Transcriptional Regulation of the ant Operon, Encoding Two-Component Anthranilate 1,2-Dioxygenase, on the Carbazole-Degradative Plasmid pCAR1 of Pseudomonas resinovorans Strain CA10

ABSTRACT

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 σ⁷⁰ recognition sequence. Hence the promoter of the ant operon was designated P_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_ant. Luciferase expression from P_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 kb upstream of the antA gene. We revealed that the product of ORF23, designated AntR, is indispensable for the stimulation of P_ant in Pseudomonas putida cells. Northern hybridization and RT-PCR analyses revealed that another copy of P_ant, 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.