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Applied and Environmental Microbiology, March 2001, p. 1107-1115, Vol. 67, No. 3
0099-2240/01/$04.00+0   DOI: 10.1128/AEM.67.3.1107-1115.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

Genetic Diversity among 3-Chloroaniline- and Aniline-Degrading Strains of the Comamonadaceae

Nico Boon,1 Johan Goris,2 Paul De Vos,2 Willy Verstraete,1 and Eva M. Top1,*

Laboratory of Microbial Ecology and Technology1 and Laboratory of Microbiology,2 Ghent University, B-9000 Ghent, Belgium

Received 17 July 2000/Accepted 5 December 2000

We examined the diversity of the plasmids and of the gene tdnQ, involved in the oxidative deamination of aniline, in five bacterial strains that are able to metabolize both aniline and 3-chloroaniline (3-CA). Three strains have been described and identified previously, i.e., Comamonas testosteroni I2 and Delftia acidovorans CA28 and BN3.1. Strains LME1 and B8c were isolated in this study from linuron-treated soil and from a wastewater treatment plant, respectively, and were both identified as D. acidovorans. Both Delftia and Comamonas belong to the family Comamonadaceae. All five strains possess a large plasmid of ca. 100 kb, but the plasmids from only four strains could be transferred to a recipient strain by selection on aniline or 3-CA as a sole source of carbon and/or nitrogen. Plasmid transfer experiments and Southern hybridization revealed that the plasmid of strain I2 was responsible for total aniline but not 3-CA degradation, while the plasmids of strains LME1 and B8c were responsible only for the oxidative deamination of aniline. Several transconjugant clones that had received the plasmid from strain CA28 showed different degradative capacities: all transconjugants could use aniline as a nitrogen source, while only some of the transconjugants could deaminate 3-CA. For all four plasmids, the IS1071 insertion sequence of Tn5271 was found to be located on a 1.4-kb restriction fragment, which also hybridized with the tdnQ probe. This result suggests the involvement of this insertion sequence element in the dissemination of aniline degradation genes in the environment. By use of specific primers for the tdnQ gene from Pseudomonas putida UCC22, the diversity of the PCR-amplified fragments in the five strains was examined by denaturing gradient gel electrophoresis (DGGE). With DGGE, three different clusters of the tdnQ fragment could be distinguished. Sequencing data showed that the tdnQ sequences of I2, LME1, B8c, and CA28 were very closely related, while the tdnQ sequences of BN3.1 and P. putida UCC22 were only about 83% identical to the other sequences. Northern hybridization revealed that the tdnQ gene is transcribed only in the presence of aniline and not when only 3-CA is present.

* Corresponding author. Mailing address: Ghent University, Faculty of Agricultural and Applied Biological Sciences, Laboratory of Microbial Ecology and Technology (LabMET), Coupure Links 653, B-9000 Ghent, Belgium. Phone: 32 (0)9 264 59 76. Fax: 32 (0)9 264 62 48. E-mail: Eva.Top{at}rug.ac.be.

Applied and Environmental Microbiology, March 2001, p. 1107-1115, Vol. 67, No. 3
0099-2240/01/$04.00+0   DOI: 10.1128/AEM.67.3.1107-1115.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.


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Copyright © 2001 by the American Society for Microbiology. All rights reserved.