Journal of Environmental Biotechnology
Vol. 9, No. 1, 43–50, 2009

Original paper (regular paper)

Cloning and Characterization of sigA and sigB Genes from Rhodococcus
opacus Strain B4: Involvement of sigB in Organic Solvent Tolerance
AKIHISA KITA, NOBORU TAKIGUCHI and JUNICHI KATO*
Department of Molecular Biotechnology, Graduate School of Advanced Sciences of Matter, Hiroshima University,
Higashi-Hiroshima, Hiroshima 739–8530, Japan* TEL: +81–(0)82–424–7757 FAX: +81–(0)82–424–7047* E-mail: jun@hiroshima-u.ac.jp(Received; 22 December, 2008/Accepted; 17 January, 2009)
We cloned and sequenced the sigA and sigB genes from organic solvent-tolerant Rhodococcus opacus strain B4. The
predicted SigA and SigB proteins showed high similarity with the mycobacterial principal sigma factor, σA proteins, and the
mycobacterial alternative sigma factor, σB proteins, respectively. The sigA gene was transcribed almost constitutively during
growth and after environmental stress, while transcription of the sigB gene was induced during transition from exponential
growth phase to stationary phase and after environmental stress including heat shock and ethanol stress. The sigB mutant of
R. opacus strain B4 showed decreased growth at higher temperature and in the presence of ethanol. In addition, the sigB
mutant increased susceptibility to benzene, toluene, and n-hexane, although it was as tolerant to other organic solvents as
the parental strain. Our results indicate that SigB is involved in cell tolerance to specific organic solvents in R. opacus strain
B4. It was found that SigB is required for expression of benzene dioxygenase operon. However, genetic analysis revealed thatorganic solvent degradation is not involved in organic solvent-tolerance even under the sigB deficient background.
Key words: solvent tolerance, Rhodococcus opacus, sigA, sigB, RpoD family

1. Introduction
It was 1989 when Inoue and Horikoshi first reported thepresence of a bacterium which can thrive in high concentrations of toluene13). Since then, several organic solvent-tolerant
bacteria have been isolated35). They include Pseudomonas
putida strains, Bacillus spp., Flavobacterium strain DS711, Arthrobacter ST-1, and Rhodococcus sp. strain 33(reference 4, and see references cited in reference 35). We
isolated Rhodococcus opacus strain B4 from gasolinecontaminated soil as a benzene-tolerant bacterium24). This
strain not only tolerates a wide variety of aliphatic andaromatic hydrocarbons but also utilizes them as a solesource of carbon and energy. It was demonstrated that thisstrain is able to survive and exhibit biocatalytic activity inanhydrous organic solvents for at least 5 days33,39). Therefore, R. opacus strain B4 is expected to be a good candidate of a host strain for bioproduction of value addedhydrophobic chemicals.
The mechanisms underlying bacterial tolerance to organic
solvents have been investigated intensively with Gram-negative bacteria P. putida and Escherichia coli35). They include
modifications in cell envelope to increase cell membrane
rigidity and decrease permeability9,10,15), solvent-inactivating
enzymes 5), active efflux of solvents by solvent efflux
pumps16,19,31), and release of membrane vehicles with solvent

molecules adhering to cells1). However, little is known about
solvent-tolerant mechanisms in Gram-positive bacteria. Although Iwabuchi et al. reported involvement of extracellularpolysaccharide in organic solvent-tolerance in Rhodococcus
rhodochrous14), it is unclear even whether Gram-positivebacteria possess solvent-tolerant mechanisms similar to
those of Gram-negative bacteria. Sardessai and Bhosle paid
attention to sigma B in Bacillus subtilis as a factor involved in solvent tolerance because this secondary sigma
factor is induced by environmental stress including ethanol
shock and involved in bacterial survival after exposure toethanol35). They also pointed out that SigB had been shown
to regulate expression of bmrUR operon encoding proteinsthat may contribute to resistance to multidrug compounds28).
However, this speculation has not yet been verified experimentally.
Rhodococcus, together with Mycobacterium, Corynebacterium, Nocardia, and Gordonia, belongs to the mycolata, a broad and diverse group of mycolic-acid-containing
actinomycetes. In Mycobacterium tuberculosis, Corynebacterium glutamicum, and Brevibacterium flavum, two70
AB
σ family proteins, σ and σ , have been identified and2,12,26). The maycobacterial σA protein is a princharacterized
cipal and essential sigma factor, while mycobacterial σB isan alternative sigma factor and plays roles similar to thoseof B. subtilis sigma B. Transcription of the σB gene is in-KITA et al.

