DNA rearrangement has occurred in the carbazole-degradative plasmid pCAR1 and the chromosome of its unsuitable host, Pseudomonas fluorescens Pf0-1

The carbazole-degradative plasmid pCAR1 carries the class II transposon Tn4676, which contains the car and ant genes, essential for conversion of carbazole into anthranilate, and anthranilate into catechol, respectively. In our previous study, DNA rearrangements in pCAR1 were frequently detected in the host Pseudomonas fluorescens Pf0-1 in the presence of carbazole, resulting in the improvement of host survivability. Several Pf0-1 mutants harbouring pCAR1 were isolated, and deletion of DNA in the plasmid ant gene was found. Here, we compared genome sequences of the parent strain Pf0-1L(pCAR1 : : rfp) and one of its mutants, 5EP83, to assess whether other DNA rearrangements occurred in either the plasmid or the host chromosome. We found transposition of Tn4676 into the 5EP83 chromosome. In addition, ISPre1 had transposed into the car gene intergenic region on the pCAR1-derivative plasmid of 5EP83, which inhibited car transcription. As a result of these transpositions, 5EP83 was able to metabolize carbazole due to the Tn4676 on its chromosome, although the car genes on its plasmid were non-functional. We also found that one copy of duplicate carAa genes had been deleted, and that ISPre4 had transposed into both the host chromosome and the plasmid. Our findings suggest that Pf0-1 harbouring pCAR1 is subjected to DNA rearrangements not only on the plasmid but also on its chromosome in the presence of carbazole.

Evolution of the IncP-7 carbazole-degradative plasmid pCAR1 improves survival of its host Pseudomonas fluorescens Pf0-1 in artificial water microcosms

In our previous study, Pseudomonas fluorescens Pf0-1L, harbouring the IncP-7 carbazole-degradative plasmid pCAR1 : : rfp, was shown to be undetectable within 5 days post-inoculation in carbazole-contaminated artificial freshwater microcosms containing several plasmid-free bacteria in addition to Pf0-1L(pCAR1 : : rfp). Fourteen days after the inoculation, carbazole degraders become detectable. Here, we revealed that these isolates were not pCAR1 transconjugants, but Pf0-1L(pCAR1 : : rfp) mutants, based on RFLP and BOX-A1R-based repetitive extragenic palindromic-PCR (BOX-PCR) analysis. Notably, the mutants displayed more rapid initiation of carbazole degradation than the parent strain Pf0-1L(pCAR1 : : rfp). The mutants were unable to degrade anthranilate due to a 163 bp deletion in the antA gene, which was overcome by their transformation with a wild-type antABC-expressing plasmid. Quantitative RT-PCR analysis indicated that the transcriptional induction of carbazole-, anthranilate- and catechol-degradative genes was comparable in both parent and mutant strains. The deletion mutants became dominant in the artificial water microcosm. The mutation caused anthranilate to accumulate instead of catechol, a toxic compound for the parent strain, and may be beneficial to host survival in artificial microcosms.