH 8.0])

c) 1 mM phenylmethylsulfonyl

Extracted and washed bacterial cells were pelleted by centrifugation and

embedded within low-melting-point agarose in a 1-ml syringe. The agarose plug was

then extruded from the syringe and incubated in 10 ml of lysis buffer 1 h at 37°C. The

plug was transferred into 40 ml of ESP buffer and incubated for 16 h at 55°C,

followed by inactivation of proteinase K with 1 mM phenylmethylsulfonyl fluoride

from a fresh phenylmethylsulfonyl fluoride stock in isopropanol with 1 h of

incubation at room temperature. After three 10-min washes in TE, plugs were stored

at 4°C in 10 mM Tris-HCl with 50 mM EDTA (pH 8.0).

Pang’s protocol

Reagents required

a) Extraction buffer


28

TrisCl 100mM (pH8)

Na-EDTA 100mM (pH8)

NaCl 1.5M

b) Lysis buffer

SDS 20%

Lysozyme 10mg/ml

Proteinase K 20mg/ml

c) Polyethylene glycol (30%)

Soil (20 g) was suspended in 50 ml of DNA extraction buffer along with 1 ml

of Lysozyme (10 mg/ml) and incubated at 37ºC for 1 h. Sample was further incubated

at 65ºC with SDS (2 ml, 20%, w/v) and proteinase K (15 μl, 20 mg/ml) for 2 h and

then centrifuged at 6,000 rpm for 10 min to remove soil residue. Supernatant was

transferred into a clean tube, and then precipitated by using half-volume of PEG

(30%, w/v) and incubated at room temperature for another 2 h. DNA was pelleted and

resuspended in milliQ.

Inceoglu’s protocol (modified version of the method of Zhou et al. (1996).

Briefly, 2.5 g soil samples were mixed with 30 ml of EDTA, 50 mM Tris

buffer (pH 8.3) and centrifuged (6,000 x g, 4ºC, 30 min), after which the supernatant

was discarded. Following this initial wash, 2.5 ml of 500 mM NaCl, 50 mM Tris, 50

mM EDTA (pH 8.3) was added to the soil pellets. Lysozyme was then added at 5

mg/ml and the suspension incubated for 1 h at 37°C. Then, 140 μl of 20% SDS was

added, along with 1 mg of proteinase K, for a further 1h digestion. After incubation, 5

ml of 500 mM NaCl, 300 mM succinic acid, 10 mM EDTA (pH 5.7) were added,

followed by 700 μl of 20% SDS. The mixtures were incubated for 30 min at 65°C and

centrifuged (15,000 x g for 30 min), after which the soil pellets were discarded.

Aliquots (8 ml) of supernatant were then transferred to 50-mL Falcon tubes and

supplemented with 1 ml of 5 M NaCl followed by 1 ml of 10% cetyl trimethyl

ammonium bromide (CTAB). The suspensions were gently mixed and incubated for

30 minutes at 65ºC. Five ml of 50% polyethylene glycol - 8000 (PEG- 8000) were

then added before overnight incubation at 4ºC, centrifugation (40,000 x g, 30 min,


29

4ºC) and discarding of the supernatant. The resulting pellets were resuspended in 240

µl TE buffer (10 mM Tris, 1 mM EDTA; pH 8.0) in Eppendorf tubes. Then, these

were extracted with one Vol. of phenol/chloroform/isoamylalcohol (25:24:1) and

subsequently with one Vol. of chloroform/isoamylalcohol (24:1). Humic acids were

further removed by adding CaCl2. 2H2O at 27 mM, incubating at 65ºC for 1 h, and

centrifuging at 14,000 x g for 20 min. The resulting supernatants, containing the soil

DNA, were precipitated with 3 M potassium acetate, centrifuged (12,000 x g, 10 min),

and the resulting pellets washed with 70% ethanol and dissolved in 20 μl TE buffer

3.3.2(b) PCR amplification

DNA extracted from multiple methods (n=3) was pooled and diluted (1/100

dilutions) with final concentration of 20 ng/µl and used as template for PCR

amplification.