44

duced upon entry into the stationary phase and by environmental stress7,11), and involved in cell responses to environmental stress including ethanol stress8). In this paper, wecloned and characterized genes encoding σA and σB from R.
opacus strain B4. We also constructed the sigB mutant of R.
opacus strain B4 and examined it for its responses to organic
solvents to investigate the role of SigB in solvent tolerance
in R. opacus strain B4.

2. Materials and Methods
2.1. Bacterial strains and growth conditions
Rhodococcus opacus strain B424) and its derivatives wereused in this study. R. opacus strains were grown at 28°C
with shaking in MSB medium24) with 0.5% glucose andtryptic soy broth (TSB; Difco Laboratory, Detroit, MI,
USA). For solid media, 2% agar was added. Whenevernecessary, media were supplemented with 5 mg/l of gentamycin, 25 mg/l of chloramphenicol, and 20% sucrose. E.
coli MV118438), which was used for plasmid construction
and DNA manipulation, was grown at 37°C with shaking in
2x YT medium32) supplemented with appropriate antibiotics.
2.2. DNA manipulation and sequencing
Standard procedures were used for plasmid DNA preparations, restriction enzyme digestions, ligations, transformations, agarose gel electrophoresis, and Southern hybridization32). Polymerase chain reactions (PCRs) were carried out

using KOD plus DNA polymerase (Toyobo, Tokyo, Japan)
according to the manufacturer’s instructions. Oligonucleotides used for PCR are listed in Table 1. Before cloning,
PCR products were electrophoresed and purified from the
1% agarose gel using the GenecleanII kit (Bio101, Carlsbad, CA, USA). R. opacus was transformed by electroporation as described previously25). The nucleotide sequence wasdetermined by the dideoxynucleotide sequencing method34).
Nucleotide sequence similarities were determined using
BLAST (National Center for Biotechnology Information
databases).
2.3. Cloning and sequencing of the sigA and sigB genes
from R. opacus strain B4
The genome sequence of Rhodococcus sp. strain RHA1,
which is closely related to R. opacus strain B4, has been
completely sequenced21). We designed PCR primer sets
B4sigAf/B4sigAr and B4sigBf/B4sigBr (Table 1) basedon nucleotide sequences of the sigA and sigB genes from
Rhodococcus sp. strain RHA1 (accession number NC
008268, locus tags RHA1 ro06823 [sigA] and RHA1
ro06813 [sigB]). The sigA and sigB genes were amplified
from the R. opacus strain B4 genome by PCR using theseprimer sets and the resulting RCR products were cloned
into the SmaI site of pBluescript II KS+ (Stratagene, TX,
USA). Then, the R. opacus strain B4 genome was analyzed
by Southern hybridization with the cloned R. opacus strain
B4 sigA and sigB sequences as probes. The sigA probe

Table 1. Oligonucleotides used for PCR and qRT-PCR
PrimerPrimers for PCR
B4sigAf
B4sigAr
B4sigBf
B4sigBr
sigB1fsigB1rsigB2fsigB2rsigB3fsigB3rPrimers for qRT-PCR(For measurement of sigA mRNA)
sigA-RT-F
sigA-RT-R(For measurement of sigB mRNA)
sigB-RT-F
sigB-RT-R(For measurement of bnzA1 mRNA)
bnzA1-RT-F
bnzA1-RT-R(For measurement of gyrB mRNA)
gyrB-RT-F
gyrB-RT-R

