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The IncP-7 plasmid pCAR1 of Pseudomonas resinovorans CA10 confers the ability to degrade carbazole upon transfer to the recipient strain P. putida KT2440. We designed a customized whole-genome oligonucleotide microarray to study the coordinated expression of pCAR1 and the chromosome in the transconjugant strain KT2440(pCAR1). First, the transcriptome of KT2440(pCAR1) during growth with carbazole as the sole carbon source was compared to that during growth with succinate. The carbazole catabolic car and ant operons were induced, along with the chromosomal eat and pea genes involved in the catechol branch of the [beta]-ketoadipate pathway. Additionally, the regulatory gene antR encoding the AraC/XylS family transcriptional activator specific for car and ant operons was upregulated. The characterization of the antR promoter revealed that antR is transcribed from an RpoN-dependent promoter, suggesting that the successful expression of the carbazole catabolic operons depends on whether the chromosome contains the specific RpoN-dependent activator. Next, to analyze whether the horizontal transfer of a plasmid alters the transcription network of its host chromosome, we compared the chromosomal transcriptomes of KT2440(pCAR1) and KT2440 under the same growth conditions. Only subtle changes were caused by the transfer of pCAR1, except for the significant induction of the hypothetical gene PP3700, designated parI, which encodes a putative ParA-like ATPase with an N-terminal Xre-type DNA-binding motif. Further transcriptional analyses showed that the parI promoter was positively regulated by ParI itself and the pCAR1-encoded protein ParA.

The carbazole-degradative plasmid pCAR1 of Pseudomonas resinovorans strain CA10 has two gene clusters, carAaAaBaBbCAcAdDFE and antABC, which are involved in the conversions of carbazole to anthranilate and anthranilate to catechol, respectively. We proved that the antABC gene cluster, encoding two-component anthranilate 1,2-dioxygenase, constitutes a single transcriptional unit through Northern hybridization and reverse transcription-PCR (RT-PCR) analyses. The transcription start point of antA was mapped at 53 bp upstream point of its translation start point, and the-10 and-35 boxes were homologous to conserved [[sigma].sup.70] recognition sequence. Hence the promoter of the ant operon was designated [P.sub.ant]. 5' Deletion analyses using luciferase as a reporter showed that the region up to at least 70 bp from the transcription start point of antA was necessary for the activation of [P.sub.ant] Luciferase expression from [P.sub.ant] was induced by anthranilate itself, but not by catechol. Two probable AraC/XylS-type regulatory genes found on pCAR1, open reading frame 22 (ORF22) and ORF23, are tandemly located 3.2 Ida upstream of the antA gene. We revealed that the product of ORF23, designated AntR, is indispensable for the stimulation of [P.sub.ant] in Pseudomonas putida cells. Northern hybridization and RT-PCR analyses revealed that another copy of Pant, which is thought to be translocated about 2.1 kb upstream of the carAa gene as a consequence of the transposition of ISPre1, actually drives transcription of the carAa gene in the presence of anthranilate, indicating that both ant and car operons are simultaneously regulated by AntR.