CHARACTERIZATION OF SOIL AND LEAF ISOPRENE-DEGRADING COMMUNITIES AND THEIR

CONTRIBUTION IN ENVIRONMENTAL BIOTECHNOLOGY

Panteliana Ioannou

School of Biological Sciences, University of Essex

Final Year Project BSc, Biomedical Sciences

Dr Terry McGenity

20th March 2014

WORD COUNTS: ABSTRACT: 245

INTRODUCTION: 1,255

METHODS, RESULTS AND DISCUSSION: 4,400

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ACKNOWLEDGEMENTS

I would like to gratefully and sincerely thank my supervisor Dr Terry McGenity for

his excellent guidance, understanding, patience and most importantly his support

during my project research. His mentorship was paramount in providing a well-

rounded experience consistent my long-term career goals. I would also like to

thank Mr Farid Benyahia and Mr Gordon Murphy for their assistance and guidance

throughout my practical work.

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CONTENTS

Abbreviations…………………………………………………………………......... 5-6

Abstract…………………………………………………………………………….... 7

1. Introduction…………………………………………………………………….. 8-12

1.1 An introduction to isoprene. ……………………………………………….. 8

1.2. Isoprene’s biological roles ………………………………………………... 8-9

1.3. Isoprene’s contribution to regional tropospheric chemistry………........ 9

1.4. Isoprene degradation………………………………………………………. 10

1.5. Isoprene degradation in Rhodococcus AD45……………………………. 10-11

1.6. The genus Rhodococcus ………………………………………………….. 11-12

1.7. Aims of the present study………………………………………………….. 12

2. Materials and methods………………………………………………………… 13-16

2.1. Source of the organisms, media and growth conditions………………. 13

2.2. DNA extraction……………………………………………………………… 13-14

2.3. PCR amplification………………………………………………………….. 14

2.4. Agarose gel electrophoresis………………………………………………. 14-15

2.5. PCR purification………………………………………………………......... 15

2.6. DNA sequencing……………………………………………………………. 15

2.7. Phylogenetic analysis……………………………………………………… 15

2.8. Isoprene degradation and gas analysis………………………………….. 15-16

2.9. Statistical analysis………………………………………………………….. 16

2.10. Physiological and biochemical characterization……………………….. 16

3. Results……………………………………………………………………………. 17-28

3.1. PCR analysis and agarose gel electrophoresis…………………………. 17

3.2. PCR purification analysis and agarose gel electrophoresis…………… 17-18

3.3. Isoprene degradation and gas analysis………………………………….. 19-21

3.4. Phylogenetic analysis………………………………………………............ 21-24

3.5. Morphological characteristics…………………………………………….... 24-25

3.6. Physiological and biochemical characteristics…………………………… 25-28

4. Discussion……………………………………………………………………...... 29-36

4.1. Overview.…………………………………………………………………….. 29

4.2. Phylogenetic analysis………………………………………………………. 29-30

4.3. Degradation of isoprene and its relation to catabolic plasmids……… 31-32

4.4. Comparison of Rhodococcus bW1 and Rhodococcus bE1b with

phylogenetic neighbours…….………………………………………………..... 32

4.5. Comparison of Arthrobacter p12 with phylogenetic neighbours……… 32-33

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4.6. Rhodococcus and Arthrobacter species in environmental

biotechnology…………………………………………………………………….. 33-34

4.7. Importance of identifying new species…………………………………… 34

4.8. Research needs and further studies……………………………………… 35

4.9. Conclusion……………………………………………………………………

5. References……………………………………………………………………….

Appendices……………………………………………………………………………

35-36

36-39

40-42

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ABBREVIATIONS

A MA. Arthrobacter MgSO4∙ magnesium sulfate

AIDS Acquired Immune Deficiency

Syndrome

MUSCLE Multiple sequence

comparison by Log-

Expectation

ANOVA Analysis of variance MgCl2 magnesium chloride

B NBLAST Basic Local Alignment Search Tool NOx nitrogen oxide (n=x)

bp base pairs NaCl sodium chloride

C NH4NO3 ammonium nitrate

CH4 methane Na2HPO4 disodium phosphate

CO carbon monoxide ND Not Determined

CoA coenzyme A OCaCl2 calcium chloride OD Optical Density

CTAB cetyl trimethylammonium bromide OH hydroxide

D PDNA deoxyribonucleic acid PCR Polymerase chain

reaction

dNTP Deoxyribonucleotide triphosphate RE R. Rhodococcus

EtBr Ethidium Bromide RT Room Temperature

e-value expect value rRNA Ribosomal

ribonucleic acid

F SFID Flame Ionisation Detector sp. species

G TGMBA 2-glutathionyl-2-methyl-3-butenoic

acid

TAE Trisbase, acetic acid

GC Gas Chromatography UK UV ultraviolet

KH2PO4 monopotassium phosphate WL w Weakly positive

LB Lysogeny broth

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Units of Measurecfu colony-forming unit

g grams

‘g’ gravity

h hour

kb kilobyte

m meter

mg milligram

min minute

ml millilitre

µl microlitre

M molar

mM millimolar

µM micromolar

r.p.m revolutions per minute

s second

V volt

Amino acidsAla Alanine

Lys Lysine

Ser Serine

Thr Threonine

NucleobasesA Adenine

C Cytosine

G Guanine

T Thymine

Y Pyrimidine

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ABSTRACT

Isoprene (2-methyl-1,3 butadiene) is a volatile compound with an atmospheric

concentration similar to that of methane. Isoprene is produced in high quantities by

vegetation and plays a significant role in regulating atmospheric chemistry. In the present

study, three bacterial isolates from soil and leaf isoprene-enrichments were examined by

a polyphasic approach to establish their taxonomic position. Two of the isolates defined

as strains bW1 and bE1b, were obtained from soil samples, whereas the third isolate,

strain p12 was derived from a leaf sample. Characterization of the strains with respect to

16S rRNA gene sequence analysis as well as morphological and biochemical

characteristics demonstrated that the unknown strains bW1 and bE1b represent a distinct

line of descent within the genus Rhodococcus, most closely related to Rhodococcus

globerulus. Likewise, strain p12 was found to share similarities with the genus

Arthrobacter and more specifically with Arthrobacter ilicis, Arthrobacter nicotinovorans

and Arthrobacter nitroguajacolicus. The identified bacteria were then examined for their

ability to degrade isoprene by gas chromatography analysis with a flame ionization

detector. The resultant data indicated that strains bW1 and bE1b were the only strains

capable of degrading isoprene. These findings may provide a significant biological sink for

atmospheric isoprene and also information on the cell structure of microorganisms

capable of degradation, as they may contain specifically evolved enzymes encoded by

certain catabolic plasmids. Furthermore, identification of new species may contribute to

environmental biotechnology.