Sequence (5’ to 3’)
GTGGCAGCCACCGATACACGTCAGA
GCAGGTGCTGCGCGACTACCTGGACATGACAAGCCCCAGCACCACTACTCCGAGCGGCTGCGCTCCTACGCCAGC
GGAAGCTTACCGGTGGGTGCTCTCCGACCTCGAGGAGATCTGACTTGCCTCCTCATCGGTGGTGTCGGAGATCTTCTCTCCCTCCGCATCAGCGCCGGT
GGTCTAGATTCGACTGTGCGCTGCTGTTCGGGT
AACTGATCGAACTTCTCGCTGCCGC
GCCGAGGGTGCGCTGGTCACCGATTGACATGGACGACATCGAC
CTTGTCCTTCTCGGACGCACCACTACTCGCATTCGACAGTTCCACCTCGTCTGTGTACGAACAGGAACTGGAG
ATCCTCGCCCATGTAGTTCGTCTACAGCTTCGAGACGTGACGACCTCTTCGGTG

sigA AND sigB FROM R. OPACUS STRAIN B4

detected 1.1-kb and 0.6-kb BamHI genomic fragments,
while the sigB probe detected a 4.7-kb genomic fragment.
These BamHI and NotI fragments were cloned into pBluescript II KS+. The PCR products and cloned BamHI and
NotI fragments were sequenced to determine nucleotide
sequences of the sigA and sigB genes in R. opacus strain B4.
2.4. Plasmid construction
PCR using primer sets sigB1f/sigB1r and sigB2f/sigB2r(Table 1) was conducted to amplify 1.3-kb regions upstream
and downstream of sigB from the R. opacus strain B4
genome, respectively. Amplified upstream and downstream
regions were digested with HindIII-BglII and BglII-XbaI,
respectively, and ligated with the backbone of HindIIIXbaI-digested pG19II22) to obtain pG19-dsigB. Plasmid
pG19-Pkm-bnz17) was used to exchange a promoter regionof the bnz operon in R. opacus strain B4 with a promoterof kanamycin resistant (Kmr) gene from pUC4K37). The
multi-cloning site from pBluescript II KS+ (Stratagene,
Cedar Creek, TX, USA) was introduced into RhodococcusE. coli shuttle vector pKNR01.125) to obtain pKNR10.1.
The sigB gene was amplified from R. opacus strain B4
genome by PCR using a primer set sigB3f/sigB3r and the
PCR product was cloned into pKNR10.1 to construct
pKNR-sigB.
2.5. Unmarked genomic gene modification
Plasmid pG19II22) was used for the unmarked gene modification in R. opacus strain B4. pG19II was originally constructed for the insertion-deletion mutation of chromosomal
genes in Pseudomonas aeruginosa. This plasmid contains
gentamycin resistant (Gmr) gene as a selectable marker,
sacB (conferring sucrose sensitive phenotype) as a counterselectable marker, the multi-cloning sites, an E. coli replication origin, and an origin of transfer. To delete a genomic
gene, a derivative of pG19II was constructed by cloning
upstream and downstream regions of the target gene into
pG19II. Strain B4 was transformed with the pG19II derivative and Gmr transformant cells were obtained. The Gmr
transformants were grown in TSB without gentamycin toallow the second homologous chromosomal recombination.
After cultivation for 6 h, culture broth was spread on TSB
agar plates containing 20% sucrose and the plates were incubated at 28°C to select sucrose resistant colonies. Theoretically, half of the sucrose resistant colonies were unmarked deletion mutants and the other half has a parental
genotype. Unmarked gene modification in derivatives from
R. opacus strain B4 was confirmed by PCR analysis.
2.6. Organic solvent-sensitive growth assays
R. opacus strains were cultivated with shaking in TSB.
Overnight cultures were diluted with TSB and adjusted to
optical density at 600 nm (OD600)=0.1. To assay sensitivity
to organic solvents, 5 μl of 10-fold serial dilutions of thediluted cultures were spotted onto TSB agar plates. The
plates were incubated at 28°C in a desiccator with a beaker45