PCR amplification of 16S rRNA for assessment of bacterial diversity

Small region (V1-V3) of the 16S rRNA gene was amplified from the total soil

DNA by PCR using bacterial Universal primers set 27F5`-

AGAGTTTGATCCTGGCTCAG-3` and 519R 5`-ATTACCGCGGCTGCTGGCA-

3`) (Kumar et al. 2011a). 0.5µl (10- 50 ng/ µl) of DNA was used per 10µl of PCR

reaction volume and the PCR reaction mix is given in Table 8. The PCR program

included initial denaturation at 94°C for 5 min followed by 30 cycles of denaturation

at 94°C for 45s, annealing at 55

°C for 45s and extension at 72

°C for 1 min and 30s

followed by a final extension at 72°C for 10 min

PCR amplification of ITS for assessment of fungal diversity

Inter transcribed spacer region (ITS1-ITS4) was amplified using Universal

primer pair ITS1f (5’-TCCGTAGGTGAACCTGCGG-3’) and ITS4r (5’-

TCCTCCGCTTATTGATATGC-3’) (White et al. 1990). DNA was amplified

according to the PCR program as Denaturation at 95°C for 5min followed by 30

cycles of denaturation at 95°C for 45s, annealing at 55

°C for 30s, extension at 72

°C for

60s and final extension at 72°C for 10min. PCR reaction volume and the PCR reaction

mix is given in Table 8. The amplicon was further purified from agarose gel by gel


30

elution column (Qiagen). The purified amplicons thus obtained (16S and ITS) were

send for pyrosequencing at research and testing laboratory (Lubbock, TX, USA)

3.3.2(c) Analysis of data of 454 pyrosequencing

Bar-coded pyrosequencing

Bacterial and fungal tag-encoded FLX-Titanium amplicon pyrosequencing

(bTEFAP and fTEFAP) and data processing were performed at the Research and

Testing Laboratory (Lubbock, TX) (www.research and testing.com). The bacterial

primers based on V1-V3 region were used and sequencing was performed, extended

from 27F numbered in relation to Escherichia coli 16S ribosome gene (forward 27F

5`- AGAGTTTGATCCTGGCTCAG-3`). Single step PCR reaction (35 cycles) was

used and 1 U of Hot Star High fidelity Polymerase was added to each reaction

(Qiagen, Valencia, CA). The fungal primers were based on the ITS region and

sequencing was performed forward from 458F in relation to Candida albicans (Dowd

et al. 2008).

Quality filtering and Phylogenetic analysis

Uchime tool was used to remove the chimeric sequences (Edgar et al. 2011).

Quality trimmed sequences were clustered into operational taxonomic units (OTUs)

using CD-hit (Balzer et al. 2013) with a cut-off value of 99% sequence identity.

Candidate OTUs were assigned to phylogeny using RDP (Cole et al. 2009; Krober et

al. 2009) scheme set at 80% confidence value. Rarefaction curves and diversity

indices were calculated by using RDP pyrosequencing pipeline (Cole et al. 2009).

Pvclust tool was used to obtain the Bootstrap Probability (BP) value the

Approximately Unbiased (AU) (Shimodaira and Hasegawa 2001, Suzuki 2006).

Cluster dendrogram was constructed with AU/BP values (%).

3.3.2(d) Reference datasets

Bacterial data comparison

A total of four reference datasets was obtained from NCBI. Two reference

datasets were selected randomly from the Antarctic study with accession no

SRX206452, SRX206985 and remaining two reference datasets were selected

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randomly from the Arctic study with accession no SRX017110. The sources of

Antarctic and Arctic samples were from Grove Mountains, East Antarctic and

Foreland of MidreLoven glacier, respectively.

Fungal data comparison

A total of two reference datasets was obtained from NCBI with accession no

ERX253153 for Arctic dataset. The data for Antarctic were not submitted by the

authors on NCBI-SRA, so sequences were obtained through mail on request. The

source of Antarctic sample data were McMurdo Dry Valleys (Dreesens et al. 2014)

and the source of Arctic sample were Alaskan permafrost (Penton et al. 2013)

4.4 Diversity of Lipolytic genes

4.4.1 Screening cultivable bacteria for production of lipolytic enzymes

Agar medium (1.5% w/v) containing substrate 0.4% (w/v) tributyrin for

esterase and olive oil (1%) for lipases were inoculated with freshly grown cultures

and incubated at 4°C, 10°C, 20°C, 30°C for one week (Gangwar et al. 2009). For

screening lipases, syringe filtrated olive oil (1%) and a florescent dye rhodamine B

(0.001% w/v) was added to the autoclaved cooled growth medium with vigorous

stirring. The plates containing bacterial cultures were observed for an orange

fluorescence under UV light at 350nm (Ranjitha et al. 2009).

Tributyrin medium:

Tryptone 1%

Yeast extract 0.5%

NaCl 1%

Tributyrin 1%

Gumacacia 1%

pH 7.0

Agar 1.5%

Lipase screening media was stirred for 30 minutes to make tributyrin emulsion.

Autoclaving of above media was done at 15 psi for 20 minutes at 7.0 pH.

