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Applied and Environmental Microbiology, September 2006, p. 6183-6193, Vol. 72, No. 9
0099-2240/06/$08.00+0     doi:10.1128/AEM.00947-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Transcriptomic Assessment of Isozymes in the Biphenyl Pathway of Rhodococcus sp. Strain RHA1

Edmilson R. Gonçalves, Hirofumi Hara, Daisuke Miyazawa, Julian E. Davies, Lindsay D. Eltis, and William W. Mohn^*

Department of Microbiology and Immunology, University of British Columbia, Vancouver, British Columbia V6T 1Z3, Canada¹

Received 20 April 2006/ Accepted 21 June 2006

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.

Supplemental material for this article may be found at http://aem.asm.org/.

Present address: Pontificia Universidade Catolica de Campinas, Centro de Ciencias da Vida—Faculdade de Ciencias Biologicas, Av. John Boyd Dunlop, s/n, Campinas-São Paulo—CEP 13.059-900, Brazil.

Present address: Department of Applied Biotechnology, Graduate School of Agriculture and Life Science, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan.

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