Three of the Seven bphC Genes of Rhodococcus erythropolis TA421 ...

APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Aug. 1997, p. 3282–3285 0099-2240/97/$04.0010 Copyright © 1997, American Society for Microbiology

Vol. 63, No. 8

Three of the Seven bphC Genes of Rhodococcus erythropolis TA421, Isolated from a Termite Ecosystem, Are Located on an Indigenous Plasmid Associated with Biphenyl Degradation SAORI KOSONO,1 MICHIHISA MAEDA,2† FUMIE FUJI,2‡ HIROYUKI ARAI,1 1,2 AND TOSHIAKI KUDO * The Institute of Physical and Chemical Research (RIKEN)1 and Research Development Corporation of Japan (JRDC),2 Wako, Saitama, Japan Received 30 December 1996/Accepted 19 May 1997

Rhodococcus erythropolis TA421, a polychlorinated biphenyl and biphenyl degrader isolated from a termite ecosystem, has seven bphC genes expressing 2,3-dihydroxybiphenyl dioxygenase activity. R. erythropolis TA421 harbored a large and probably linear plasmid on which three (bphC2, bphC3, and bphC4) of the seven bphC genes were located. A non-biphenyl-degrading mutant, designated strain TA422, was obtained spontaneously from R. erythropolis TA421. TA422 lacked the plasmid, suggesting that the three bphC genes were involved in the degradation of biphenyl. Southern blot analyses showed that R. erythropolis TA421 and Rhodococcus globerulus P6 have a similar set of bphC genes and that the genes for biphenyl catabolism are located on plasmids of different sizes. These results indicated that the genes encoding the biphenyl catabolic pathway in Rhodococcus strains are borne on plasmids. isolation and characterization of four of these genes (bphC1 to bphC4) have been reported (15), and the other three genes have recently been isolated (14a). An enzyme assay with cell extract of Escherichia coli carrying each bphC-cloned plasmid indicated that all of the seven bphC gene products exhibited much higher substrate specificities for 2,3-DHBP than for catechol, 3-methylcatechol, or 4-methylcatechol (reference 15 and unpublished result), and thus these gene products are primarily involved in the ring cleavage of 2,3-DHBP. According to the classification of extradiol dioxygenases (9), all bphC-encoded proteins of R. erythropolis TA421, except for BphC2, belong to the two-domain extradiol dioxygenase family. The bphC2-encoded protein belongs to the single-domain extradiol dioxygenase family. Here, we report the isolation and characterization of a nonbiphenyl-degrading spontaneous mutant derived from R. erythropolis TA421 and show that three bphC genes, bphC2, bphC3, and bphC4, of the seven bphC genes are located on a plasmid associated with biphenyl degradation. We also show that R. globerulus P6 has a set of bphC genes similar to those of R. erythropolis TA421 and that the genes for biphenyl degradation are also located on plasmids in R. globerulus P6. Isolation of non-biphenyl-degrading R. erythropolis TA422. R. erythropolis TA421 expresses 2,3-DHBD activity on a LuriaBertani (LB) plate, and this activity can be visualized by spraying 2,3-DHBP due to the yellow color of the meta-cleavage product. From R. erythropolis TA421, we obtained spontaneous mutants which lacked 2,3-DHBD activity as follows. R. erythropolis TA421 was grown in liquid C medium (7) containing 0.5% biphenyl. Appropriate dilutions of the culture were spread onto LB plates. Then a 2,3-DHBP solution was sprayed. White colonies were obtained at a frequency of 2%. They had lost the ability to utilize biphenyl as a sole carbon source. Dot-blot hybridization analysis showed that all of the spontaneous mutants had lost the bphC2, bphC3, and bphC4 genes (data not shown). A single colony was selected from the spontaneous derivatives and designated strain TA422. The partial