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Rhodamine agar media

Nutrient agar 2.8%

NaCl 0.4%

Rhodamine B 0.1mg

Olive oil 3.1ml

pH 7.0

Agar 1.5%

Nutrient agar media was autoclaved separately at 121°C, 15 psi for 20 minutes.

Separate autoclaved tube was used to prepare rhodamine B solution dissolved in ethanol.

Rhodamine B solution was sterilized by passing it through 0.2μm filter. After autoclaving

the nutrient agar media was cooled at room temperature and rhodamine solution and olive

oil was added respectively. The lipase positive cultures were visualised on UV

transilluminator (Bangalore Genei).

4.4.2 Diversity of lipolytic genes by cultivation independent approach.

4.4.2 (a) PCR amplification

Metagenomic DNA amplified by using degenerate primers LIPF (LIPF 5-

GACCRATYGTSCTSGTVCAYGG–3’) and LIPR2 (5’-GCCRCCSTGRCTRTGR

CC – 3’) that target oxynion hole and active site. The PCR reactions (10-20 μl)

contained between 10-50 ng genomic DNA and the PCR reaction recipe is given in

Table 9. Touch down PCR was run. Initial denaturation at 94°C for 5 min, followed

by 4 cycles of denaturation at 94°C for 30s, annealing at 65°C for 1 min, and

elongation at 72°C for 2 min. Cycling conditions were then altered: the same

denaturation and elongation conditions were used, but the annealing temperature was

reduced to 64°C with a reduction of 1°C every cycle for 14 cycles. Cycling conditions

were again adjusted: the denaturation and elongation conditions were maintained, but

the annealing temperature was set at 50°C for 20 cycles. Following the last cycle, a

final elongation step at 72°C for 3 min was performed. For Aliquots of the PCR

reaction mixtures were subsequently analyzed by 1.5 % agarose gel electrophoresis.

Primer pair lip LipF and LipR2

yielded a single band. The band excised, eluted and

send for Pyrosequencing (research and testing lab).


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4.4.2(b) Bioinformatic analysis

OTU filtering was done with CD-hit 454 software (Balzer et al. 2013).

BLASTp was performed against Lipase Engineering Database (Widmann et al. 2010;

Jimenez et al. 2015) to assign the class to lipolytic sequences. BLASTp was also

performed (available in the National Centre for Biotechnology Information) to assign

phylogeny.

Chapter – 4

Results &

Discussions

Chapter 4 Results and Discussion

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4. RESULTS AND DISCUSSION

The present study was undertaken with an aim to explore diversity of

phylogenetic and lipolytic genes from the soil metagenome of Drass located in

Ladakh region of Jammu and Kashmir. Both approaches cultivation independent and

cultivation dependent were used to achieve the aim.

4.1. Sampling site

Drass, a town in the Kargil district of Ladakh region (Jammu and

Kashmir) India at 34.45°N, 75.77°E with an average elevation of 3,280 meters

(10,764 feet) is the second coldest inhabited place in the world. Drass is characterized

by hostile climatic conditions and remoteness. Summers are warm with temperature

up to 35°C with cool nights and winters are long and cold with temperatures often

dropping to −40°C (Sagwal 1997).

4.2. Physio- Chemical analysis of soil

Drass soil is sandy, coarse, slightly alkaline and nutrient-poor with 0.72%

organic content (Table10). Low organic content of the soil is reported due to lack of

vegetation, poor microbial activities, coarse sediments and low humus (Kastovska et

al. 2005; Rawat and Adhikari 2005; Sagwal 1997). High altitude cold desert soils,

have been reported to be originated from weathered rocks with large proportion of

sand gravel and stone with low water (Dwivedi et al. 2005). Coarse alkaline sandy

soils have been reported from Ladakh (Namgail et al. 2012; Charan et al. 2013) and

other Himalayan cold environments (Pradhan et al. 2010; Shivaji et al. 2011) Cold

desert high altitude soils have poor water and nutrient holding capacity (Charan et al.

2013) and so is true for Drass. Electric conductivity (EC) of the Drass soil is 12 ds/m

which suggests that the soil is extremely saline or salt rich. Organic carbon content,

availability, soil texture and salinity influences microbial community composition and

diversity (Fierer et al. 2003; Fierer et al. 2007). Physicochemical profile of the soil

shows that it is not supportive of high microbial activity as is true of other similar

soils (Pradhan et al. 2010; Shivaji et al. 2011).

