Plasmid pCAR3 contains multiple gene sets involved in the conversion of carbazole to anthranilate

Abstract

The carbazole degradative car-I gene cluster (carAaIBaIBbICIAcI) of Sphingomonas sp. strain KA1 is located on the 254-kb circular plasmid pCAR3. Carbazole conversion to anthranilate is catalyzed by carbazole 1,9a-dioxygenase (CARDO; CarAaIAcI), meta-cleavage enzyme (CarBaIBbI), and hydrolase (CarCI). CARDO is a three-component dioxygenase, and CarAaI and CarAcI are its terminal oxygenase and ferredoxin components. The car-I gene cluster lacks the gene encoding the ferredoxin reductase component of CARDO. In the present study, based on the draft sequence of pCAR3, we found multiple carbazole degradation genes dispersed in four loci on pCAR3, including a second copy of the car gene cluster (carAaIIBaIIBbIICIIAcII) and the ferredoxin/reductase genes fdxI-fdrI and fdrII. Biotransformation experiments showed that FdrI (or FdrII) could drive the electron transfer chain from NAD(P)H to CarAaI (or CarAaII) with the aid of ferredoxin (CarAcI, CarAcII, or FdxI). Because this electron transfer chain showed phylogenetic relatedness to that consisting of putidaredoxin and putidaredoxin reductase of the P450cam monooxygenase system of Pseudomonas putida, CARDO systems of KA1 can be classified in the class IIA Rieske non-heme iron oxygenase system. Reverse transcription-PCR (RT-PCR) and quantitative RT-PCR analyses revealed that two car gene clusters constituted operons, and their expression was induced when KA1 was exposed to carbazole, although the fdxI-fdrI and fdrII genes were expressed constitutively. Both terminal oxygenases of KA1 showed roughly the same substrate specificity as that from the well-characterized carbazole degrader Pseudomonas resinovorans CA10, although slight differences were observed.

FIG. 1.

Carbazole degradation by Car enzymes harbored by various carbazole degraders. The product of angular dioxygenation of carbazole shown in brackets is unstable and has not been detected directly. Enzyme (or protein) names: terminal oxygenase (CARDO-O), ferredoxin (CARDO-F), and reductase (CARDO-R) components of CARDO; meta-cleavage enzyme (CarBaBb); meta-cleavage compound hydrolase (CarC). ox. and red., oxidized and reduced states of the CARDO components, respectively. Compounds: I, carbazole; II, 2′-aminobiphenyl-2,3-diol; III, 2-hydroxy-6-oxo-6-(2′-aminobiphenyl)-hexa-2,4-dienoic acid; IV, anthranilic acid.

FIG. 2.

Genetic organization of the car-I gene cluster (locus A), car-II gene cluster (locus B), fdxIfdrI locus (locus C), and fdrII locus (locus D) encompassing the pCAR3 genes involved in the degradation of carbazole by Sphingomonas sp. strain KA1. The genetic organization of the car gene cluster responsible for carbazole degradation in P. resinovorans CA10 is also shown. The carAa gene of CA10 is tandemly duplicated (36). The arrows in the physical maps indicate the size, location, and direction of transcription of the ORFs derived from the nucleotide sequence data.