Rhodococcus is a taxon of aerobic gram-positive bacteria belonging to the nocardioform actinomycetes (10, 18). This taxon is able to transform a wide range of xenobiotic compounds including polychlorinated biphenyls (PCBs) (7, 11, 19) and is considered to play a critical role in the biodegradation of toxic pollutants in soil (22). Rhodococcus erythropolis TA421 can use biphenyl as a sole carbon source and can cometabolize a wide spectrum of PCBs (7). R. erythropolis TA421 was isolated from the ecosystem of wood-feeding termites, suggesting that the termite ecosystem is one of the possible habitats for PCB- or biphenyl-degrading bacteria because of the abundant biphenyl moieties resulting from the breakdown of lignin (7, 15). The initial process for the oxidative degradation of PCBs or biphenyl in aerobic bacteria consists of four steps and gives rise to chlorobenzoate or benzoate and 2-hydroxypenta-2,4-dienoic acid via dihydrodiol compound, 2,3-dihydroxybiphenyl (2,3DHBP) and 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoic acid. Four enzymes, i.e., multicomponent biphenyl dioxygenase (BphA), dihydrodiol dehydrogenase (BphB), 2,3-dihydroxybiphenyl dioxygenase (2,3-DHBD) (BphC), and hydrolase (BphD), catalyze these steps (1). Usually, the genes for these enzymes are clustered and transcribed as an operon (2, 12, 16, 20). Rhodococcus globerulus P6 (3) (previously designated Acinetobacter sp. strain P6 [11] or Corynebacterium sp. strain MB1 [5]) was reported to have three bphC genes (4), two of which are not clustered with the other bph genes (4). R. erythropolis TA421 has seven bphC genes, designated genes bphC1 to bphC7. The

* Corresponding author. Mailing address: Laboratory of Microbiology, The Institute of Physical and Chemical Research (RIKEN), 2-1 Hirosawa, Wako, Saitama 351-01, Japan. Phone: 81-48-467-9544. Fax: 81-48-462-4672. E-mail: [email protected]. † Present address: School of Agriculture, Meiji University, Tama-ku, Kawasaki, Kanagawa 214, Japan. ‡ Present address: Japan Marine Science and Technology Center (JAMSTEC), Yokosuka, Kanagawa 237, Japan. 3282

RHODOCOCCUS bphC GENES ARE ON A PLASMID

VOL. 63, 1997 TABLE 1. Growth of R. erythropolis TA421 and TA422 on various aromatic compounds as sole carbon sources Growth of straina: Carbon source

Biphenyl Naphthalene Benzoate 2-Hydroxybenzoate 3-Hydroxybenzoate 4-Hydroxybenzoate Protocatechuic acid Pyrocatechol Phenol Benzene Toluene Xylene Toluic acid 4-Cymene 4-Cumic acid Ethylbenzene

TA421

TA422

1 2 1 2 2 1 1 1 1 2 2 2 2 2 2 2

2 2 1 2 2 1 1 1 1 2 2 2 2 2 2 2

a Overnight culture was inoculated at 1% and cultured for 7 days at 30°C. 1, growth; 2, no growth.

sequence of 16S ribosomal DNA from TA422 was completely identical with that of R. erythropolis TA421 (data not shown), indicating that TA422 was derived from R. erythropolis TA421. We determined the utilization of various aromatic compounds by R. erythropolis TA421 and TA422 (Table 1). An overnight culture was inoculated at 1% into C medium containing each carbon source at a final concentration of 1, 5, and 10 mM. The growth of the two strains was improved by increasing of the concentrations of benzoate, 4-hydroxybenzoate, protocatechuate, pyrocatechol, and phenol. In the utilization of all other compounds except biphenyl, there was no difference between R. erythropolis TA421 and TA422. The bphC2, bphC3, and bphC4 genes are located on a plasmid associated with the biphenyl degradation of R. erythropolis TA421. To determine the genetic differences between the seven bphC genes of R. erythropolis TA421 and TA422, we conducted a Southern blot analysis with total DNAs from the two strains. The total DNAs were digested with the same restriction enzymes as those used for probe preparation and then were separated by electrophoresis. The seven bphC genes of R. erythropolis TA421 were individually used as probes. Hybridization was performed at 42°C in the presence of 50% formamide–53 SSC (13 SSC is 0.15 M NaCl plus 0.015 M sodium citrate). The probes did not cross-hybridize under this condition. All of the bphC probes gave signal bands of the expected size (Fig. 1, lanes 1). No difference in the hybridization patterns for TA421 and TA422 was observed when bphC1, bphC5, bphC6, or bphC7 was used as the probe. However, bphC2, bphC3, and bphC4 did not hybridize with the total DNA of TA422 (Fig. 1, lanes 1 and 2), confirming the result of dot-blot hybridization. Non-biphenyl-degrading R. erythropolis TA422, lacking bphC2, bphC3, and bphC4, was obtained spontaneously from R. erythropolis TA421 with a high frequency, suggesting that these genes are associated with an unstable element, such as a plasmid. Recently, linear structures have often been found in bacterial genomes, particularly in the genomes of actinomycetes (6, 8, 13, 14). To investigate the possibility that the three bphC genes are located on a linear plasmid, we performed pulsedfield gel electrophoresis (PFGE) of undigested total DNA of R. erythropolis TA421 and TA422. The DNA sample for PFGE was prepared as described by