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The soil contains ferrous, magnessium, aluminium, iron, potassium and

calcium in abundance whereas tracer elements such as cobalt, copper, nickel, arsenic,

lead and zinc are present in low concentration. Metals play an important role in

biological process as essential micro-elements. Heavy metals at elevated

concentrations are known to effect soil microbial population and their associated

activities (Ahmed et al. 2005; Anyanwu et al. 2011).

4.3 Microbial Diversity

4.3.1 Cultivation dependent approach (bacteria)

4.3.1.1 Bacterial isolation and characterization

Both oligotrophic and nutrient rich media were selected to obtain maximum

cultivable bacteria. About 600 isolates were randomly selected (100 each from six

different media: Nutrient agar, LB agar, King,s B agar, TSA, Minimal media, R2A agar)

used in the study. Since the average summer and winter temperature varies between 4°C-

30°C, the bacteria were isolated within this temperature range. The growth pattern of

individual bacterial culture were studied and placed into psychrophilic (4–20°C),

psychrotrophic (4–30°C), and psychrotolerant mesophilic (4–37°C), mesophilic (25–

40°C) groups (Sahay et al. 2013) (Table 13). Maximum bacterial load (including

pigmented and non-pigmented 5.0± 0.07x 106

CFU/ml at 30°C CFU/ml was obtained

using NA (Table 11) but maximum number of pigmented bacteria 2.9±0.17x106

were

obtained with R2A media (Fig. 2-3). Pigment production was intense at 4°C and

decreased with increase in incubation temperature which is in accordance with earlier

studies on bacterial diversity of Puruogangri ice core (Zhang et al. 2008b) and

Himalayas (Venkatachalam et al. 2014). R2A is a oligotrophic medium and allows

cultivation of many pigmented bacteria in particular that will not readily grow on fuller,

complex organic media. R2A has been used to isolate bacteria from various cold

environments e.g glaciers (Foght et al. 2004), marine surface waters (Agogue et al.

2005), ice cores (Zhang et al. 2008b) and Antarctic soils (Dieser et al. 2010; Peeters et

al. 2012). The pigments produced by these bacteria are reported to be carotenoids and

has been co-related with cold adaptation of microorganisms by many workers

(McDougald et al. 1998; Cho et al. 2000; Daniela et al. 2012; Mojib et al. 2013).


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4.3.1.2 Diversity measures

Diversity indices were used to compare between the communities obtained by

using different media. More community complexity was found using R2A media (Table

12). Overall Shannon-Wiener index (H) was 3.2 that is in accordance with previous

reports from Himalayan bacterial diversity (Pradhan et al. 2010; Shivaji et al. 2011;

Yadav et al. 2014).

4.3.1.3 Molecular and phylogenetic analysis of 16S rDNA sequences of isolates

Bacterial isolates were screened for duplicacy by colony/cell morphology

analysis, pigmentation, conventional biochemical tests that narrowed the 600 isolates

into 99 isolates. These selected isolates were subjected to 16S rRNA gene amplification

followed by restriction digestion with (Alu I and Hha I). On the basis of ARDRA

profiling, representative isolate from each cluster were sequenced and the nucleotide

sequences were deposited in the NCBI GenBank database (Accession numbers:

JX978884-JX978891, JX978892-JX978896, JN088486, JN088488, JN088491,

KF555604-KF555606, KF555608- KF555624, KF682428, KF682429 and HE774268,

KM188063). The nearest phylogenetic neighbor of all the 40 representative isolates were

identified through BLAST analysis of the 16S rRNA gene sequences against nucleotide

database available in the National Centre for Biotechnology Information (NCBI) (Table

13).

Drass isolates represented both Gram-positive and Gram-negative heterotrophic

bacteria belonging to three major phylogenetic groups organized into three clusters

Proteobacteria (37.5%), Firmicutes (32.5%) and Actinobacteria (30%) (Fig. 4).

Proteobacteria dominates (37.5%) the culturable bacterial diversity of Drass with

Gammaproteobacteria (35%) as the dominant class represented by genera Pseudomonas,

Acinetobacter, Serratia, Pantoea. Pseudomonads represented the dominant genera

among Gammaproteobacterium. Alphaproteobacteria is however represented by single

genera i.e Paracoccus (Dr32) (Fig. 5). The results are in accordance to the previous

studies on Himalayan that reports Firmicutes, Actinobacteria, and Proteobacteria as the

most common phylum (Shivaji et al. 2011).

Bacterial isolates showed 99% similarity with the reference sequences in the

Genbank except for Dr 46 that showed 96% similarity with Pantoea agglomerans (Fig.


