Journal of Environmental Biotechnology
Vol. 9, No. 1, 25–29, 2009

Original paper (short paper)

Development of Unmarked Gene Modification System in Organic
Solvent-Tolerant Rhodococcus opacus Strain B4
AKIHISA KITA, NOBORU TAKIGUCHI and JUNICHI KATO*
Department of Molecular Biotechnology, Hiroshima University, Higashi-Hiroshima, Hiroshima 739–8530, Japan* TEL: +81–82–424–7757 FAX: +81–82–424–7047* E-mail: jun@hiroshima-u.ac.jp(Received; 29 October, 2008/Accepted; 29 December, 2008)
Unmarked gene modification system in organic solvent tolerant Rhodococcus opacus strain B4 has been developed. Upstream and downstream regions of a target site for deletion are tandemly cloned into pG19II, a suicide plasmid which was
originally constructed for gene disruption in Pseudomonas aeruginosa and contains gentamycin resistant gene as a selective
marker and sacB as a counter-selective marker. Unmarked deletion mutant of strain B4 can be obtained after two homologous recombination events between the resulting plasmid and the B4 genome. This unmarked gene modification system can beused for unmarked insertion mutation in R. opacus strain B4.
Key words: unmarked gene modification, solvent-tolerant bacterium, Rhodococcus opacus

Rhodococcus opacus strain B4, isolated from a soil sample,
tolerates various organic solvents including benzene, toluene, xylene, ethylebenzene, n-octane, and n-decane1). This
strain has a high hydrophobicity and exhibits a high aﬃnity
for hydrocarbons. Strain B4 not only survived for at least
5 days in organic solvents, but also exhibited toluene dioxygenase activity in anhydrous organic solvents2). This solventtolerant and hydrophobic strain is a potential host for the
production of chemical products from hydrophobic substrates. To construct biocatalytic cells for bioproduction, theestablishment of a genetic transformation system for strain
B4 is necessary. In the previous study, we constructed E. coliRhodococcus shuttle plasmid vectors using a 4.4-kb endogenous plasmid in strain B4 and established a genetic transformation system by electroporation3). In the industrial
bioproduction process, the genome modification (deletionof genomic genes and insertion of exogenous genes into the
genome) are favored over the genetic modification using
plasmid vectors due to its genetic stability. Especially, unmarked genomic modification is a technology desirable forthe construction of biocatalytic cells because unlimited
times of genomic modifications are possible theoretically.
Although van der Geize et al. reported application of unmarked gene modification technique to deletion of 3-ketosteroid Δ1-dehydrogenase gene (kstD) in Rhodococcus
erythropolis SQ17), there was quite limited reports about
the construction of unmarked gene deletion mutants in thegenus Rhodococcus. In this report, we describe development of an unmarked gene modification system in solventtolerant R. opacus strain B4.
R. opacus strains were grown in tryptic soy broth (TSB)

medium (Difco) at 28°C with shaking. TSB agar was prepared by adding 20 g/l of agar to TSB medium. Whenevernecessary, TSB medium and TSB agar were supplemented
with 5 mg/l of gentamycin or 25 mg/l of chloramphenicol.
Plasmid pG19II4) 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 Escherichia
coli replication origin, and an origin of transfer. Plasmids
were introduced into R. opacus strains by electroporation
as described by Na and colleagues3). Polymerase chain reactions (PCR) were carried out using KOD DNA polymerase(Toyobo, Tokyo, Japan) according to the manufacturer’s instructions.
HS buﬀer (252 mM sucrose, 7 mM HEPES [pH 7.0])
was used for cell suspension buﬀer in the previous study3).
In this study, buﬀer containing no sucrose was examined forcell suspension buﬀer of electroporation because pG19II
contains sucrose sensitive determinant (sacB). HG buﬀer(0.3 M glycerol, 7 mM HEPES [pH 7.0]) was used for cellsuspension buﬀer instead of HS buﬀer, and strain B4 wastransformed with E. coli-Rhodococcus shuttle vector
pKNR01.13). Since the transformation eﬃciency (approximately 0.7×104 transformants per μg of plasmid DNA) was
comparable to that with HS buﬀer, we used HG buﬀer ascell suspension buﬀer for electroporation.
Since pG19II contains only the E. coli replication origin
but not the Rhodococcus replication origin, it should notKITA et al.