Keywords; Isoprene, biodegradation, taxonomy, 16S rRNA gene sequencing, phylogeny,

Rhodococcus, Arthrobacter, biotechnology

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1. INTRODUCTION

1.1. An introduction to isoprene

Isoprene is a volatile hydrocarbon with an atmospheric concentration nearly equal to that

of methane (CH4) (Alvarez et al., 2009). Isoprene is emitted mainly by vegetation reaching

almost 5 × 1014 g/year, which is similar to annual global methane emissions (Monson et

al., 1991). Isoprene is the second most abundant biogenic hydrocarbon emitted into the

atmosphere (Muller et al., 2008) by bacteria (Kuzma et al., 1995), algae and animals, but

the main source of its production being terrestrial plants (Sharkey et al., 2008). Such

plants include poplars, willows and oaks, which emit isoprene at 0.1 to 3% of the rate of

carbon assimilation (Logan et al., 1999). When released into the atmosphere, isoprene is

transformed by free radicals and ozone, into several species, for example aldehydes,

hydroperoxides, organic nitrates and epoxides that mix into water droplets creating

aerosols and haze (Claeys et al., 2004). Isoprene (C5H8) is the monomer of natural rubber

as well as a common structure motif in biological systems. The isoprenoids (e.g.

carotenes) are derivatives of isoprene. Molecular formula of isoprenoids is multiples of

isoprene in the form of (C5H8)n, which is called the isoprene rule. The precursors to

isoprene units in biological systems are dimethylallyl diphosphate (DMAD) and its isomer

isopentenyl diphosphate (IDP) (Heinz-Hermann, 2000).

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Fig. 1. Chemical structure

of isoprene C5H8.

Picture taken from

Freeman and Beattie

(2008).

1.2. Isoprene’s biological roles

In plants

Additionally, many plants emit isoprene in order to protect themselves against heat stress

providing tolerance to heat spikes (Siwko et al., 2007). Sharkey and Singsaas (2000)

proposed that isoprene plays an important role in thermotolerance. They argued that

isoprene protects the photosynthetic system from thermal damage by preventing the

formation of non-lamellar aggregates and contributing in the stabilisation of the

photosynthetic complexes anchored in thylakoid membrane. Due to its high volatility,

isoprene protects plant membranes against transient exposure to high temperatures, as

the residence time of isoprene in the thylakoid membrane is short.

In humans

Isoprene is abundant in the breath of humans and is thought to serve as a non-invasive

indicator for identifying certain metabolic effects in the human body (Karl et al., 2001).

Such effects include cholesterologenesis, the biosynthesis of cholesterol, which was

suggested by experiments with liver extracts by Deneris et al. (1984). More recent studies

though, stated that isoprene might also be linked to subjects treated with a cholesterol-

lowering drug, which have a resulting effect of reducing isoprene production in human

breath (Stone et al., 1993). Due to these findings there is a raised possibility that

measurements of breath isoprene might represent a non-invasive means of assessing

body cholesterol status since isoprene excretion is directly proportional to high levels of

cholesterol (Taucher et al., 1997).

1.3. Isoprene’s contribution to regional tropospheric chemistry

Physiological and biochemical processes in bacteria can have significant effects on

atmospheric chemistry. It has been argued by Thompson (1992) that isoprene reacts with

the OH radical in the presence of NOx to form ozone, which becomes toxic to humans and

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moreover causes reduction in crop yield. Decomposition of isoprene reduces the

concentration of the atmospheric OH radicals required for degradation of methane. This

leads to deterioration of air pollution in areas where NOx pollution is high. Moreover,

isoprene oxidation in the atmosphere produces CO (Cicerone, 1994), a toxic gas that may

cause poisoning in humans and animals when inhaled at high concentrations.

1.4. Isoprene degradation

Kuzma et al. (1995) reported that soil was found to contain high levels of isoprene-

producing bacteria, especially bacilli. According to Cleveland and Yavitt (1997), the ability

of bacteria to degrade isoprene in soil and freshwater sediments is a fact. By measuring

isoprene uptake, they determined that soil from various ecosystems take up isoprene in a

temperature-dependent manner. The highest rate of isoprene degradation is seen mainly

in temperate forest soils and in soils pre-exposed to isoprene. In forest soil however,

levels of culturable isoprene degraders have been recorded as 5.8×105 cfu/g of dry weight

and enrichment soil cultures resulted in the isolation and identification of certain

microorganisms. Examples of such microorganisms include Nocardia sp., Xanthobacter

sp., Arthrobacter sp. and Rhodococcus sp. that were related to the genus Arthrobacter.

Moreover, Van Ginkel et al. (1987) have proved that pure cultures of a Nocardia sp. were

able to degrade isoprene and use it as sole source of carbon and energy. Furthermore,

Hou et al. (1981) proposed that a Xanthobacter sp. grown on propene was able to

epoxidize isoprene but not use it as a carbon or energy source. Also, as stated by van

Hylckama Vlieg et al. (2000), the pathway of isoprene degradation in Rhodococcus

species is described in detail.

1.5. Isoprene degradation in Rhodococcus AD45

Based on information presented by van Hylckama Vlieg et al. (2000), Rhodococcus

AD45 was the only bacterium isolated, whose pathway has been investigated elaborately,

by using isoprene as sole carbon source. The pathway of isoprene degradation in

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Rhodococcus has been shown by in Copley and Fall (2000). The pathway begins with

conversion of isoprene to 1,2-epoxy-1-methyl-3-butene, a reaction catalysed by mono-

oxygenase encoded by isoABCDEF (Fig. 1). The molecule 1,2-epoxy-1-methyl-3-butene

is then converted to a glutathione conjugate by a glutathione-S-transferase encoded by

IsoI. This enzyme catalyses glutathione, yielding the glutathione conjugate, 1-hydroxy-2-

glutathionyl-2-methyl-3-butene. Two oxidation steps by IsoH in which the hydroxyl is

oxidised to a carboxylate then lead to the formation of 2-glutathionyl-2-methyl-3-butenoic

acid (GMBA). GMBA is then converted to the corresponding CoA thioester by an

unidentified ligase, before removal of glutathione by IsoJ. This proposition is based upon

the presence of an additional gene, IsoG, adjacent to IsoH in the isoprene degradation

gene cluster (van Hylckama Vlieg et al., 2000).

1.6. The genus

Rhodococcus

1.6. The genus Rhodococcus

The genus name ‘Rhodococcus’ was first used by Zopf in 1891, but it was then redefined

in 1977 to accommodate the ‘rhodochrous’ complex. Rhodococcus is a genus of aerobic,

non-motile, non-sporulating and Gram-positive bacteria (Bell et al., 1998) that is closely

related to Mycobacteria and Corynebacteria. It embraces more than 50 strains of bacteria,

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Fig. 2. Scheme for

isoprene degradation

in Rhodococcus

AD45, based on

information

presented in van

Hylckama Vlieg et al.