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McClelland et al. (17) with the following exceptions. Rhodococcus cells were grown to mid- or late log phase in LB medium containing 10% sucrose and 2% glycine. The cells were treated with 2 mg of lysozyme per ml in SPE buffer (10% sucrose, 10 mM EDTA, 10 mM sodium phosphate [pH 7]) before being embedded in agarose. Electrophoresis was performed using a CHEF Mapper system (Bio-Rad) with a gel of 1% SeaKem GTG agarose (FMC, Bioproducts) in 0.53 Trisborate-EDTA. Gels were run with a 6-V/cm electric field at 14°C for 28 h. The pulse time was increased from 13 to 90 s with a linear gradient, and the field angle was 120°. A large DNA molecule of approximately 500 kb was detected in R. erythropolis TA421 but not in TA422 (Fig. 2A). The DNA molecule was separated from the undigested total DNA by PFGE, and the migration pattern was found to be reproducible in comparison with the Saccharomyces cerevisiae chromosome standard marker, suggesting that R. erythropolis TA421 has a linear plasmid. We designated this plasmid pTA421. After PFGE, the gel was blotted onto a nylon membrane and Southern hybridization was performed with the seven bphC probes under the same conditions described above. As shown in Fig. 2B, the bphC2, bphC3, and bphC4 probes hybridized to the plasmid, indicating that these genes are located on the plasmid. In contrast, the bphC1, bphC5, bphC6, and bphC7 probes hybridized with the total DNA (Fig. 1) but not with the plasmid of R. erythropolis TA421 (Fig. 2B), suggesting that these four genes are located on the chromosome. The Southern blot and PFGE analyses have suggested that the plasmid-borne bphC2, bphC3, and bphC4 genes are asso-

FIG. 1. Southern blot analyses of total DNAs from Rhodococcus strains with each of the seven bphC genes from R. erythropolis TA421. Total DNAs were from R. erythropolis TA421 (lanes 1), R. erythropolis TA422 (lanes 2), R. erythropolis TA431 (lanes 3), and R. globerulus P6 (lanes 4). Probes used for hybridization were as follows: the 2.0-kb BamHI-SacI fragment of pCY1 (15) (bphC1 probe); the 1.8-kb SacI-EcoRI fragment of pCM1 (15) (bphC2 probe); the 2.2-kb SalI fragment of pTC4 (15) (bphC3 probe); the 2.1-kb XbaI-HindIII fragment of pTC5 (15) (bphC4 probe); the 2.4-kb BamHI fragment of the bphC5 region (bphC5 probe); the 0.84-kb EcoRI fragment of the bphC6 region (bphC6 probe); the 1.6-kb SmaI fragment of the bphC7 region (bphC7 probe). The sizes of molecular standards of a mixture of HindIII-digested l and HaeIII-digested fX174 DNA (Toyobo) are indicated in bases (lanes M). Notice that for bphC4 and bphC7 the sizes of the signal bands are different from those of their probes because the partial regions to be detected were used for probes.

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FIG. 2. Locations of the bphC genes in Rhodococcus PCB or biphenyl degraders. (A) PFGE; (B) Southern blot analysis. Lane M, S. cerevisiae PFGE size standard marker (Bio-Rad); lanes 1, R. erythropolis TA421; lanes 2, R. erythropolis TA422; lanes 3, R. erythropolis TA431; lanes 4, R. globerulus P6. The sizes of molecular standards are indicated in kilobases. Preparation of the bphC2, bphC3, and bphC4 probes was as described in the legend for Fig. 1.