26

be replicated in R. opacus B4. As expected, no Gmr transformants were obtained when strain B4 was transformed
with pG19II. pG19II derivatives harboring genomic fragments of strain B4 are expected to be integrated into the B4
genome by homologous recombination and it generates
Gmr transformants of strain B4. Actually, Gmr transformants were obtained when strain B4 was transformed with
pG19 derivative harboring a 1.5-kb upstream region of ben-zene dioxygenase (bnz) operon (accession number
AB193045) of strain B4 (Fig. 1). We then investigated eﬀects
of lengths of B4 genomic DNA fragments cloned in pG19II
derivatives on transformation eﬃciency of strain B4. PCR
was conducted to amplify 0.2-, 0.5-, 1.0-, 1.5-, and 2.0-kb
upstream regions of bnz operon and the PCR products werecloned into the SmaI site of pG19II to construct pG19-0.2
to pG19-2.0 (Fig. 1). Strain B4 was transformed with theTable 1. Oligonucleotides used for PCR
PrimerSequence (5’ to 3’)bnzA-200-F
bnzA-500-F
bnzA-1000-F
bnzA-1500-F
bnzA-2000-F
bnzA-H-R1
bnzA1500-R
bnzS0-2F
bnzS0-2RbnzA-kp-F12
bnzA-kp-R12
bnzA-kp-F2
bnzA-kp-R2
pk-F
pk-R
bnzA-RT-F
bnzA-RT-R
gyrB-RT-F
gyrB-RT-R

TGGAAGCTTAGCCGTTGGTATCGAACCTGGAAGCTTCCATCGCTGGAAGTACCTGGAAGCTTCGTAGTCGTGGTCAAGTCTGGAAGCTTAGTGACAACTCGACCAGGTGGAAGCTTATCCGACCCAACACCTG
ACGTTTAAACAACCAATATGGTCGTGATCGGTGGTTCGGATTGGCATGAGAAGACTGCAGAACAACAGCGAACGACACCAGG
AGAAGAAGCTTTCGCGTTTCCTCCATCGCTATCACGAAGCTTGGTCATCAATCCGATCATTGC
ACTGGTACCATGCAGGTGATGGCGTG
ACGTCTAGACACTAACGACCGTACAAGTG
ACGTTTAAAGTTCTGTTCAGGTGGAGAC
ACTGGTACCTGTGGAATTGTGAGCGGATAAC
ACGTCTAGAGTATTACTGTTTATGTAAGCAGACAGTGTACGAACAGGAACTGGAG
ATCCTCGCCCATGTAGTTCGTCTACAGCTTCGAGACGTGACGACCTCTTCGGTG

Fig. 1. Physical map of the bnz operon region of R. opacus strain B4 and pG19II derivatives used in this study.
Upstream regions of bnz operon were amplified from the R. opacus strain B4 genome by PCR. PCR primer sets bnzA-200-F/bnzA-H-R1,
bnzA-500-F/bnzA-H-R1, bnzA-1000-F/bnzA-H-R1, bnzA-1500-F/bnzA-H-R1, and bnzA-2000-F/bnzA-H-R1 (Table 1) were used toamplify 0.2-, 0.5-, 1.0-, 1.5-, and 2.0-kb regions upstream of bnz operon, respectively. The PCR products were cloned into the SmaI siteof pG19II to construct pG19-0.2 to pG19-2.0. A region downstream of bnz operon was amplified with a primer set bnzS0-2F/bnzS0-2R.
The 1.5-kb product was cloned into pG19-1.5 after digestion with PstI and HindIII to construct pG19-dbnz. Upstream and downstream
regions of the bnz operon promoter and the Kmr gene promoter from pUC4K were amplified with PCR primer sets bnzA-kp-F12/bnzAkp-R12, bnzA-kp-F2/bnzA-kp-R2, and pk-F/pk-R, respectively. The amplified Kmr promoter region was digested with KpnI and XbaI,
while the amplified upstream and downstream regions were digested with HindIII and KpnI, and XbaI and DraI, respectively. These
digested PCR products were ligated with the backbone of HindIII-SmaI-digested pG19II to obtain pG19-Pkm-bnz. The locations of PCRprimers used for confirmation of unmarked gene modifications are indicated by thin arrows.