(2000). Box,

proposed pathway for

further GMBA

metabolism (data not

shown).

isolated from a broad range of environments including soil, water and eukaryotic cells.

Most of Rhodococcus species are non-pathogenic, but nevertheless few pathogenic

strains exist such as R. fascians and R. equi. The causative agent of foal pneumonia is

Rhodococcus equi that may also affect pigs, cattle and especially immunocompromised

patients, such as AIDS patients and those on immunosuppressive therapy (Muscatello et

al., 2007). Rhodococcus fascians is a major pathogen of tobacco plants, contributing to

economic significance. Moreover, Rhodococcus strains have the ability of catabolizing a

wide range of compounds and producing bioactive steroids, acrylamide and acrylic acid

and they are also involved in fossil fuel biodesulfurization (McLeod et al., 2006). Also,

many Rhodococcus strains have the ability to degrade toxic aromatic compounds such as

chlorinated phenols, dinitrophenol and naphthalene, contributing in bioremediation.

1.7. Aims of the present study

This work presents a study of the occurrence of isoprene in both soil and leaf enrichments

in one of the Eastern regions of England (Essex). The major aims of this study are:

i. to determine and characterize the exact phylogenetic position of three unknown

strains isolated from both soil (bW1 and bE1b) and leaf (p12) enrichments, by

using 16S rRNA gene sequencing and phylogenetic analysis;

ii. to examine the ability of these strains in degrading isoprene using Gas

Chromatography (GC) with a Flame Ionisation Detector (FID);

iii. to measure the optical density (OD) of these strains after utilizing various carbon

compounds as a sole of carbon and energy sources using spectrophotometry;

iv. to evaluate the role of these isoprene sources in environmental biotechnology

(bioremediation and biodegradation of pollutants) and the importance of

identifying new species.

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2. MATERIALS AND METHODS

2.1. Source of the organisms, media and growth conditions

Soil and plant samples were taken from selective enrichments in Essex, England. One

gram of each sample was incubated in 9 ml of minimal medium containing (per litre) 0.5

NaCl, 0.5 g MgSO4·7H2O, 0.1 g CaCl2·H2O, 1 g NH4NO3, 1.1 g Na2HPO4, 0.25 g KH2PO4,

50 mg cyclohexamide (10 mg/ml), 1 ml trace element solution (x1000 stock), 10 ml

vitamin solution (x100 stock) and 688 ml of Milli-Q water supplemented with 1 ml of

isoprene stock headspace and 15 g of agar. This medium was incubated at 20 oC on a

horizontal shaker at 110 r.p.m. The appearance of colonies was monitored on a regular

basis and pure cultures of the bacteria were established. Two of the pure cultures that

have been established, were pale-white in colour and were designated bW1 and bE1b,

whereas the third pure culture that has been established was dark-yellow in colour and

was designated p12.

2.2. DNA extraction

Bacterial genomic DNA was extracted from these enrichments by using a standard

method. A small amount was retrieved from each culture and transferred into three tubes

of 0.5 ml hexadecyltrimethylammonium bromide extraction buffer (CTAB). In those three

tubes, 0.5 ml of phenol-chloroform-isoamylalcohol was added and samples were then

transferred in silica bead tubes and lysed for 30 s at a machine speed setting of 5.5 m/s

and the aqueous phase containing the nucleic acids was separated by centrifugation

(1140 × g) for 5 min. Aqueous phase was then extracted and phenol was removed by

mixing with an equal volume of chloroform-isoamyl alcohol followed by centrifugation.

Nucleic acids were then precipitated from the extracted aqueous layer with two volumes

of 1 ml of 30% polyethylene glycol-1.6M NaCl. After left overnight at RT, samples were

centrifuged, and pelleted nucleic acids were washed in 1 ml of ice-cold 70% (vol/vol)

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ethanol (-20 oC). Ethanol was then removed and samples were air-dried for 30 min and

resuspended in 50 μl of water.

2.3. PCR amplification

During the second part of the experiment PCR amplification was carried out, where 1 μl of

DNA extracts (bW1, bE1b and p12) and positive control (E. coli K12 JM 109) were added

to four different PCR tubes (plus one for negative control). Afterwards, a master mix was

made, which contained 30 μl of 10x Taq buffer (containing 15mM MgCl2), 12 μl of 1mM

dNTP mix (from Fermentas), 12 μl of 0.4 M forward primer [27F (AGA GTT TGA TCC

TGG CTC AG)], 12 μl of 0.4 M reverse primer [1492R (TAC GGY TAC CTT GTT ACG

ACT T)], 1.50 μl of the TopTaq DNA polymerase enzyme and 226.50 μl of RNase free

water. Then 49 μl of the master mix were transferred into each PCR tube except into the

one with the negative control that 50 μl was added. The solutions were then loaded on the

PCR machine for 2 h. The amplification was performed in a thermocycler in a

programmable block by using 30 reaction cycles, each consisting of a 1-min denaturation

step at 94 oC, a 1-min annealing step at 60 oC and a 2-min elongation step at 72 oC.

2.4. Agarose gel electrophoresis

Firstly, 200 ml of TAE (Tris, Acetate and EDTA) and 1.60 g of agarose powder (Fisher)

were added in a cylindrical flask and placed in the microwave for 1-3 min, until the

agarose was completely dissolved. Once the solution was solidified, the 0.8% agarose gel

was placed into a gel box. The gel box was then filled with the solution up to a certain

level and 5 μl of a molecular weight DNA ladder (#SM 0333-Fermentas) were loaded into

the first lane of the gel. Afterwards, 1 μl of the loading dye (Fermentas) was mixed with 5

μl of the PCR products and loaded into the additional wells of the gel. The gel was then

run at 120 V until the dye lines were approximately 80% of the way down the gel. A typical

run time of a gel is about 45 min-1 h. The gel was then removed from the gel box and

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placed into 20 mg/ml of Ethidium Bromide (EtBr) (Sigma). Finally, using software that has

UV light, the DNA fragments were visualized.

2.5. PCR purification

PCR products were purified by using a method called GenEluteTM PCR Clean-up Kit

(catalog number NA1020) as described by SIGMA manufacturing company (see

Appendix. 1).

2.6. DNA sequencing

Sequencing of the purified PCR products was carried out by a method called Sanger

Sequencing. Amplified PCR products and 1492R primer were sent for DNA sequencing to

a company called Source BioScience.

2.7. Phylogenetic analysis

The generated DNA sequences were then analyzed and edited by Chromas LITE

software. The 16S rRNA gene sequences of strains bW1, bE1b and p12 were aligned

with their closest relatives and strains selected from Nucleotide BLAST (Basic Local

Alignment Search Tool) database. By using MUSCLE (multiple sequence comparison by

log-expectation) alignment, the pairwise evolutionary distances for the above sequences

were computed using the DNADIST program with Kimura’s two-parameter model. A

phylogenetic tree was constructed by using neighbour-joining method. Also the stability of

relationships was assessed by a bootstrap analysis of 900 replicates.