containing each of liquid organic solvents. Colony growth
was inspected after 72 h of incubation.
2.7. Quantitative reverse transcription PCR (qRT-PCR)
R. opacus strains were cultivated in TSB in the absence
and presence of toluene in the vapor phase. Cells were harvested by centrifugation at 4°C for 10 min. Total RNA wasextracted from R. opacus cells using an RNeasy kit (QIAGEN, Inc., Valencia, CA) with a RNAprotect bacteria reagent(QIAGEN) for the stabilization of RNA. RNA samples
were treated with an RNase-free DNase set (QIAGEN) toeliminate contaminating DNA. Complete removal of DNA
was confirmed by PCR. A One Step SYBR PrimeScript RTPCR kit (Takara Bio, Inc., Shiga, Japan) was used for
cDNA generation and qRT-PCR. qRT-PCR was carried out
using a LightCycler system (Roche Diagnostics) with anreverse transcription at 42°C for 15 s followed by cycles ofdenaturation at 95°C for 5 s, primer annealing at 60°C for
10 s, and extension at 72°C for 6 s. Primer sets used in
qRT-PCR were listed in Table 1. The gyrB mRNA was alsomeasured as the standard using primer set gyrB-RT-F/gyrBRT-R and used for normalization.
2.8. Nucleotide sequence accession numbers
The DDBJ accession numbers of the sigA and sigB genesof B-4 are AB477528, and AB477529, respectively.

3. Results and Discussion
3.1. Cloning and sequence analysis of sigA and sigBfrom R. opacus strain B4
The sigA and sigB genes from R. opacus strain B4 werecloned and sequenced as described in “Materials andMethods” section. Sequence analysis revealed that the sigA
gene of 1371 bp and the sigB gene of 969 bp could codefor proteins of 457 and 323 amino acid residues having
calculated molecular weights of 50.0 and 36.0 kDa, respectively. Blastp analysis indicated that R. opacus strain B4
SigA shows a high similarity to mycobacterial principal
sigma factors (σ70 family group 1), σA proteins. In the σ70
family proteins, regions 1.2, 2, 3, and 4 are conserved20) ,and in these regions, R. opacus strain B4 SigA shares 88–
97% identical amino acid residues with Mycobacterium
tuberculosis H37Rv MysA (NP 212719), Mycobacterium
smegmatis strain MC2 155 SigA (YP 887090), and Corynebacterium glutamicum ATCC 13032 SigA (NP 601117).
Especially in the highly conserved regions (RpoD box inregion 2.3, 14-mer sequence in region 2.4, and 20-mer
sequence in region 4.220)), R. opacus strain B4 SigA showsa perfect match with M. tuberculosis H37Rv MysA. In
contrast, there is much lower similarity among these sigma
factors in region 1.1 which is poorly conserved in the principal sigma factors although R. opacus strain B4 SigA isalmost identical to Rhodococcus sp. strain RHA1 SigA inthis region. The conserved regions (regions 1.2, 2, 3, and 4)
were also found in R. opacus strain B4 SigB. However, this46

KITA et al.

protein lacks region1.1. Moreover, R. opacus strain B4
SigB has higher similarity (79–88% identity) to mycobacterial σ B proteins (M. tuberculosis H37Rv MysB [NP217226], C. glutamicum ATCC 13032 SigB [NP 601125],
and M. smegmatis strain MC2 155 SigB [YP 887083]) thanto σA proteins. These results suggest that R. opacus strain
B4 SigB belongs to the mycobacterial σB.
3.2. Transcriptional analysis of sigA and sigB
Mycobacterial σA proteins are principal and essential
sigma factors and responsible for most RNA synthesis inexponentially growing cells6,7,11,20,27). The σA genes are transcribed almost constitutively during growth and after stressconditions. Mycobacterial σB proteins are dispensable alternative sigma factors and thought to play roles similar to
those of E. coli RpoS8,11,18,23). Transcription of the σB genes
is induced during transition from exponential growth phaseto stationary phase and after several stress conditions suchas acid stress, ethanol shock, heat shock, and H2O2 exposure8,11,23,27). To confirm that R. opacus strain B4 SigA and
SigB belong to mycobacterial σA and σB, respectively, transcriptional analysis was conducted for sigA and sigB by
qRT-PCR.
Fig. 1 shows time course data of sigA and sigB transcription when R. opacus strain B4 was cultivated in TSB withshaking at 28°C. The level of transcription of sigA wasalmost constant during diﬀerent growth phase, suggestingthat sigA is constitutively transcribed. Transcription patternof sigB is similar to those of other mycobacterial σB genes
and the level of sigB transcription increased during thetransition from exponential to stationary growth phase. We
then investigated eﬀects of environmental stress on transcription of sigA and sigB. R. opacus strain B4 cells at thelate exponential growth phase were subjected to heat shock