Unmarked Gene Modification in Rhodococcus opacus

resulting plasmids and spread on TSB agar plates with gentamycin. Transformation efficiency was determined bycounting Gmr colonies. Although pG19II derivatives (pG190.5 to pG19-2.0) contained diﬀerent lengths of genomic
fragments of strain B4, transformation eﬃciency was almost
same (15–20 transformants per μg of plasmid DNA). When
pG19-0.2 was used for transformation, transformants wereobtained but its transformation eﬃciency (approximately 5transformants per μg of plasmid DNA) was somewhat lower
than that by pG19-0.5 to pG19-2.0. Therefore, we used
1.5–2-kb B4 genomic DNA fragments to construct pG19II
derivatives for unmarked gene modification.
We examined concentrations of sucrose for counter-selection by sacB in strain B4. Gmr transformant cells obtainedby transforming strain B4 with pG19-1.5 were streaked on
TSB agar plates with gentamycin and 0–20% sucrose andthe plates were incubated at 28°C for 2 days. The transformant showed normal growth on TSB agar containing up to
10% sucrose, while 20% sucrose severely inhibited colony
formation of the transformant (data not shown). Growth of
the wild-type B4 was not aﬀected by 20% sucrose. Therefore, we used 20% sucrose for counter-selection by sacB instrain B4.
The scheme of unmarked gene deletion is shown in Fig. 2.
DNA regions upstream and downstream of the target site
for deletion are cloned in tandem into pG19II to construct
the suicide plasmid for unmarked gene deletion. Single
crossover recombination between homologous regions ofgenomic DNA and the plasmid results in integration of theplasmid into the genome. Phenotype of cells containing theplasmid in their genome should be Gmr and sucrose sensitive
and they can be selectively obtained as Gmr cells. The second
single crossover recombination generates two types of cells.

27

When the recombination occurs between regions “a” (Fig. 2),
cells return to the wild type cells. The recombination between regions “b” generates unmarked deletion mutant cells.
After the second single crossover recombination, phenotype
of cells is Gms and sucrose resistant and they can be selectedby growth in the presence of 20% sucrose.
To delete the whole bnz operon, we constructed pG19dbnz by cloning upstream and downstream regions of bnz
operon into pG19II (Fig. 1). Strain B4 was transformed
with pG19-dbnz and Gmr and sucrose-sensitive transformant cells were obtained. Transformation eﬃciency was 20transformants per μg of plasmid DNA. The transformant
cells were cultivated in TSB medium with shaking and timecourse of occurrence of sucrose resistant cells was examined.
Six hour incubation in TSB medium was enough to obtain
sucrose resistant cells (data not shown). One hundred sucrose
resistant colonies were randomly selected to examine foroccurrence of the second single crossover recombination.
All of them were Gms. About half of colonies were able togrow on toluene as a sole source of carbon, while the remaining colonies failed to grow on toluene. PCR analysis
using PCR primers specific to upstream and downstreamsites of bnz operon confirmed that colonies able to grow ontoluene contained the wild type bnz operon and that bnz
operon was deleted in colonies which could not grow ontoluene (Fig. 3). These results clearly demonstrate that unmarked gene deletion is possible in R. opacus strain B4.
Unmarked gene modification depicted in Fig. 2 may be
used not only for unmarked deletion mutation but also for
unmarked insertion mutation. pG19II derivatives used forunmarked insertion mutation should contain an inserted
fragment consisting of upstream region-a fragment to be inserted-downstream region. To confirm that pG19II can be

Fig. 2. Scheme of unmarked deletion mutation.
See the text for details.

28

KITA et al.