2.8. Isoprene degradation and gas analysis

The capability of strains bW1, bE1b and p12 to degrade isoprene was determined in liquid

cultures, where 0.1 ml of cells grown in LB broth were inoculated in 9.9 ml of minimal

medium and 1 ml of isoprene stock headspace. Isoprene degradation was measured on a

gas chromatograph equipped with a flame ionization detector. Gas samples were injected

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on-column by split-less injection. Column (capillary) temperature was 120 oC and the

retention time of the isoprene was 0.6 min. Isoprene concentration was determined by

peak area measured with a peak integrator.

2.9. Statistical analysis

Further statistical tests were also carried out in IBM SPSS Statistics program using one-

way ANOVA and Dunnett’s test for comparing the significant differences (p-value)

between control and each of the other strains in order to confirm isoprene degradation.

2.10. Physiological and biochemical characterization

Five different carbon compounds were used to examine the ability of the cultures to utilize

a carbon compound provided as the sole carbon source, using minimal medium

containing 1% (w/v) of the carbon source. The samples were inoculated in 8.9 ml of

minimal medium, 1 ml of carbon source and 0.1 ml of cells grown in LB broth. Samples

were then incubated at 20 oC, shaken in the dark at 110 r.p.m. The utilization results were

checked over a period of two weeks by determining the OD600 using spectrophotometry.

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3. RESULTS

3.1. PCR analysis and agarose gel electrophoresis

The agarose gel electrophoresis patterns generated after DNA extraction and PCR

amplification can be seen in Fig. 2A. DNA bands from strains bW1, bE1b and p12 indicate

successful amplification of the target sequences. The gel also shows a DNA ladder

containing different DNA fragments of known length for sizing the bands, a positive

control and a negative control. The size of PCR products can be estimated by comparison

with a DNA ladder, which in this case is #SM 0333 and thus the molecular size of an

unknown band of DNA can be determined. The patterns of strains bW1, bE1b and p12

were identical and according to Fig. 2A, the size of their DNA bands was 1,500 base pairs

(bp). Moreover, except of the major bands that can be seen, gels usually show and some

other fainter bands in the same lane.

3.2. PCR purification analysis and agarose gel electrophoresis

Therefore in order to get distinguishable and clear DNA bands, PCR purification was

performed. Distinguishable DNA bands of 1,500 bp (Fig. 2B) indicated successful PCR

purification as no faint bands could be seen in the same lane. Remaining primers, dNTPs,

enzymes, short-failed PCR products and salts from PRC fragments over 100 bp were

removed by PCR purification.

The gene target used in this study was 16S rRNA, which is a ~1,500 base pair gene that

codes for a portion of the 30S ribosome. Universal primers, 1492 R and 27 F are usually

chosen as complementary to the conserved regions at the beginning of the gene (0 bp) or

at the end of the sequence (1,550 bp). The sequence of the variable region in between is

used for the comparative taxonomy.

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Fig. 4. Ethidium bromide-stained PCR products of strains bW1, bE1b and p12 after

agarose gel electrophoresis. (A) Agarose gel displaying a DNA ladder (#SM 0333)

containing DNA fragments of known length for sizing the bands, three 1,500 bp DNA

bands of the PCR products from strains bW1, bE1b and p12, positive (E coli K12 JM109)

and negative controls and also the DNA products extracted from strains bW1, bE1b and

p12. (B) Agarose gel showing a DNA ladder and three 1,500 bp DNA bands of the

purified PCR products from strains bW1, bE1b and p12.

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Fig. 3. A schematic for 16S rRNA, located on the small ribosomal subunit (30S).

Circles represent conserved regions that serve as gene targets for PCR amplification

and DNA sequencing of bacteria and arrows the primers used in this study. Picture

taken from Reller et al., (2007).

3.3. Isoprene degradation and gas analysis

Time-course of isoprene degradation at 0, 45, 140 164, 216 and 384 hours was examined

for strains bW1, bE1b and p12 and is displayed on Fig.3. A decrease in isoprene levels

was observed in strains bW1 and bE1b, whereas strain p12 showed no significant

isoprene loss. Also, these observations can be more clearly derived from Fig. 4, where

the percentage of isoprene degradation was calculated in relation to control. The

decrease in isoprene levels (0% remaining isoprene compared to control) indicated that

both bW1 and bE1b strains degraded completely the isoprene, whereas p12 (99.5 %

remaining isoprene compared to control) did not.

Over and above, standard deviation was calculated in order to be aware of the differences

of each observation from the mean. Standard deviation can be expressed as an error bar

on either a scatter plot or a bar chart and its length indicates the uncertainty in a particular

value. The larger the error bars, the more spread the distribution of points is and also the

smaller the error bars the tighter the distribution between the points.

Also, p-value, the statistically significance probability was calculated by using one-way

ANOVA and Dunnett’s test, displaying the p-values of strains bW1, bE1b and p12

compared to control. P-value of strain bW1 was found to be 0.033 and 0.037 for strain

bE1b, both numbers lower than 0.05, showing that the hypothesis that mean relative area

for isoprene degradation is the same can be rejected, indicating that strains bW1 and

bE1b degraded isoprene. On the other hand, strain p12 presented a p-value greater than

0.05, showing that there is no evidence to reject the hypothesis that the mean relative

area is the same as control, suggesting that strain p12 is not able to degrade isoprene

(Table 1).

Rhodococcus seems to be the main organism responsible for isoprene degradation in soil

samples studied in the specific investigation, as phylogenetic analysis carried out by

neighbour-joining method and Kimura-2-parameter model based on the 16S rRNA gene

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sequence showed that both bW1 and bE1b isolates belong to the genus Rhodococcus,

which is a part of the phylum Actinobacteria (Fig.5). Moreover, another actinobacterium

was studied in this research involving an Arthrobacter species, which was also isolated.

Strain p12 though displayed incapability to degrade isoprene. Therefore, it is obvious that

isoprene-degrading ability or disability is widespread in a variety of phyla. This brings into

question whether such microorganisms are specialists isoprene-degraded or generalists.

In order to find out the answer to that, their ability to grow on a wide range of carbon

sources, such as sugars (glucose, fructose and maltose), organic acids (pyruvate) and

glycerol was tested (Fig.7).

Fig. 5. Time course of isoprene degradation in the cells of Rhodococcus strains bW1 and

bE1b, Arthrobacter strain p12 and sterile control. Data are the means and ± standard

deviation for six different times carried out in triplicates (n=3).

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Fig. 6. Time-course of isoprene degradation percentage remaining compared to control.

Data are the percentage means in respect to sterile control.

Table 1. Statistically significant differences between control and each of the other strains

using one-way ANOVA and Dunnett’s test.

