Characterization of biphenyl catabolic genes of gram-positive polychlorinated biphenyl degrader Rhodococcus sp. strain RHA1

Abstract

Rhodococcus sp. strain RHA1 is a gram-positive polychlorinated biphenyl (PCB) degrader which can degrade 10 ppm of PCB48 (equivalent to Aroclor1248), including tri-, tetra-, and pentachlorobiphenyls, in a few days. We isolated the 7.6-kb EcoRI-BamHI fragment carrying the biphenyl catabolic genes of RHA1 and determined their nucleotide sequence. On the basis of deduced amino acid sequence homology, we identified six bph genes, bphA1A2A3A4, bphB, and bphC, that are responsible for the initial three steps of biphenyl degradation. The order of bph genes in RHA1 is bphA1A2A3A4-bphC-bphB. This gene order differs from that of other PCB degraders reported previously. The amino acid sequences deduced from the RHA1 bph genes have a higher degree of homology with the tod genes from Pseudomonas putida F1 (49 to 79%) than with the bph genes of Pseudomonas sp. strains KF707 and KKS102 (30 to 65%). In Escherichia coli, bphA gene activity was not observed even when expression vectors were used. The activities of bphB and bphC, however, were confirmed by observing the transformation of biphenyl to a meta-cleavage compound with the aid of benzene dioxygenase activity that complemented the bphA gene activity (S. Irie, S. Doi, T. Yorifuji, M. Takagi, and K. Yano, J. Bacteriol. 169:5174-5179, 1987). The expected products of the cloned bph genes, except bphA3, were observed in E. coli in an in vitro transcription-translation system. Insertion mutations of bphA1 and bphC of Rhodococcus sp. strain RHA1 were constructed by gene replacement with cloned gene fragments. The bphA1 and bphC insertion mutants lost the ability to grow on biphenyl, demonstrating that the cloned bph genes are essential for biphenyl catabolism in this strain.

Isolation and characterization of a thermophilic Bacillus sp. JF8 capable of degrading polychlorinated biphenyls and naphthalene

“…pUH3 (a gift from M. Fukuda, Nagaoka University of Technology, Japan), containing the bph genes of Rhodococcus sp. strain RHA1 [15], was digested with BglII and SalI to give a 1.5-kb region containing the bphA1 gene, which was used as a probe. The third probe was a 2.1-kb PstI fragment from pPTS3 with the pahA1A2 genes of Pseudomonas aeruginosa PaK1 [16].…”

Cloning of a Sphingomonas paucimobilis SYK-6 Gene Encoding a Novel Oxygenase That Cleaves Lignin-Related Biphenyl and Characterization of the Enzyme

“…A ring cleavage enzyme for OH-DDVA has been thought to be involved in this pathway because the production of 5-CVA from OH-DDVA resembles the formation of benzoic acid from biphenyl by 2,3dihydroxybiphenyl through the sequential action of a meta cleavage enzyme and a meta-cleavage compound hydrolase ( Fig. 1B) (1,9,13,18,21,28).…”

Characterization and functional analysis of a novel gene cluster involved in biphenyl degradation in Rhodococcus sp. strain R04

“…Based on the BLAST searches, seven open reading frames (ORFs), which showed homology with the previously characterized bph genes, were identified in the plasmid DNA of pBS01. One of ORFs was identified as the bphC (2,3‐dihydroxybiphenyl 1,2‐dioxygenase gene) on the basis of the screening of activity and the sequence similarity with other bphC genes reported previously (Masai et al. 1995).…”

“…Since Lunt and Evans first isolated micro‐organisms capable of growing on biphenyl as a sole source of carbon and energy (Lunt and Evans 1970), many other PCB degrading micro‐organisms have been found (Ahmad et al. 1990; Masai et al. 1995), and PCB degradation by these micro‐organisms has been extensively studied (Brenner et al.…”

“…strain RHA1 and Achromobacter georgiopolitanum KKS102 (Furukawa and Miyazaki 1986; Mondello 1989; Peloquin and Greer 1993; Kikuchi et al. 1994; Masai et al. 1995; Ohtsubo et al.…”