Fig. 3. PCR analysis of unmarked deletion and insertion mutantsof R. opacus strain B4.
(A) Unmarked deletion of genomic bnz operon was performed
for strain B4 using pG19-dbnz. Resulting Gms and sucrose resistant colonies after the second recombination were subjected
to PCR analysis with primers specific to sequences upstream
and downstream of bnz operon (bnzA-2000-F and bnzS0-2R[Fig. 1]). M, λ/HindIII size marker; lanes 1–5, Gms sucrose
resistant colonies; C, parental strain. Colonies No. 1, 2, 4, andthe wild type B4 could grow on toluene, while colonies 3 and5 failed to grow on toluene. A PCR product 6.6 kb shorter
than that from the wild type B4 was obtained from the colonies which could not grow on toluene. (B) Unmarked insertionof the Kmr gene promoter into the upstream region of bnz
operon was performed for strain B4 with pG19-Pkm-bnz. Resulting Gms and sucrose resistant colonies after the second recombination were subjected to PCR analysis with primers specific to sequences of Kmr promoter and bnzA2 (pk-F andbnzA1500-R [Fig. 1]). This PCR should produce 1.8-kb amplified product from the insertion mutant strain, but not fromthe wild type strain. Colonies No. 2 and 5 were expected to beinsertion mutant strains.

used for unmarked insertion mutation, we tried to exchange
the bnz promoter with the promoter of kanamycin resistant(Kmr) gene from pUC4K5). The bnz promoter is inducible
and toluene, benzene, and ethylbenzene are inducers3), while
the Kmr promoter shows constitutive expression. Therefore,
unmarked insertion mutant strain harboring the Kmr genepromoter-bnz oepron is expected to constitutively express
bnz operon. To obtain the unmarked insertion mutant, weconstructed pG19-Pkm-bnz by cloning the upstream regionof the bnz promoter region, the promoter region of Kmr
gene from pUC4K, and the downstream region of the bnz
promoter region into pG19II as shown in Fig.1. We performed the procedures shown in Fig. 2 with pG19-Pkm-bnz
and obtained Gms and sucrose resistant colonies where thesecond recombination was supposed to have occurred.
Genomic DNA was extracted from five Gms and sucrose-resistant colonies and subjected to PCR analysis. PCR analysis
revealed that two of the five colonies harbored insertion of
the Kmr gene promoter in the region upstream of bnz operon(Fig. 3). To further confirm chromosomal insertion of Kmr
gene promoter, we measured bnzA1 transcription in theinsertion mutant and the wild type strains by quantitative
reverse transcription PCR (Fig. 4). As described in our previous study, transcription of bnzA1 was induced by toluenein the wild type strain, whereas the bnzA gene was constitutively transcribed in the Kmr gene promoter insertion mutant.

Fig. 4. Transcription of the bnzA1 gene in the unmarked insertion
mutant and parental strains of R. opacus B4.
The Kmr promoter insertion mutant and parental strains were
cultivated in TSB with and without toluene in the vapor phase,and total RNA was extracted from cells by an RNeasy kit(QIAGEN, Inc., Valencia, CA). A One Step SYBR PrimeScript RT-PCR kit (Takara Bio, Inc., Shiga, Japan) was used
for cDNA generation and quantitative RT-PCR (qRT-PCR).
Quantitative RT-PCR was carried out using a LightCycler system (Roche Diagnostics). For qRT-PCR analysis of the bnzA1
mRNA, a primer set bnzA-RT-F/bnzA-RT-R was used. The
gyrB mRNA was also measured as the standard using primer
set gyrB-RT-F/gyrB-RT-R and used for normalization of bnzA1
transcription.

These results confirmed that pG19II can be used for unmarked
insertion mutation in strain B4.
In summary, we have developed unmarked gene modification system for R. opacus strain B4 using the Pseudomonas
suicide vector pG19II. It was demonstrated that this system
can be used for introduction of both unmarked deletion
and insertion mutation in the R. opacus strain B4 genome.
This technology developed in this paper will enable not only
future cell engineering of the strain, but also protein tagging
for easy purification of any given protein and signal sequence
fusion to secrete any give protein in R. opacus strain B4.

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 Ministryof Economy, Trade, and Industry (METI), which was entrusted
by New Energy and Industrial Technology Development
Organization (NEDO).

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