Multiple Comparisons (Dunnett t (2-sided)a)

(I) Strains (J) Strains

Mean Difference

(I-J) Std. Error Sig.

95% Confidence Interval

Lower Bound Upper Bound

bW1 control -134591.5000* 49109.9572 .033 -259349.043 -9833.957

bE1b control -131779.5556* 49109.9572 .037 -256537.098 -7022.013

p12 control -7212.9444 49109.9572 .998 -131970.487 117544.598

3.4. Phylogenetic analysis

To investigate the phylogenetic relationships between strains bW1 and bE1b and

Rhodococcus species, the 16S rRNA gene sequence was compared with those of

representative members of the genus Rhodococcus. A tree showing the phylogenetic

affinity of the new isolates to other members of the genus Rhodococcus is shown in Fig. 3

and shows that the unknown bacterial species represent a lineage within the genus

Rhodococcus. The species most closely related to the two strains are R. globerulus, R.

jostii, R.marinonascens, R. tukisamuensis and R. maanshanensis. It is apparent from Fig.

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3 though that strains bW1 and bE1b form distinct evolutionary line in the R. globerulus

sub-clade having a bootstrap percentage, which indicate the reliability of clusters of 96%.

The 16S rRNA gene sequence of p12 isolate was also determined and revealed a 92%

bootstrap percentage between p12 strain and A. ilicis, A. nicotinovorans and A.

nitroguajacolicus showing their high phylogenetic relatedness. The topology of the

consensus phylogenetic tree (Fig. 4) displays the phylogenetic position of the p12 strain

next to its closest phylogenetic neighbours. Also, bootstrap percentage indicates the

reliability of the cluster descending from that node so the higher the percentage number,

the more reliable is the estimate of the taxa.

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Fig. 7. Phylogenetic tree derived from analysis of 16S rRNA gene sequences of strains

bW1 and bE1b compared to the other species of the genera Rhodococcus,

Mycobacterium and Gordonia. The tree was constructed by using the neighbour-joining

method and the evolutionary distances were computed using the Kimura two-parameter

model. Numbers at nodes indicate the percentages of bootstrap support, derived from

900 replications. The bar represents one substitution per 100 nucleotides. Mycobacterium

species and Gordonia species are used as outgroups.

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Fig. 8. Phylogenetic tree derived from analysis of 16S rRNA gene sequences of strain

p12 compared to the other species of the genera Arthrobacter and Mycobacterium. The

tree was constructed by using the neighbour-joining method and the evolutionary

distances were computed using the Kimura two-parameter model. Numbers at nodes

indicate the percentages of bootstrap support, derived from 900 replications. The bar

represents one substitution per 100 nucleotides. Mycobacterium species are used as

outgroups.

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Fig. 9. Growth of strains bW1, bE1b and p12 on different carbon sources: glucose,

fructose, maltose, glycerol and pyruvate and on control at 96 h of incubation. Data are the

means and ± standard deviation at 96 h carried out in triplicates (n=3).

3.5. Morphological characteristics

For the investigation of phenotypical characteristics of strains bW1, bE1b and p12, their

cultures grown on minimal medium agar plates were observed. The appearance of

colonies was monitored on a regular basis and cultures of the bacteria were established.

Two of the three isolates, bW1 and bE1b produced pale white-coloured, glistening,

mucoid and entire colonies up to 6mm in diameter. Strain bW1 produced irregularly

wrinkled and raised margins, whereas bE1b flat wrinkled margins. Colonies grew

aerobically on minimal medium agar at 20 oC, which was an optimal temperature for them

to grow.

The third isolate, p12, produced dark yellow-coloured colonies. Strain p12 was also

incubated at 20 oC and was cultured on minimal medium agar plates in an isoprene rich

environment. The dark yellow colonies of p12 were 0.5-1 mm in diameter, circular,

umbonate and shiny with moist and entire margins.

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3.6. Physiological and biochemical characteristics

Growth and physiological characteristics as well as some other chemotaxonomic

properties of the strains bW1 and bE1b are shown in Table 2; details relating to colony

morphology, temperature requirement, utilization as sole carbon sources and isolation

source. The chemotaxonomic and phenotypic features showed that strains bW1 and

bE1b should be allocated to the genus Rhodococcus.

The characteristics of strain p12 were observed to be consistent with the description of

the genus Arthrobacter with respect to various phenotypic properties including cell

morphology, temperature requirements and utilization as sole carbon sources (Table 3).

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Table 2. Physiological characteristics that differentiate strains bW1 and bE1b from other Rhodococcus species

Taxa are identified as: 1, R. globerulus DSM 43954; 2, R. jostii IFO 15295; 3, R. marinonascens DSM 43752; 4, R. tukisamuensis JCM 11308; 5, R. maanshanensis JCM 11374; 6, strain bW1; 7, strain bE1b. +, Positive; -, negative; w, weakly positive; ND, not determined.

Table 3. Differential characteristics of some Arthrobacter species and strain p12

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Characteristic 1 2 3 4 5 6 7

Colony morphology

Pink-coloured with rough and entire margins

Light pink-coloured with irregular margins

Cream-coloured with a tinge of pink and lobate margins

Cream-coloured with irregular margins

Cream-coloured with a tinge of pink

Pale white-coloured with raised, mucoid and entire margins

Pale white-coloured with moist and entire margins

Temperature requirements

10-40 oC 10-30 oC 18-20 oC 15-45 oC 25-30 oC 20 oC 20 oC

Utilization as sole carbon sources:

Glucose + + + + + + + Fructose + + + w + + + Maltose + + - + w + + Glycerol + - ND + - + + Pyruvate + ND ND ND ND + + Alanine ND ND + ND + ND ND Succinate + + ND ND ND ND ND Inulin + ND + + w ND ND

Isolated from: Soil (polluted site - The Netherlands)

Femur (remains of a grave - Czech Republic)

Marine bottom sediments

Soil (Japan)

Soil (Maanshan mountain, China)

Soil (willow tree)

Soil (no tree)

Characteristic 1 2 3 4 5 6 7

Colony morphology

Yellow-coloured, circular and smooth

White-coloured with entire margins

Yellow-coloured, opaque and convex

Grey-coloured, circular and convex

Grey-coloured, smooth and opaque

Grey-coloured with moist and shiny margins

Dark yellow-coloured with moist and entire margins

Temperature requirements

20-30 oC 20-30 oC 25-30 oC 15-30 oC 15-30 oC 15-30 oC 20 oC

Utilization as sole carbon sources:

Glucose + + + + + + + Fructose + ND ND + ND ND + Maltose + ND ND + ND ND + Glycerol + ND ND + ND ND + Pyruvate ND ND ND + ND ND + Arginine + + + ND + + ND Galactose + + + + + + ND Inositol + + ND ND + + ND

Isolated from: Leaf (American holly-ilex opaca)

Soil Soil (spill site containing herbicide atrazine)

Forest soil

Sandy dune soil after app. of atrazine

Sugar-disclosed soil

Leaf (poplar tree)

Taxa are identified as: 1, A. ilicis DSM 20138; 2, A. histidinolovorans DSM 20115; 3, A. aurescens DSM 20116; 4, A. nitroguajacolicus CCM 4924; 5, A. nicotinovorans DSM 420; 6, A. ureafaciens DSM 20126; 7, strain p12. +, Positive; -, negative; w, weakly positive; ND, not determined.