Fig. 1. Time course of transcription levels of sigA and sigB during
growth in MSB medium with glucose.
R. opacus strain B4 was cultivated in MSB medium with glucose. Bacterial cells were sampled at 6, 12, 18, 21, 24, and28 hours of incubation and total RNA was isolated from cells.
Transcription levels of sigA and sigB were measured by qRTPCR. Normalized mRNA values were obtained by dividing themRNA values by that of the gyrB mRNA value for the same
template. Open circles, bacterial growth; open bars, normalized sigA mRNA values; grey bars, normalized sigB mRNAvalues.

stress (33°C) and ethanol stress (3% ethanol) and transcription levels of sigA and sigB in cells were measured. As controls, transcription levels of sigA and sigB were measured incells which were not exposed to the environmental stress.
Heat shock and ethanol stress induced the transcription of
the sigB mRNA (Fig. 2). Compared with controls, 1.7-fold
and 4.8-fold induction of the sigB mRNA was observed
after exposure to heat shock and ethanol stress, respectively.
In contrast, transcription of sigA was not aﬀected by heatshock and ethanol stress. There was no significant diﬀerencein transcriptional level of the sigA gene between control
cells and stressed cells. Thus, it was shown that transcriptional responses of R. opacus strain B4 sigA and sigB to
growth rate and environmental stress are similar to those of
mycobacterial σA and σB genes, respectively.
3.3. Construction of unmarked sigB deletion mutant of
R. opacus strain B4
We further characterized R. opacus strain B4 SigB. To
investigate the role of SigB in R. opacus strain B4 cells, anunmarked sigB deletion mutant of R. opacus strain B4,
designated R. opacus strain dsigB, was constructed using
pG19-dsigB as described in “Materials and Methods” section.
Deletion mutation of the genomic sigB gene was confirmed
by PCR analysis (data not shown). Strain dsigB showednormal growth when it was cultivated under normal conditions (in MSB medium with glucose at 28°C with shaking)(Fig. 3A). To investigate the influence of sigB deletion on
responses to environmental stress, we examined strain dsigB
for its growth under diﬀerent environmental stress. Strain

Fig. 2. Effects of ethanol stress and heat shock on transcription of
sigA and sigB in R. opacus strain B4.
R. opacus strain B4 was grown in MSB medium with glucose.
When OD600 of cultures reached at 1.0, ethanol was added to
cultures at a concentration of 3% (ethanol stress) or cultivation temperature was shifted from 28°C to 33°C (heat shock).
After one hour of incubation under stressed conditions, total
RNA was isolated and transcription of sigA and sigB was
measured by qRT-PCR. Relative expression of sigA and sigB
was obtained by dividing normalized mRNA values after one
hour of incubation under stressed conditions by normalized
mRNA values just before exposure to stress. Values represent
the mean and standard deviation of at least three observations. Open bars, relative expression in unstressed cells; grey
bars, relative expression in stressed cells.

