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Journal of Bacteriology, March 2007, p. 2007-2020, Vol. 189, No. 5
0021-9193/07/$08.00+0     doi:10.1128/JB.01486-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

The Sphingomonas Plasmid pCAR3 Is Involved in Complete Mineralization of Carbazole{triangledown} ,{dagger}

Masaki Shintani, Masaaki Urata, Kengo Inoue, Kaori Eto, Hiroshi Habe, Toshio Omori,{ddagger} Hisakazu Yamane, and Hideaki Nojiri*

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

Received 29 October 2006/ Accepted 6 December 2006

We determined the complete 254,797-bp nucleotide sequence of the plasmid pCAR3, a carbazole-degradative plasmid from Sphingomonas sp. strain KA1. A region of about 65 kb involved in replication and conjugative transfer showed similarity to a region of plasmid pNL1 isolated from the aromatic-degrading Novosphingobium aromaticivorans strain F199. The presence of many insertion sequences, transposons, repeat sequences, and their remnants suggest plasticity of this plasmid in genetic structure. Although pCAR3 is thought to carry clustered genes for conjugative transfer, a filter-mating assay between KA1 and a pCAR3-cured strain (KA1W) was unsuccessful, indicating that pCAR3 might be deficient in conjugative transfer. Several degradative genes were found on pCAR3, including two kinds of carbazole-degradative gene clusters (car-I and car-II), and genes for electron transfer components of initial oxygenase for carbazole (fdxI, fdrI, and fdrII). Putative genes were identified for the degradation of anthranilate (and), catechol (cat), 2-hydroxypenta-2,4-dienoate (carDFE), dibenzofuran/fluorene (dbf/fln), protocatechuate (lig), and phthalate (oph). It appears that pCAR3 may carry clustered genes (car-I, car-II, fdxI, fdrI, fdrII, and, and cat) for the degradation of carbazole into tricarboxylic acid cycle intermediates; KA1W completely lost the ability to grow on carbazole, and the carbazole-degradative genes listed above were all expressed when KA1 was grown on carbazole. Reverse transcription-PCR analysis also revealed that the transcription of car-I, car-II, and cat genes was induced by carbazole or its metabolic intermediate. Southern hybridization analyses with probes prepared from car-I, car-II, repA, parA, traI, and traD genes indicated that several Sphingomonas carbazole degraders have DNA regions similar to parts of pCAR3.

* Corresponding author. Mailing address: Biotechnology Research Center, 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 15 December 2006.

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

{ddagger} Present address: Department of Industrial Chemistry, Shibaura Institute of Technology, 3-9-14 Shibaura, Minato-ku, Tokyo 108-8548, Japan.

Journal of Bacteriology, March 2007, p. 2007-2020, Vol. 189, No. 5
0021-9193/07/$08.00+0     doi:10.1128/JB.01486-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.



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