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4. DISCUSSION

4.1. Overview

This study aimed to identify and characterize cultures of isoprene-degrading soil bacteria

isolated from soil near trees or/and lake and leaf enrichments. Phylogenetic analysis based

on the 16S rRNA gene sequencing was used in order to identify these bacteria. The

identified strains were then aligned with their closest relatives selected from BLAST database

and a phylogenetic tree was then constructed. The phylogenetic relationships between the

strains sequenced in this report and a few other reference strains of the genera Mycobacteria

and Gordonia; both selected from literature, are shown on the phylogenetic trees. The

identified bacteria were then cultured and their ability to degrade isoprene was examined by

a method called GC. Moreover five different carbon sources were used to investigate the

ability of these strains in utilizing carbon compounds.

4.2. Phylogenetic analysis

Phylogenetic inference based on 16S rRNA sequences indicated clearly that strains bW1

and bE1b belong to the genus Rhodococcus and more specifically as members of the group

IV, which is the largest Rhodococcus cluster, including R. globerulus and R. marinonascens

(Rainey et al., 1995). Strains bW1 and bE1b exhibited their highest homology to

Rhodococcus species forming a coherent cluster with Rhodococcus globerulus, R. jostii, R.

marinonascens, R. tukisamuensis and R. maanshanensis. It is apparent from Fig. 5 though

that both R. bW1 and R. bE1b form a distinct evolutionary line in the R. globerulus subclade

possessing high statistical significance.

Similarly, phylogenetic analysis showed that strain p12 belongs to the radiation of the genus

Arthrobacter and more specifically as a member of the group I. The highest binary 16S rRNA

similarity value was found with Arthrobacter ilicis, A. nicotinovorans and A. nitroguajacolicus.

The topology of the consensus phylogenetic tree (Fig. 6) displays the phylogenetic position

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of strain p12 next to its closest phylogenetic neighbours, also possessing high statistical

significance.

The software used for sequence comparison and retrieval from databases in this study is

BLAST. BLAST uses statistical theory to produce a bit score and e-value (expect value) for

each alignment pair. The higher the bit score, the better the alignment will be and the lower

the e-value, the more significant the hit will be. Generally the bit score gives an indication of

how good the alignment is and e-value the statistical significance (Kerfeld and Scott, 2011).

MUSCLE alignment is the one used throughout this experiment, which uses a progressive

alignment strategy and refinement steps in order to improve accuracy.

Phylogenetic trees shown in Fig. 5 and Fig. 6 were constructed using the neighbor-joining

method. Neighbour-joining is a clustering method for the creation of phonetic trees

(phenograms). The main advantage of neighbor-joining is its speed and ease as well as its

capability to analyze large data sets and bootstrapping, compared to other means of analysis

that may be computationally prohibitive (Saitou and Nei, 1987).

Evolutionary distances are also fundamental for the study of molecular evolution and are

very useful for phylogenetic reconstructions and the estimation of divergence times. The one

that was used in the present study was the Kimura 2-parameter model, which takes into

consideration transitional and transversional substitution rates (Kimura, 1980).

Estimating the reliability of the tree is also a very important factor on phylogenetic

reconstructions. A way to estimate this reliability is by bootstrap method. Once the number of

bootstrap replicates is set between 100 and 2,000, a tree will appear with numbers on every

node. The numbers on the nodes, also called bootstrap percentages, indicate the reliability of

the cluster descending from that node. The higher the number, the more reliable is the

estimate of the taxa (Hall, 2013).

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4.3. Degradation of isoprene and its relation to catabolic plasmids

It is the aim of this work to examine whether Rhodococcus bW1, Rhodococcus bE1b and

Arthrobacter p12 degrade isoprene or not. The results of investigations on isoprene

consumption by these soil-microorganisms suggested that only Rhodococcus bW1 and

Rhodococcus bE1b were able to degrade isoprene. This discovery may provide an important

biological sink for atmospheric isoprene.

Degradation occurs mainly by microorganisms and especially bacteria, under many

enzymatic processes. For soil bacteria, these enzymatic processes are encoded by large

groups of genes clustered on the main chromosome or on catabolic plasmids. Catabolic

plasmids are usually 80 to >200 kb with one or more clusters of multicistronic transcriptional

units, having 10-15 degradative genes. Most of the times, catabolic plasmids are self-

transmissible by cell-to-cell contact and are transferred throughout diverse soil bacteria,

resulting to novel combinations of degradative genes. Such genes are competent of

degrading the most complex, recalcitrant and persistent of synthetic molecules (Pemberton

and Schmidt, 2001). In some cases, these gene clusters occur within transposable elements,

also called transposons, having the ability of changing their position within the genome,

between plasmids and the main chromosome.

In bacteria, transposons can move from chromosomal DNA to plasmid DNA and back,

providing powerful mechanisms for the evolution of bacteria, leading to their ability of

degrading and also recycling mutagenic, carcinogenic and/or teratogenic chemicals.

(Pemberton and Schmidt, 2001).

Furthermore, bacteria that are capable of degrading aromatic hydrocarbons are mainly found

in soil, in marine sediments, on the surfaces of plants and in the gut of organisms that feed

on plants. Such bacteria include members of the genera Rhodococcus, Pseudomonas,

Bacillus, Streptomyces, Alcaligenes, Burkholderia, Ralstonia, Xanthomonas, etc. In some

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instances, many soil microorganisms have specifically evolved enzymes that degrade

chlorinated substrates via modified ortho cleavage pathway enzymes encoded by catabolic

plasmids (Pemberton and Schmidt, 2001). Therefore, the conclusion derived from the study

that was conducted, confirmed that both Rhodococcus bW1 and Rhodococcus bE1b consist

of specifically evolved enzymes, which are responsible for the degradation of isoprene.

Conversely, Arthrobacter p12 do not consist of these specific enzymes, therefore resulting to

the inability of the strain to degrade isoprene.

