This document summarizes a student research project characterizing soil and leaf bacteria capable of degrading the volatile compound isoprene. Three bacterial isolates were obtained from soil and leaf samples exposed to isoprene. Based on 16S rRNA gene sequencing and phenotypic characterization, two soil isolates were identified as Rhodococcus species closely related to R. globerulus, while the leaf isolate was identified as an Arthrobacter species closely related to A. ilicis, A. nicotinovorans, and A. nitroguajacolicus. Gas chromatography analysis showed the Rhodococcus isolates were able to degrade isoprene. Identifying these isoprene-degrading bacteria provides insight into atmospheric chemistry and potential applications in

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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

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4.5. Comparison of Arthrobacter p12 with phylogenetic neighbours……… 32-33
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 M
A. Arthrobacter MgSO4∙ magnesium sulfate
AIDS Acquired Immune Deficiency
Syndrome
MUSCLE Multiple sequence
comparison by Log-
Expectation
ANOVA Analysis of variance MgCl2 magnesium chloride
B N
BLAST 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 O
CaCl2 calcium chloride OD Optical Density
CTAB cetyl trimethylammonium bromide OH hydroxide
D P
DNA deoxyribonucleic acid PCR Polymerase chain
reaction
dNTP Deoxyribonucleotide triphosphate R
E R. Rhodococcus
EtBr Ethidium Bromide RT Room Temperature
e-value expect value rRNA Ribosomal
ribonucleic acid
F S
FID Flame Ionisation Detector sp. species
G T
GMBA 2-glutathionyl-2-methyl-3-butenoic
acid
TAE Trisbase, acetic acid
GC Gas Chromatography U
K UV ultraviolet
KH2PO4 monopotassium phosphate W
L w Weakly positive
LB Lysogeny broth

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Units of Measure
cfu 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 acids
Ala Alanine
Lys Lysine
Ser Serine
Thr Threonine
Nucleobases
A 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).
Fig. 1. Chemical structure
of isoprene C5H8.
Picture taken from
Freeman and Beattie
(2008).

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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,
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).

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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 o
C 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 o
C). 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 o
C, a 1-min annealing step at 60 o
C and a 2-min elongation step at 72 o
C.
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 o
C 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 o
C, 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.
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).

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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 o
C, 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 o
C 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.
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)

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

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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

30. 30 | P a g e
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

32. 32 | P a g e
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 o
C, whereas R. globerulus grows at a comparatively wide range of
temperatures (10-40 o
C). 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.

33. 33 | P a g e
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 o
C.
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

34. 34 | P a g e
(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.

35. 35 | P a g e
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.

36. 40 | P a g e
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.

37. 41 | P a g e
Appendix 3. Rhodococcus strains analyzed, with their 16S rRNA accession numbers
Organism Strain Accession number
Gordonia aichiensis DSM 43978 X80633
Gordonia bronchialis DSM 43247 X79287
Gordonia rubripertincta DSM 43248 AY995557
Gordonia terrae DSM 43249 X79286
Mycobacterium bovis ATCC 12910 X55589
Mycobacterium chlorophenolicus ATCC 43826 X79292
Mycobacterium tuberculosis ATCC 27294 X52917
Rhodococcus aetherivorans 10bc312 AF447391
Rhodococcus artemisae YIM 65754 GU367155
Rhodococcus aurantiacus ATCC 25938 AF283282
Rhodococcus baikonurensis GTC 1041 AB071951
Rhodococcus canchipurensis MBRL 353 JN164649
Rhodococcus cerastrii C5 FR714842
Rhodococcus cercidiphylli YIM 65003 EU325542
Rhodococcus chubuensis DSM 44019 X80627
Rhodococcus coprophilus DSM 43347 X80626
Rhodococcus corynebacterioides DSM 20151 AF430066
Rhodococcus equi DSM 20307 AF490539
Rhodococcus equi DSM 43199 X80613
Rhodococcus erythropolis DSM 43066 X79289
Rhodococcus erythropolis DSM 43188 X80618
Rhodococcus fascians DSM 20669 X79186
Rhodococcus globerulus DSM 43954 X80619
Rhodococcus gordoniae W4937 AY233201
Rhodococcus imtechensis RKJ 300 AY525785
Rhodococcus jialingiae djl-6-2 DQ185597
Rhodococcus jostii IFO 16295 AB046357
Rhodococcus koreensis DNP 505 AF124342
Rhodococcus kroppenstedtii K07-23 AY726605
Rhodococcus kunmingensis YIM 45607 DQ997045
Rhodococcus kyotonensis DS 472 AB269261
Rhodococcus luteus DSM 43673 X79187
Rhodococcus maanshanensis M712 AF416566
Rhodococcus marinonascens DSM 43752 X80617
Rhodococcus maris DSM 43672 FJ468333
Rhodococcus nanhaiensis SCS10 10187 JN582175
Rhodococcus opacus DSM 43205 X80630
Rhodococcus percolatus DSM 44240 X92114
Rhodococcus phenolicus DSM 44812 AY533293
Rhodococcus pyridinivorans PDB9 AF173005
Rhodococcus qingshengii djl-6 DQ185597
Rhodococcus rhodnii DSM 43336 X80621
Rhodococcus rhodochrous DSM 43241 X79288
Rhodococcus rhodochrous DSM 43274 X80624
Rhodococcus roseus DSM 43274 X80624
Rhodococcus ruber DSM 43338 X80626
Rhodococcus triatomae IMMIB RIV-095 AJ854055
Rhodococcus trifolii T8 FR714843
Rhodococcus tukisamuensis MB8 AB067734
Rhodococcus wratislaviensis CCM 4930 AJ786666
Rhodococcus yunnanensis YIM 70056 AY602219
Rhodococcus zopfii DSM 44108 AF191343