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6). DNA-DNA hybridization will be carried with close relatives to confirm and publish

as novel species. The bacteria isolated and characterized from Drass soil have been

reported from other cold environments also. The genera Arthrobacter, Bacillus,

Sporosarcina, Rhodococcus, Pseudomonas were reported in the culturable bacterial

diversity of Pindari glacier (Shivaji et al. 2011). The genera Acinetobacter, Bacillus,

Pseudomonas were reported in the culturable bacterial diversity of Kafni glacier

(Srinivas et al. 2011b). The genus Arthrobacter is the dominant bacteria in Qinghai-

Tibet Plateau permafrost (Zhang et al. 2007a), Brevibacterium and Acinetobacter are

present in abundance in Dry Valley soils of Antarctica (Cary et al. 2010),

Planomicrobium, Mycetocola, Rhodococcus, Sporosarcina have been reported from

Himalayan soils in India and Nepal (Venkatachalam et al. 2014) and an Arctic glacier

(Reddy et al. 2009). Exiguobacterium (Gram positive and facultatively anaerobic) have

been repeatedly isolated from ancient Siberian permafrost (Rodrigues et al. 2009).

Members of genera Exiguobacterium are adapted to long- term freezing at temperatures

as low as -12oC where intracellular water is not frozen and grow at subzero temperatures,

displaying several feature of psychrophiles, such as membranes composition. Genus

Pantoea (Selvakumar et al. 2008; Venkatachalam et al. 2014), Dietzia (Mayilraj et al.

2006), Staphylococcus and Citricoccus (Yadav et al. 2015) have been reported from

Indian Himalayas. Members of the genus Paracoccus have been reported from Qinghai-

Tibet Plateau permafrost (Zhu et al. 2013).


Only three metagenomic DNA extraction protocols, developed by Zhou and

coworkers (1996); Wechter and coworkers (2003), Pang and coworkers (2008) worked

efficiently on the soil of Drass (Fig. 7). DNA extracted using these three protocols were

pooled and used as a template for PCR amplification. Multiple DNA extraction

protocols were employed since no single method of metagenomic DNA isolation is

efficient enough to represent all the bacterial diversity (Delmont et al. 2011).

4.3.2.1 Assessment of bacterial diversity by 454 pyrosequencing

Metagenomic DNA was diluted 1/100 times and used as template for PCR

amplification.The hypervariable region V1-V3 of 16SrRNA genes were amplified from

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the extracted metagenomic DNA using the universal bacterial primers 27F 5`-

AGAGTTTGATCCTGGCTCAG-3`and 519R 5`-ATTACCGCGGCTGCTGGCA-3

(Dowd et al. 2008). Targeting V1-V3 regions of bacterial 16S rRNA genes provide two

advantages: first, the V1–V3 regions are more divergent and thus can provide more

phylogenetic resolution than other regions. Secondly, RDP and other databases store

maximum sequences that correspond to the V1–V3 region of 16SrRNA gene

(Chakravorty et al. 2007). Hence more sequences are available for comparison and it

facilitates the correct phylogenetic analysis from the phylum to the genus level (Jeraldo

et al. 2011; Eren et al. 2014). Short pyrosequencing reads also assess the microbial

diversity reliably as near-full-length sequences if appropriate primers are chosen (Will C

et al. 2010). The 500 bp amplicon was gel eluted and sent for pyrosequencing in

research and testing laboratory (Lubbock, TX, USA) (www.researchandtesting.

com/next-generation-sequencing-service.html (Fig. 8)

Bacterial diversity in metagenome of Drass soil

The amplicons were pyrosequenced and 3000 sequence reads were generated.

Downstream quality filtering resulted in 2819 high quality sequences with average

read length of ≥ 200 bp. Phylogenetic analysis revealed Acidobacteria,

Proteobacteria, and Actinobacteria as highly abundant in the soil of Drass

representing 39% of the total bacterial sequences. In addition, Chloroflexi,

Bacteroidetes, Verrucomicrobia, Planctomycetes, Firmicutes, Nitrospira,

Armatimonadetes (former candidate division (OP10), Gemmatimonadetes,

Cyanobacteria, were also identified at the relatively low abundance (


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However, this framework would be benefitted via deeply sequenced longitudinal time

series datasets.

Comparative account of bacterial diversity across Drass, Antarctic and Arctic

soil samples

The 16S rRNA gene sequences from the present study (Drass soil) and those

retrieved from NCBI (Arctic and Antarctic soil) were clustered at 99% sequence

identity (Egge et al. 2013). A Significant proportion (2.2%, 2.8% and 1.9%) of the

total sequences from Drass and Antarctic (ANT1 & ANT2) sa

Diversity of phylogenetic and lipolytic genes from soil...

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