4.4. Comparison of Rhodococcus bW1 and Rhodococcus bE1b with phylogenetic neighbours

In addition to the results obtained by 16S rRNA gene sequence analysis, strains bW1 and

bE1b can also be distinguished from their closest relatives based on a number of other

characteristics (Table 2). The finding that strains bW1 and bE1b are more closely related to

Rhodococcus globerulus than to R. jostii, R. marinonascens, R. tukisamuensis and R.

maanshanensis is supported by the findings that strains bW1 and bE1b, alike R. globerulus,

are capable of utilizing glucose, fructose, maltose, glycerol and pyruvate. However, strains

bW1 and bE1b differ from R. globerulus in that they do not form a pink pigment but a pale-

white one. The temperature growth range also differs between the strains (Table 2). Strains

bW1 and bE1b grow at 20 oC, whereas R. globerulus grows at a comparatively wide range of

temperatures (10-40 oC). Finally, the sources of isolation are similar as strains bW1 and

bE1b as well as R. globerulus derived from soil enrichment. In order to be able to say that R.

bW1 and R. bE1b do not differ from R. globerulus, more tests should have been carried out

such as determination of their peptidoglycan type.

4.5. Comparison of Arthrobacter p12 with phylogenetic neighbours

Apart from the 16S rRNA gene sequencing, strain p12 can also be differentiated from its

closest relatives with respect to various phenotypic and biochemical characteristics (Table 3).

The findings from 16S rRNA sequence data that strain p12 is more closely related to A. ilicis,

A. nitroguajacolicus and A. nicotinovorans than to A. aurescens, A. histidinolovorans and A.

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ureafaciens are supported by the findings that strain p12, alike A. ilicis and A.

nitroguajacolicus are capable of growing on glucose, fructose, maltose, glycerol and

pyruvate. However, strain p12 differs from A. nitroguajacolicus and A. nicotinovorans in that

they do not form a grey pigment but a yellow one, which is more similar to A. ilicis. The genus

Arthrobacter includes a few species that are unpigmented (white-coloured colonies) such as

A. histidinolovorans, but a majority of them produce a range of pigments, for example, A.

ilicis, A. aurescens and A. nicotianae that produce a yellow pigment, A. nicotinovorans, A.

nitroguajacolicus and A. ureafaciens a grey to yellow pigment, A. agilis a blue to black

pigment and A. atrocyaneus a red pigment (Reddy et al., 2000). In order to distinguish

Arthrobacter p12 from the rest yellow-pigmented Arthrobacter species, more tests should

have been performed such as determination of its peptidoglycan type. This would be very

useful in identifying the exact position of A. p12 as it has already been established as a

member of group I, which includes those species containing the peptidoglycan Lys-Ser-Thr-

Ala (A. oxydans and A. polychromogenes) and those with Lys-Ala-Thr-Ala (A. aurescens, A.

ilicis, A. ureafaciens, A. histidinolovorans and A. nicotinovorans) (Reddy et al., 2000).

Moreover, the temperature growth range is the same as all of the strains grow at 20 oC.

Finally, the sources of isolation differ, as A. nitroguajacolicus and A. nicotinovorans were

isolated from soil, whereas strain p12 and A. ilicis were isolated from leaves.

4.6. Rhodococcus and Arthrobacter in environmental biotechnology (Bioremediation and

biodegradation of pollutants)

Generally, rhodococcus cells are hydrophobic because of the aliphatic chains of mycolic

acids in their cell walls. Thus, degradation of hydrophobic pollutants can occur by allowing

cells to adhere to oil or water interphases. Moreover, some rhodococcus strains are

psychrotrophic, which may contribute to effective bioremediation in cold climates and high

pHs. However, high pHs can effectively prevent bioremediation, but it has been shown that

some strains of rhodococci were able to degrade benzene in an alkaline environment up to

pH 10 (Fahy et al., 2008). Rhodococci are also candidate organisms for use as inocula

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(immobilised cells) in many treatments, promising in laboratory stimulations. Furthermore,

rhodococci exhibit high tolerance to many toxic substrates and solvents playing a part in

mediating remediation in environments with high concentration of pollutants (Hennessee and

Li, 2010).

As for Arthrobacter species, recent studies on cold and chemical tolerance indicated that

Arthrobacter strains are capable of adapting well in cold temperature bioremediation

operations (Hennessee and Li, 2010).

4.7. Importance of identifying new species

Identification of new species of bacteria can be accomplished by possible biotechnological

applications, which may be useful in determining the physiological and biochemical

properties of the microorganisms contributing to the selection of the most appropriate cloning

vectors for genetic manipulation. Furthermore, methods for the identification of Rhodococcus

species may be important in environmental biotechnologies, such as in monitoring bacterial

populations during bioremediation studies. For instance, R. coprophilus can be used as an

indicator of animal faecal pollution of waterways and R. rubber or R. rhodochrous as

indicators in oil prospecting and identification of nocardioforms used in foaming in activated

sludge systems (Bell et al., 1998).

Also, a number of problems have been identified in the diagnosis of R. equi infections in

humans. The specific microorganism is not well recognized as a pathogen and it may be

dismissed as a commensal diphtheroid rather than an infecting agent when it is tested in a

clinical sample. It is also possible to be confused with Mycobacterium tuberculosis due to

their similar appearances.

4.8. Research needs and further studies

Degradation is carried out mainly by soil microorganisms after the completion of various

enzymatic steps. These steps are encoded by genes clusters on catabolic plasmids.

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Research contributing to an understanding of the regulation of these genes in response to

different substrates and conditions is needed. Further studies of understanding their cellular

physiology and mechanisms for adaptation to new substrates for a wider number of strains

are also required. Also, Rhodococcus strain’s abilities relative to biodegradation and

tolerance of extreme conditions and toxic substrates have yet to be discovered (Larkin et al.,

2010).

Furthermore, studies on genes and proteins involved in degradation of hydrocarbons by

Arthrobacter strains are required, particularly at a molecular level. Their adaptive

mechanisms used to tolerate harsh environments and utilize recalcitrant substrates will also

need further study. However, research leading to the understanding of catabolism of

hydrocarbons and halogenated compounds as well as biodesulphurization and physiological

adaptations to chemical stress may enhance their importance and usefulness in

biotechnology (Hennessee and Li, 2010).

4.9. Conclusion

In conclusion, this study has revealed the phylogenetic position of strains bW1, bE1b and

p12. Results have indicated that strains bW1 and bE1b are closely related to Rhodococcus

sp. showing higher similarity to Rhodococcus globerulus. Likewise, strain p12 represented a

distinct line within the genus Arthrobacter and more specifically to Arthrobacter ilicis, A.

nicotinovorans and A. nitroguajacolicus. Rhodococcus bW1, Rhodococcus bE1b and

Arthrobacter p12 were then examined for their ability to degrade isoprene and also to grow

on various carbon sources. Based on the observations of isoprene, it has been proved that

only Rhodococcus bW1 and Rhodococcus bE1b have the ability of degrading isoprene.

Therefore, it can be assumed that their degradative capabilities can be further investigated

regarding to their specific evolved enzymes encoded by certain plasmids. Furthermore the

identification of new species may significantly contribute in environmental biotechnology.

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APPENDICES

Appendix 1. GenEluteTM PCR Clean-Up Kit by SIGMA manufacturing company.

