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 70 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.Among the toxic and persistent compounds that have been released in the environment by human action, the aromatic compounds are of major concern. In spite of their recalcitrance to degradation by most organisms, bacterial strains have adapted to them in consequence of the acquisition of the necessary catabolic abilities to utilize aromatic compounds, and thus rapidly adapting bacteria are regarded as the experimental systems of choice in understanding how catabolic genes end up with regulated expression (8).Carbazole is a recalcitrant N-heterocyclic aromatic compound derived from coal tar and shale oil and possesses mutagenic and toxic activity (1). Pseudomonas resinovorans strain CA10, a gram-negative bacterium isolated from activated sludge, degrades carbazole as the sole source of carbon, nitrogen, and energy (27). In strain CA10, carbazole is initially converted into anthranilate and 2-hydroxypenta-2,4-dienoate through a three-step upper pathway catalyzed by carbazole 1,9a-dioxygenase (CarA), the meta-cleavage enzyme (CarB), and the meta-cleavage compound hydrolase (CarC) (Fig. 1A) (32,33). CarA is a multicomponent dioxygenase system composed of terminal oxygenase component CarAa, ferredoxin component CarAc, and ferredoxin reductase component CarAd. meta-Cleavage enzyme CarB is a heterotetrameric enzyme (␣ 2 ␤ 2 ) composed of CarBa and CarBb subunits. The resulting metabolite, anthranilate, is a naturally occ...

The Behavior and Significance of Degradative Plasmids Belonging to Inc Groups in <i>Pseudomonas</i> within Natural Environments and Microcosms

“…In contrast, antR was lost from pCAR1 in the pure culture, but not in the artificial microcosms. Absence of the antR gene will slow down carbazole's degradation since it encodes a transcriptional regulator of the car and ant genes in response to anthranilate (58,119). In the pure culture, delaying the degradation of carbazole is probably necessary to prevent catechol accumulation because no other catechol metabolizing strains were present.…”

The Sphingomonas Plasmid pCAR3 Is Involved in Complete Mineralization of Carbazole

“…1). In the case of DBO1, it appears that the And enzyme consists of a three-component system (9), and it is largely different from the two-component anthranilate dioxygenase encoded by the ant operon on another CAR-degradative plasmid, pCAR1 (43,63). Similar to the and operon of DBO1, we also found a gene encoding a putative transcriptional activator of the operon on pCAR3, although the direction was opposite to that of DBO1, and it was designated andR (see Fig.…”

Properties, environmental fate and biodegradation of carbazole

“…This anthranilate degradative gene cluster is a putative composite transposon flanked by two homologous insertion sequences ISPre1 and ISPre2. Furthermore, antR gene encoding a transcriptional regulator of the ant operon was found outside the putative composite transposon containing antABC, which regulates the inducible expression of the car gene cluster (Urata et al 2004). Tn5 mutagenesis was used to isolate the b-ketoadipate pathway (ortho-cleavage pathway) genes involved in catechol mineralization from strain CA10 genome (Kimura et al 1996).…”