Genetic characterization and evolutionary implications of a car gene cluster in the carbazole degrader Pseudomonas sp strain CA10

The nucleotide sequences of the 27,939 bp-long upstream and 9,448-bp-long d ownstream regions of the carAaAaBaBbCAc(ORF7)Ad genes of carbazole degradin g Pseudomonas sp, strain CA10 were determined. Thirty-two open reading fram es (ORFs) were identified, and the car gene cluster was consequently reveal ed to consist of 10 genes (carAaAaBaBbCAcAdDFE) encoding the enzymes for th e three-step conversion of carbazole to anthranilate and the degradation of 2-hydroxypenta-2,4-dienoate. The high identities (68 to 83%) with the enzy mes involved in 3-(3-hydroxyphenyl)propionic acid degradation were observed only for CarFE. This observation, together with the fact that two ORFs are inserted between carD and carFE, makes it quite likely that the carFE gene s were recruited from another locus. In the 21-kb region upstream from carA a, aromatic-ring-hydroxylating dioxygenase genes (ORF26, ORF27, and ORF28) were found. Inductive expression in carbazole-grown cells and the results o f homology searching indicate that these genes encode the anthranilate 1,2- dioxygenase involved in carbazole degradation. Therefore, these ORFs were d esignated antABC. Four homologous insertion sequences, IS5car1 to IS5car4, were identified in the neighboring regions of car and ant genes. IS5car2 an d IS5car3 constituted the putative composite transposon containing antABC. One-ended transposition of IS5car2 together with the 5 ' portion of antA in to the region immediately upstream of carAa had resulted in the formation o f IS4car1 and ORF9, In addition to the insertion sequence-dependent recombi nation, gene duplications and presumed gene fusion were observed. In conclu sion, through the above gene rearrangement, the novel genetic structure of the car gene cluster has been constructed. In addition, it was also reveale d that the car and ant gene clusters are located on the megaplasmid pCAR1.