This part of the experiment involved where GenElute plasmid mini spin columns were

inserted into collection tubes and column preparation solution was then added to each of

them and centrifuged at 1140 g for 30 s-1 min. The eluate was then discarded and 250 μl of

binding solution were added to 50 μl of the PCR reaction and mixed. Solution was then

transferred to binding column, centrifuged and discarded. Binding column was then replaced

into the collection tubes and 0.5 ml of diluted wash solution was added. Columns were then

replaced into the collection tubes, centrifuged to remove any excess ethanol and discarded.

Afterwards, columns were transferred to fresh 1.5 ml collection tubes and 50 μl of water were

added and then incubated at RT for a min. For eluting the DNA, tubes were centrifuged for

one min. The purified PCR products were used directly for DNA sequencing after they were

run through agarose gel electrophoresis.

Appendix 2. Isoprene degradation in the cells of Rhodococcus strains bW1 and bE1b,

Arthrobacter strain p12 and sterile control using IBM SPSS program. Data are the means

carried out in triplicates.

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Appendix 3. Rhodococcus strains analyzed, with their 16S rRNA accession numbers

Organism Strain Accession numberGordonia aichiensis DSM 43978 X80633Gordonia bronchialis DSM 43247 X79287Gordonia rubripertincta DSM 43248 AY995557Gordonia terrae DSM 43249 X79286Mycobacterium bovis ATCC 12910 X55589Mycobacterium chlorophenolicus ATCC 43826 X79292Mycobacterium tuberculosis ATCC 27294 X52917Rhodococcus aetherivorans 10bc312 AF447391Rhodococcus artemisae YIM 65754 GU367155Rhodococcus aurantiacus ATCC 25938 AF283282Rhodococcus baikonurensis GTC 1041 AB071951Rhodococcus canchipurensis MBRL 353 JN164649Rhodococcus cerastrii C5 FR714842Rhodococcus cercidiphylli YIM 65003 EU325542Rhodococcus chubuensis DSM 44019 X80627Rhodococcus coprophilus DSM 43347 X80626Rhodococcus corynebacterioides DSM 20151 AF430066Rhodococcus equi DSM 20307 AF490539Rhodococcus equi DSM 43199 X80613Rhodococcus erythropolis DSM 43066 X79289Rhodococcus erythropolis DSM 43188 X80618Rhodococcus fascians DSM 20669 X79186Rhodococcus globerulus DSM 43954 X80619Rhodococcus gordoniae W4937 AY233201Rhodococcus imtechensis RKJ 300 AY525785Rhodococcus jialingiae djl-6-2 DQ185597Rhodococcus jostii IFO 16295 AB046357Rhodococcus koreensis DNP 505 AF124342Rhodococcus kroppenstedtii K07-23 AY726605Rhodococcus kunmingensis YIM 45607 DQ997045Rhodococcus kyotonensis DS 472 AB269261Rhodococcus luteus DSM 43673 X79187Rhodococcus maanshanensis M712 AF416566Rhodococcus marinonascens DSM 43752 X80617Rhodococcus maris DSM 43672 FJ468333Rhodococcus nanhaiensis SCS10 10187 JN582175Rhodococcus opacus DSM 43205 X80630Rhodococcus percolatus DSM 44240 X92114Rhodococcus phenolicus DSM 44812 AY533293Rhodococcus pyridinivorans PDB9 AF173005Rhodococcus qingshengii djl-6 DQ185597Rhodococcus rhodnii DSM 43336 X80621Rhodococcus rhodochrous DSM 43241 X79288Rhodococcus rhodochrous DSM 43274 X80624Rhodococcus roseus DSM 43274 X80624Rhodococcus ruber DSM 43338 X80626Rhodococcus triatomae IMMIB RIV-095 AJ854055Rhodococcus trifolii T8 FR714843Rhodococcus tukisamuensis MB8 AB067734Rhodococcus wratislaviensis CCM 4930 AJ786666Rhodococcus yunnanensis YIM 70056 AY602219Rhodococcus zopfii DSM 44108 AF191343

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Appendix 4. Arthrobacter strains analyzed, with their 16S rRNA accession numbers

Organism Strain Accession numberArthrobacter agilis DSM 20550 X80748Arthrobacter albidus LC-13 AB248533Arthrobacter albus DSM 13068 AJ243421Arthrobacter alkaliphilus LC-6 AB248527Arthrobacter alpinus S6-3 GQ227413Arthrobacter arilailensis DSM 16368 AJ609624Arthrobacter aurescens DSM 20116 X83405Arthrobacter bergerei DSM 16367 AJ609630Arthrobacter castelli LMG 22283 AJ639826Arthrobacter chlorophenolicus DSM 12829 AF102267Arthrobacter citreus DSM 20133 X80737Arthrobacter creatinolyticus GIFU 12498 D88211Arthrobacter crystallopoietes DSM 20117 X80738Arthrobacter cumminsii DMMZ 445 X93364Arthrobacter equi IMMIB L-1606 FN673551Arthrobacter gandavensis LMG 21287 AJ491108Arthrobacter globiformis DSM 20124 X80736Arthrobacter histidinolovorans DSM 20115 X83406Arthrobacter humicola KV-653 AB279890Arthrobacter ilicis DSM 20138 X83407Arthrobacter koreensis CA15-8 AY116496Arthrobacter luteolus DSM 13067 AJ243422Arthrobacter methylotrophus DSM 14008 AF235090Arthrobacter monumenti LMG 19502 AJ315070Arthrobacter mysorens DSM 12798 AJ617482Arthrobacter nasiphocae CCUG 42935 AJ292364Arthrobacter nicotianae DSM 20123 X80739Arthrobacter nicotinovorans DSM 420 X80743Arthrobacter nitroguajacolicus CCM 4924 AJ512504Arthrobacter oryzae KV-651 AB279889Arthrobacter oxydans DSM 20119 X83408Arthrobacter parietis LMG 22281 AJ639830Arthrobacter pascens DSM 20545 X80740Arthrobacter polychromogenes DSM 20136 X80741Arthrobacter protophormiae DSM 20168 X80745Arthrobacter psychrolactophilus ATCC 700733 AF134179Arthrobacter ramosus DSM 20546 X80742Arthrobacter rhombi CCUG 38813 Y15884Arthrobacter roseus DSM 14508 AJ278870Arthrobacter russicus A1-3 AB071950Arthrobacter sanguinis 741 EU086805Arthrobacter sulfonivorans DSM 14002 AF235091Arthrobacter sulfurous DSM 20167 X83409Arthrobacter uratoxydans DSM 20647 X83410Arthrobacter ureafaciens DSM 20126 X80744Arthrobacter woluwensis CUL 1808 X93353Mycobacterium bovis ATCC 12910 X55589Mycobacterium tuberculosis ATCC 27294 X52917

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