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Appendix 4. Arthrobacter strains analyzed, with their 16S rRNA accession numbers
Organism Strain Accession number
Arthrobacter agilis DSM 20550 X80748
Arthrobacter albidus LC-13 AB248533
Arthrobacter albus DSM 13068 AJ243421
Arthrobacter alkaliphilus LC-6 AB248527
Arthrobacter alpinus S6-3 GQ227413
Arthrobacter arilailensis DSM 16368 AJ609624
Arthrobacter aurescens DSM 20116 X83405
Arthrobacter bergerei DSM 16367 AJ609630
Arthrobacter castelli LMG 22283 AJ639826
Arthrobacter chlorophenolicus DSM 12829 AF102267
Arthrobacter citreus DSM 20133 X80737
Arthrobacter creatinolyticus GIFU 12498 D88211
Arthrobacter crystallopoietes DSM 20117 X80738
Arthrobacter cumminsii DMMZ 445 X93364
Arthrobacter equi IMMIB L-1606 FN673551
Arthrobacter gandavensis LMG 21287 AJ491108
Arthrobacter globiformis DSM 20124 X80736
Arthrobacter histidinolovorans DSM 20115 X83406
Arthrobacter humicola KV-653 AB279890
Arthrobacter ilicis DSM 20138 X83407
Arthrobacter koreensis CA15-8 AY116496
Arthrobacter luteolus DSM 13067 AJ243422
Arthrobacter methylotrophus DSM 14008 AF235090
Arthrobacter monumenti LMG 19502 AJ315070
Arthrobacter mysorens DSM 12798 AJ617482
Arthrobacter nasiphocae CCUG 42935 AJ292364
Arthrobacter nicotianae DSM 20123 X80739
Arthrobacter nicotinovorans DSM 420 X80743
Arthrobacter nitroguajacolicus CCM 4924 AJ512504
Arthrobacter oryzae KV-651 AB279889
Arthrobacter oxydans DSM 20119 X83408
Arthrobacter parietis LMG 22281 AJ639830
Arthrobacter pascens DSM 20545 X80740
Arthrobacter polychromogenes DSM 20136 X80741
Arthrobacter protophormiae DSM 20168 X80745
Arthrobacter psychrolactophilus ATCC 700733 AF134179
Arthrobacter ramosus DSM 20546 X80742
Arthrobacter rhombi CCUG 38813 Y15884
Arthrobacter roseus DSM 14508 AJ278870
Arthrobacter russicus A1-3 AB071950
Arthrobacter sanguinis 741 EU086805
Arthrobacter sulfonivorans DSM 14002 AF235091
Arthrobacter sulfurous DSM 20167 X83409
Arthrobacter uratoxydans DSM 20647 X83410
Arthrobacter ureafaciens DSM 20126 X80744
Arthrobacter woluwensis CUL 1808 X93353
Mycobacterium bovis ATCC 12910 X55589
Mycobacterium tuberculosis ATCC 27294 X52917