sigA AND sigB FROM R. OPACUS STRAIN B4

dsigB showed growth similar to that of the parental strain
under acidic conditions (pH=5.0 and 6.0) (Fig. 3B). In contrast, the sigB mutation aﬀected growth at higher temperature (33°C) and in the presence of ethanol. As shown in
Fig. 3C and D, strain dsigB showed significantly decreased
growth compared to the parental strain when they weregrown in MSB with glucose under higher temperature(33°C) or in the presence of 3–4% ethanol. Simultaneous
exposure of cells to higher temperature and ethanol stressseverely aﬀected growth of strain dsigB. Strain dsigB failed
to grow at 33°C in the presence of 3% ethanol, while theparental strain showed decreased but significant growth (Fig.
3E). These results together with transcriptional responses of
sigB to environmental stress indicate that SigB is involvedin the regulation of the gene expression in response to environmental stress.
3.4. The sigB gene is involved in tolerance to specificorganic solvents
In this study, it was demonstrated that SigB plays a rolein a response to ethanol stress in R. opacus strain B4.
Therefore, we then conducted organic solvent-sensitive
growth assays to assess involvement of sigB in tolerance toorganic solvents in R. opacus strain B4. Organic solvents
tested were aliphatic hydrocarbons (n-hexane, n-octane, andn-decane) and aromatic compounds (benzene, toluene, o-,m-, p-xylenes, ethylbenzene, o-, m-, p-cresols, dimeth-47

ylphthalate, and diethylphthalate). The sigB mutant showed
increased susceptibility to benzene, toluene, and n-hexane,
but not to other organic solvents (Fig. 4). Introduction ofpKNR-sigB containing the functional sigB gene complemented the mutant phenotype of strain dsigB, confirmingthat increased susceptibility of strain dsigB to benzene,
toluene, and n-hexane was due to the sigB mutation (Fig. 4).
It is the first demonstration that SigB is involved in bacterial
tolerance to hydrocarbons.
It was found that SigB is involved in tolerance to specificorganic solvents (i.e. benzene, toluene, and n-hexane) in R.
opacus strain B4. How can this specificity be explained? We
paid attention to inactivating (degrading) activities of organic
solvents for this explanation because both benzene andtoluene are metabolized via the common pathway, benzene
dioxygenase pathway24). In the previous study, we demonstrated that organic solvent-tolerance was not aﬀected byinactivation of benzene dioxygenase (bnz) operon in R.
opacus strain B424). However, we speculated that inactivation (degradation) of organic solvents are required fororganic solvent-tolerance in the sigB-deficient background,
where expression of some other functions required fororganic solvent-tolerance might be also defective, and that
SigB regulates expression of bnz operon. To assess this possibility, we measured transcription level of bnzA1 encoding
the benzene dioxygenase large subunit in cells grown on
TSB agar plates supplemented with toluene in the vapor

Fig. 3. Growth of the sigB mutant (strain dsigB) and parental strains of R. opacus strain B4 under various environmental stresses.
Each experiment was performed using triplicate samples. The values represent the mean and the standard deviation for each point. MSB
supplemented with glucose as a carbon source was used for growth medium. (A) Growth at 28°C and initial pH 7.0 (control), (B) growthat 28°C and initial pH 5.0 and 6.0 (low pH stress), (C) growth at 28°C and initial pH 7.0 in the presence of 3 and 4% ethanol (ethanol
stress), (D) growth at 33°C and initial pH 7.0 (heat stress), (E) growth at 33°C and initial pH 7.0 in the presence of 3% ethanol(ethanol+heat stresses). Closed symbols, the parental strain; open symbols, the sigB mutant strain.

48

KITA et al.

Fig. 4. Organic solvent-sensitive growth assays of R. opacus strain B4 and its derivatives.
Overnight cultures were diluted and adjusted to OD600=0.1. Five μl of 10-fold serial dilutions of the diluted cultures were spotted onto
TSB agar plates. The plates were incubated at 28°C in a desiccator with a beaker containing each of liquid organic solvents. Colony
growth was inspected after 72 h of incubation. B4, the parental strain; dsigB, the sigB mutant; dsigB (+sigB), strain dsigB harboringpKNR-sigB. Although data are not presented here, the sigB mutant showed growth similar to that of the parental strain when exposed toother organic solvents tested.

