Genetic Characterization and Evolutionary Implications of a car Gene Cluster in the Carbazole Degrader Pseudomonas sp. Strain CA10

Abstract

The nucleotide sequences of the 27,939-bp-long upstream and 9,448-bp-long downstream regions of the carAaAaBaBbCAc(ORF7)Ad genes of carbazole-degrading Pseudomonas sp. strain CA10 were determined. Thirty-two open reading frames (ORFs) were identified, and the car gene cluster was consequently revealed to consist of 10 genes (carAaAaBaBbCAcAdDFE) encoding the enzymes for the three-step conversion of carbazole to anthranilate and the degradation of 2-hydroxypenta-2,4-dienoate. The high identities (68 to 83%) with the enzymes 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 genes were recruited from another locus. In the 21-kb region upstream from carAa, aromaticring-hydroxylating dioxygenase genes (ORF26, ORF27, and ORF28) were found. Inductive expression in carbazole-grown cells and the results of homology searching indicate that these genes encode the anthranilate 1,2-dioxygenase involved in carbazole degradation. Therefore, these ORFs were designated antABC. Four homologous insertion sequences, IS5car1 to IS5car4, were identified in the neighboring regions of car and ant genes. IS5car2 and IS5car3 constituted the putative composite transposon containing antABC. One-ended transposition of IS5car2 together with the 5 portion of antA into the region immediately upstream of carAa had resulted in the formation of IS5car1 and ORF9. In addition to the insertion sequence-dependent recombination, gene duplications and presumed gene fusion were observed. In conclusion, through the above gene rearrangement, the novel genetic structure of the car gene cluster has been constructed. In addition, it was also revealed that the car and ant gene clusters are located on the megaplasmid pCAR1.

Properties, environmental fate and biodegradation of carbazole

“…In addition, antABC gene encoding anthranilate 1,2-dioxygenase was found in the 21-kb region upstream from carAa ( Fig. 5a) (Nojiri et al 2001). This anthranilate degradative gene cluster is a putative composite transposon flanked by two homologous insertion sequences ISPre1 and ISPre2.…”

“…Enzymes designations: CarAaAcAd, carbazole 1,9a-dioxygenase; CarBaBb, 2 0 -aminobiphenyl-2,3-diol 1,2-dioxygenase; CarC, 2-hydroxy-6-oxo-6-(2 0 -aminophenyl)-hexa-2,4-dienoate hydrolase; CarD, 2-hydroxypenta-2,4-dienoate hydratase; CarE, 4-hydroxy-2-oxovalerate aldolase; CarF, acetaldehyde dehydrogenase (acylating); AntABC, anthranilate 1,2-dioxygenase; CatA, catechol 1,2-dioxygenase; CatB, cis,cis-muconate lactonizing enzyme; CatC, muconolactone d-isomerase. Compounds: I, CAR; II, 2 0 -aminobiphenyl-2,3-diol; III, 2-hydroxy-6-oxo-6-(2 0 -aminophenyl)-hexa-2,4-dienoate; IV, anthranilic acid; V, catechol; VI, cis,cis-muconate; VII, muconolactone; VIII, b-ketoadipic acid enol-lactone; IX, 2-hydroxy-penta-2,4-dienoate; X, 4-hydroxy-2-oxovalerate; XI, pyruvate; XII, acetaldehyde; XIII, acetyl coenzyme A (Nojiri et al 2001) strain SL1, Pseudomonas sp. strain SL4 and Microbacterium esteraromaticum strain SL6, produce anthranilic acid and catechol as the major metabolites of carbazole angular dioxygenation.…”

Structural and Molecular Genetic Analyses of the Bacterial Carbazole Degradation System

“…In fact, the car gene cluster of CA10 is located on circular plasmid pCAR1. 51) We isolated pCAR1 from CA10 that had been preserved as a glycerol stock since its isolation in 1993. Its entire nucleotide sequence was determined in 2003 to elucidate the mechanism by which the car gene cluster might be distributed in nature.…”

Isolation and Description of a Stable Carbazole-Degrading Microbial Consortium Consisting of Chryseobacterium sp. NCY and Achromobacter sp. NCW

“…As the molecular mechanism for adaptation, gene recruitment is the key to establish several simultaneous degradation activities and the entire catabolic capacity for the xenobiotics to which microorganisms are exposed [13]. It is reported that some bacterial strains have the capacity to metabolize carbazole.…”

“…It is reported that some bacterial strains have the capacity to metabolize carbazole. Therefore, Pseudomonas resinovorans CA10 has been widely studied [13,15,16]. So far, these different species of carbazole degraders all appear to follow a similar carbazole degradation pathway that begins with the oxidative cleavage of the heterocyclic nitrogen ring of carbazole to form 2 0 -aminobiphenyl-2, 3-diol.…”