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Applied and Environmental Microbiology, January 2003, p. 427-433, Vol. 69, No. 1
0099-2240/03/$08.00+0     DOI: 10.1128/AEM.69.1.427-433.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

2-Hydroxypenta-2,4-dienoate Metabolic Pathway Genes in a Strong Polychlorinated Biphenyl Degrader, Rhodococcus sp. Strain RHA1

Department of Bioengineering, Nagaoka University of Technology, Kamitomioka, Nagaoka, Niigata 940-2188 Sugiyama Chemical and Industrial Laboratory, Kagetori, Totuka, Yokohama, Kanagawa 254-0064, Japan

Received 11 July 2002/ Accepted 15 October 2002

A gram-positive polychlorinated biphenyl (PCB) degrader, Rhodococcus sp. strain RHA1, metabolizes biphenyl through the 2-hydroxypenta-2,4-dienoate (HPD) and benzoate metabolic pathways. The HPD metabolic pathway genes, the HPD hydratase (bphE1), 4-hydroxy-2-oxovalerate aldolase (bphF1), and acetaldehyde dehydrogenase (acylating) (bphG) genes, were cloned from RHA1. The deduced amino acid sequences of bphGF1E1 have 30 to 58% identity with those of the HPD metabolic pathway genes of gram-negative bacteria. The order of these genes, bphG-bphF1-bphE1, differs from that of the HPD metabolic pathway genes, bphE-bphG-bphF, in gram-negative degraders of PCB, phenol, and toluene. Reverse transcription-PCR experiments indicated that the bphGF1E1 genes are inducibly cotranscribed in cells grown on biphenyl and ethylbenzene. Primer extension analysis revealed that the transcriptional initiation site exists within the bphR gene located adjacent to and upstream of bphG, which is deduced to code a transcriptional regulator. The respective enzyme activities of bphGF1E1 gene products were detected in Rhodococcus erythropolis IAM1399 carrying a bphGF1E1 plasmid. The insertional inactivation of the bphE1, bphF1, and bphG genes resulted in the loss of the corresponding enzyme activities and diminished growth on both biphenyl and ethylbenzene. Severe growth interference was observed during growth on biphenyl. The growth defects were partially restored by the introduction of plasmids containing the respective intact genes. These results indicated that the cloned bphGF1E1 genes are not only responsible for the primary metabolism of HPD during growth on both biphenyl and ethylbenzene but are also involved in preventing the accumulation of unexpected toxic metabolites, which interfere with the growth of RHA1.

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