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Transcriptomic assessment of isozymes in the biphenyl pathway of Rhodococcus sp. strain RHA1.

ABSTRACT: Rhodococcus sp. RHA1 grows on a broad range of aromatic compounds and vigorously degrades polychlorinated biphenyls (PCBs). Previous work identified RHA1 genes encoding multiple isozymes for most of the seven steps of the biphenyl (BPH) pathway, provided evidence for coexpression of some of these isozymes, and indicated the involvement of some of these enzymes in the degradation of BPH, ethylbenzene (ETB), and PCBs. To investigate the expression of these isozymes and better understand how they contribute to the robust degradative capacity of RHA1, we comprehensively analyzed the 9.7-Mb genome of RHA1 for BPH pathway genes and characterized the transcriptome of RHA1 growing on benzoate (BEN), BPH, and ETB. Sequence analyses revealed 54 potential BPH pathway genes, including 28 not previously reported. Transcriptomic analysis with a DNA microarray containing 70-mer probes for 8,213 RHA1 genes revealed a suite of 320 genes of diverse functions that were upregulated during growth both on BPH and on ETB, relative to growth on the control substrate, pyruvate. By contrast, only 65 genes were upregulated during growth on BEN. Quantitative PCR assays confirmed microarray results for selected genes and indicated that some of the catabolic genes were upregulated over 10,000-fold. Our analysis suggests that up to 22 enzymes, including 8 newly identified ones, may function in the BPH pathway of RHA1. The relative expression levels of catabolic genes did not differ for BPH and ETB, suggesting a common regulatory mechanism. This study delineated a suite of catabolic enzymes for biphenyl and alkyl-benzenes in RHA1, which is larger than previously recognized and which may serve as a model for catabolism in other environmentally important bacteria having large genomes.

SUBMITTER: Goncalves ER

PROVIDER: S-EPMC1563596 | biostudies-literature | 2006 Sep

REPOSITORIES: biostudies-literature

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Transcriptomic assessment of isozymes in the biphenyl pathway of Rhodococcus sp. strain RHA1.

Gonçalves Edmilson R ER Hara Hirofumi H Miyazawa Daisuke D Davies Julian E JE Eltis Lindsay D LD Mohn William W WW

Applied and environmental microbiology 20060901 9

Rhodococcus sp. RHA1 grows on a broad range of aromatic compounds and vigorously degrades polychlorinated biphenyls (PCBs). Previous work identified RHA1 genes encoding multiple isozymes for most of the seven steps of the biphenyl (BPH) pathway, provided evidence for coexpression of some of these isozymes, and indicated the involvement of some of these enzymes in the degradation of BPH, ethylbenzene (ETB), and PCBs. To investigate the expression of these isozymes and better understand how they c ...[more]

PMID: 16957245

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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

Transcriptomic assessment of isozymes in the biphenyl pathway of Rhodococcus sp. strain RHA1.

Publications

Transcriptomic assessment of isozymes in the biphenyl pathway of Rhodococcus sp. strain RHA1.

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