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Characterization of transcriptional regulatory genes for biphenyl degradation in Rhodococcus sp. strain RHA1.

ABSTRACT: Transcription of the bphA1A2A3A4C1B genes, which are responsible for the conversion of biphenyl and polychlorinated biphenyl to the meta-cleavage products in Rhodococcus sp. strain RHA1, was examined. The bphA1 promoter (P(bphA1)) was identified and was shown to promote transcription induction by biphenyl and ethylbenzene. An 8.8-kb HindIII fragment that promotes transcription induction of P(bphA1) in Rhodococcus erythropolis IAM1399 was isolated from the region downstream of bphB by using a reporter plasmid containing P(bphA1). Analysis of the nucleotide sequence of this fragment revealed a set of putative two-component regulatory system genes, which were designated bphS and bphT. Deletion analysis of the 8.8-kb HindIII fragment indicated that bphT is responsible for the basal activation of P(bphA1) and that both bphS and bphT are required for the elevated basal activation of and transcriptional induction by biphenyl of P(bphA1). These results support the notion that bphS and bphT encode a sensor kinase and a response regulator, respectively, of a two-component regulatory system. The bphS and bphT genes promote transcriptional induction by a variety of aromatic compounds, including biphenyl, benzene, alkylbenzenes, and chlorinated benzenes. A promoter activity assay and reverse transcription (RT)-PCR analysis revealed a weak constitutive promoter in the adjacent region upstream of bphS. RT-PCR analysis indicated that there is induced transcription of bphA1 through bphT, in which P(bphA1) is thought to take part. An insertionally inactivated bphS mutant, SDR1, did not grow on biphenyl. Growth was restored by introduction of an intact bphS gene into SDR1. These results indicate that at least bphS is indispensably responsible for the growth of RHA1 on biphenyl.

SUBMITTER: Takeda H

PROVIDER: S-EPMC374424 | biostudies-literature | 2004 Apr

REPOSITORIES: biostudies-literature

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Characterization of transcriptional regulatory genes for biphenyl degradation in Rhodococcus sp. strain RHA1.

Takeda Hisashi H Yamada Akihiro A Miyauchi Keisuke K Masai Eiji E Fukuda Masao M

Journal of bacteriology 20040401 7

Transcription of the bphA1A2A3A4C1B genes, which are responsible for the conversion of biphenyl and polychlorinated biphenyl to the meta-cleavage products in Rhodococcus sp. strain RHA1, was examined. The bphA1 promoter (P(bphA1)) was identified and was shown to promote transcription induction by biphenyl and ethylbenzene. An 8.8-kb HindIII fragment that promotes transcription induction of P(bphA1) in Rhodococcus erythropolis IAM1399 was isolated from the region downstream of bphB by using a rep ...[more]

PMID: 15028699

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Project description:The two 2-hydroxy-6-oxohepta-2,4-dienoate (HOHD) hydrolase genes, etbD1 and etbD2, were cloned from a strong polychlorinated biphenyl (PCB) degrader, Rhodococcus sp. strain RHA1, and their nucleotide sequences were determined. The etbD2 gene was located in the vicinity of bphA gene homologs and encoded an enzyme whose amino-terminal sequence was very similar to the amino-terminal sequence of the HOHD hydrolase which was purified from RHA1. Using the etbD2 gene fragment as a probe, we cloned the etbD1 gene encoding the purified HOHD hydrolase by colony hybridization. Both genes encode a product having 274 amino acid residues and containing the nucleophile motif conserved in alpha/beta hydrolase fold enzymes. The deduced amino acid sequences were quite similar to the amino acid sequences of the products of the single-ring aromatic hydrolase genes, such as dmpD, cumD, todF, and xylF, and not very similar to the amino acid sequences of the products of bphD genes from PCB degraders, including RHA1. The two HOHD hydrolase genes and the RHA1 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoate (HPDA) hydrolase gene, bphD, were expressed in Escherichia coli, and their relative enzymatic activities were examined. The product of bphD was very specific to HPDA, and the products of etbD1 and etbD2 were specific to HOHD. All of the gene products exhibited poor activities against the meta-cleavage product of catechol. These results agreed with the results obtained for BphD and EtbD1 hydrolases purified from RHA1. The three hydrolase genes exhibited similar induction patterns both in an RNA slot blot hybridization analysis and in a reporter gene assay when a promoter probe vector was used. They were induced by biphenyl, ethylbenzene, benzene, toluene, and ortho-xylene. Strain RCD1, an RHA1 mutant strain lacking both the bphD gene and the etbD2 gene, grew well on ethylbenzene. This result suggested that the etbD1 gene product is involved in the meta-cleavage metabolic pathway of ethylbenzene.

Dataset Information

Characterization of transcriptional regulatory genes for biphenyl degradation in Rhodococcus sp. strain RHA1.

Publications

Characterization of transcriptional regulatory genes for biphenyl degradation in Rhodococcus sp. strain RHA1.

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