ciated with biphenyl degradation but that the chromosomal bphC1, bphC5, bphC6, and bphC7 genes may have other roles besides biphenyl degradation. The distribution and genomic locations of the homologs of the seven bphC genes in other Rhodococcus PCB and biphenyl degraders. To determine the distribution of the seven bphC genes of R. erythropolis TA421, we also performed hybridization analyses with the total DNAs of two other Rhodococcus PCB and biphenyl degraders. R. erythropolis TA431 (7), another PCB and biphenyl degrader isolated from a termite ecosystem, had the bphC1, bphC5, bphC6, and bphC7 homologs of R. erythropolis TA421 but not the bphC2, bphC3, and bphC4 homologs (Fig. 1, lanes 3). R. globerulus P6 (Corynebacterium sp. strain MB1; ATCC 49955) (3) was found to have all of the seven bphC homologs of R. erythropolis TA421 (Fig. 1, lanes 4). R. erythropolis TA431 has one plasmid, designated pTA431, of approximately 560 kb (Fig. 2A, lane 3). R. globerulus P6 has two plasmids, designated pLP6 and pSP6, of approximately 650 and 360 kb, respectively (Fig. 2A, lane 4). Southern blot analysis revealed that in R. globerulus P6, the bphC2 homolog of R. erythropolis TA421 is located on the two plasmids, while the bphC3 and bphC4 homologs are on pSP6 (Fig. 2B, lanes 4). These results indicated that bphC2, bphC3, and bphC4 are located on plasmids of different sizes in R. erythropolis TA421 and R. globerulus P6. We summarize the distribution and genomic locations of the seven bphC genes in R. erythropolis TA421, TA422, and TA431 and R. globerulus P6 in Table 2. The observation that the bphC3 and bphC4 genes of R. erythropolis TA421 are clustered

TABLE 2. The distribution and genomic locations of the bphC genes in R. erythropolis TA421, TA422, and TA431 and R. globerulus P6 Probea

bphC1 bphC2 bphC3 bphC4 bphC5 bphC6 bphC7

Hybridizationb with the total DNA of: TA421

TA422

TA431

P6

Genomic location

1 1 1 1 1 1 1

1 2 2 2 1 1 1

1 2 2 2 1 1 1

1 1c 1d 1 1 1 1

Chromosome Plasmid Plasmid Plasmid Chromosome Chromosome Chromosome

with other bph genes enhanced the possibility of their involvement in PCB and biphenyl degradation. R. erythropolis TA431 does not have the genes corresponding to bphC2, bphC3, and bphC4, which are necessary for biphenyl degradation by R. erythropolis TA421. However, R. erythropolis TA431 can utilize biphenyl and cometabolize PCBs (7). There may be another bphC gene which contributes to PCB and biphenyl degradation by R. erythropolis TA431. Three different bphC genes in R. globerulus P6 were described by Asturias et al. (2, 4). Nucleotide sequences of the bphC2 and bphC3 genes from R. erythropolis TA421 were 94 and 99% identical to those of the bphC2 and bphC1 genes from R. globerulus P6, respectively (Table 2). The bphC2 probe hybridized with the two plasmids of R. globerulus P6, suggesting that the strain has at least two copies of the corresponding gene. The bphC4 gene of R. erythropolis TA421 is not similar to any of the three bphC genes isolated from R. globerulus P6. However, the bphC4 probe hybridized with pSP6 of R. globerulus P6, indicating that R. globerulus P6 carries a corresponding gene on the plasmid. Many examples of plasmid-borne genes for degradation of recalcitrant compounds in gram-negative bacteria are known (21). For bacterial adaptation to novel compounds, plasmids are thought to play critical roles in the development of novel catabolic pathways via horizontal gene transfer and assembly of catabolic modules (21). Because R. erythropolis TA421 and R. globerulus P6 have a set of similar genes of the bphC multigene family and because the genes for catabolism of PCB and biphenyl are encoded on plasmids, it is likely that plasmids also play an important role in the evolution of the catabolic pathway for recalcitrant compounds in gram-positive bacteria. Nucleotide sequence accession numbers. The nucleotide sequences of the bphC1 through bphC7 regions from R. erythropolis TA421 have been deposited with DDBJ under the following accession numbers: the bphC1 region, D88013; the bphC2 region, D88014; bphB and bphC3, D88015; bphA1, bphA2, bphA3, and bphA4 in the bphC3 region, D88020; bphC4 and bphD, D88016; bphA1, bphA2, bphA3, bphB, and bphA4 in the bphC4 region, D88021; the bphC5 region, D88017; the bphC6 region, D88018; the bphC7 region, D88019. We are very grateful to C. L. Koh of the University of Malaya for his critical reading of the manuscript. Support for this work was provided by the Special Postdoctoral Researchers Program from RIKEN and by CREST of the Japan Science and Technology Corporation.

a

Genes bphC1 to bphC7 of TA421 were individually used. 1, probe hybridized; 2, probe did not hybridize. Identity with bphC2 of R. globerulus P6, 94% (2). d Identity with bphC1 of R. globerulus P6, 99% (2). b c

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