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Applied and Environmental Microbiology, June 2009, p. 3920-3929, Vol. 75, No. 12
0099-2240/09/$08.00+0     doi:10.1128/AEM.02373-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Carbazole-Degradative IncP-7 Plasmid pCAR1.2 Is Structurally Unstable in Pseudomonas fluorescens Pf0-1, Which Accumulates Catechol, the Intermediate of the Carbazole Degradation Pathway{triangledown} ,{dagger}

Yurika Takahashi, Masaki Shintani, Li Li,{ddagger} Hisakazu Yamane, and Hideaki Nojiri*

Biotechnology Research Center, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan

Received 16 October 2008/ Accepted 9 April 2009

We determined the effect of the host on the function and structure of the nearly identical IncP-7 carbazole-degradative plasmids pCAR1.1 and pCAR1.2. We constructed Pseudomonas aeruginosa PAO1(pCAR1.2) and P. fluorescens Pf0-1Km(pCAR1.2) and compared their growth on carbazole- and succinate-containing media with that of P. putida KT2440(pCAR1.1). We also assessed the stability of the genetic structures of the plasmids in each of the three hosts. Pf0-1Km(pCAR1.2) showed dramatically delayed growth when carbazole was supplied as the sole carbon source, while the three strains grew at nearly the same rate on succinate. Among the carbazole-grown Pf0-1Km(pCAR1.2) cells, two types of deficient strains appeared and dominated the population; such dominance was not observed in the other two strains or for succinate-grown Pf0-1Km(pCAR1.2). Genetic analysis showed that the two deficient strains possessed pCAR1.2 derivatives in which the carbazole-degradative car operon was deleted or its regulatory gene, antR, was deleted by homologous recombination between insertion sequences. From genomic information and quantitative reverse transcription-PCR analyses of the genes involved in carbazole mineralization by Pf0-1Km(pCAR1.2), we found that the cat genes on the chromosome of Pf0-1Km, which are necessary for the degradation of catechol (a toxic intermediate in the carbazole catabolic pathway), were not induced in the presence of carbazole. The resulting accumulation of catechol may have enabled the strain that lost its carbazole-degrading ability to have overall higher fitness than the wild-type strain. These results suggest that the functions of the chromosomal genes contributed to the selection of plasmid derivatives with altered structures.

* Corresponding author. Mailing address: Biotechnology Research Center, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan. Phone: 81-3-5841-3064. Fax: 81-3-5841-8030. E-mail: anojiri{at}mail.ecc.u-tokyo.ac.jp

{triangledown} Published ahead of print on 17 April 2009.

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

{ddagger} Present address: School of Environmental Science and Engineering, Shandong University, Jinan 250100, People's Republic of China.

Applied and Environmental Microbiology, June 2009, p. 3920-3929, Vol. 75, No. 12
0099-2240/09/$08.00+0     doi:10.1128/AEM.02373-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.





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