Fig. 5. Transcription levels of bnzA1 in R. opacus strain B4 andits derivatives.
The parental strain, the sigB mutant, and the sigB mutant
harboring pKNR-sigB were cultivated on TSB agar plates withand without toluene in the vapor phase. After cultivation of
2 days, cells were harvested. Total RNA was isolated fromharvested cells and subjected to qRT-PCR to measure bnzA1
mRNA. Opened bars, cells grown on TSB agar without toluenein the vapor phase; closed bars, cells grown on TSB agar withtoluene in the vapor phase.

phase. When grown on TSB plates, toluene induced transcription of the bnzA1 gene in the parental strain (Fig. 5).
However, transcription of bnzA1 was not induced in the
sigB mutant even in the presence of toluene. Introductionof pKNR-sigB restored inducible transcription of bnzA1 inthe sigB mutant. These results clearly demonstrate that
SigB is required for inducible transcription of bnz operon.

To assess whether susceptibility to toluene in strain dsigB
is caused by failure of the degradation pathway, i.e. bnz
operon, we inserted Kmr gene promoter form pUC4K in the
upstream region of bnz operon for forced expression of bnz
operon in strain dsigB. The resulting strain was designateddsigB(Pk-bnz). Although expression of bnz operon in
dsigB(Pk-bnz) was confirmed by qRT-PCR (data notshown), it did not restore the ability of sigB mutant totolerate toluene (Fig. 6). Rather, stain dsigB(Pk-bnz)
seemed more sensitive to toluene than strain dsigB. It may
be because a metabolite(s) of toluene exerts a toxic eﬀect
on strain dsigB(Pk-bnz) cells. Considering these results andour previous result that solvent-tolerance is not aﬀected bydeletion of bnz operon24), it is unlikely that susceptibility ofstrain dsigB is due to its failure of expression of degradation
pathways.
P. putida DOT-T1E30) is a Gram negative bacterium
highly tolerant to organic solvents, and the mechanisms ofsolvent tolerance have been most extensively studied in thisstrain. In P. putida DOT-T1E, three eﬄux pumps belonging
to the Resistance-Nodulation-Cell-Division (RND) family ofbacterial transporters (TtgABC, TtgEFG, and TtgGHI) areresponsible for its solvent tolerance as well as drug resistance29). It was found that these eﬄux pumps exhibit significant diﬀerences in substrate specificity3,29,36). Although there
has been no report demonstrating involvement of eﬄux
pumps in organic solvent tolerance of Gram positive bacteria,sequence analysis predicts the presence of many multidrug

sigA AND sigB FROM R. OPACUS STRAIN B449

Fig. 6. Toluene-sensitive growth assays of R. opacus strains B4, dsigB, and dsigB(Pk-bnz).
Susceptibility of bacterial cells to toluene was assayed as described in Fig. 4.

resistant transporters in their genomes. For example, Rhodococcus strain RHA1, which is closely related to R. opacus
strain B4, is predicted to possess more than 40 putative
major facilitator superfamily multidrug resistance protein
genes and more than 10 putative multidrug ABC transporter
genes in its genome (NC 008268). Based on these data, we
speculate that R. opacus strain B4 also possesses many
genes encoding eﬄux pump in its genome and that some ofthem are responsible for organic solvent tolerance. And we
hypothesize that one of eﬄux pump is specific for toluene,benzene, and n-hexane, which is required for tolerance tothese organic solvents, and that its expression is regulated
by SigB. We are now searching candidates for eﬄux pumpgenes involved in solvent-tolerance in R. opacus strain B4
by utilizing the genome sequence data of Rhodococcus sp.
strain RHA1.

Acknowledgements
This work was carried out as part of the Project for Development of Basic Technologies for Advanced Production
Methods Using Microorganism Functions on R&D of New
Industrial Science and Technology Frontier by the Ministry of
Economy, Trade, and Industry (METI), which was entrusted
by New Energy and Industrial Technology Development
Organization (NEDO).

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