The genes coding for the conversion of carbazole to catechol are flanked by IS6100 elements in Sphingomonas sp. strain XLDN2-5

Abstract

Background: Carbazole is a recalcitrant compound with a dioxin-like structure and possesses mutagenic and toxic activities. Bacteria respond to a xenobiotic by recruiting exogenous genes to establish a pathway to degrade the xenobiotic, which is necessary for their adaptation and survival. Usually, this process is mediated by mobile genetic elements such as plasmids, transposons, and insertion sequences.

Findings: The genes encoding the enzymes responsible for the degradation of carbazole to catechol via anthranilate were cloned, sequenced, and characterized from a carbazole-degrading Sphingomonas sp. strain XLDN2-5. The car gene cluster (carRAaBaBbCAc) and fdr gene were accompanied on both sides by two copies of IS6100 elements, and organized as IS6100::ISSsp1-ORF1-carRAaBaBbCAc-ORF8-IS6100-fdr-IS6100. Carbazole was converted by carbazole 1,9a-dioxygenase (CARDO, CarAaAcFdr), meta-cleavage enzyme (CarBaBb), and hydrolase (CarC) to anthranilate and 2-hydroxypenta-2,4-dienoate. The fdr gene encoded a novel ferredoxin reductase whose absence resulted in lower transformation activity of carbazole by CarAa and CarAc. The ant gene cluster (antRAcAdAbAa) which was involved in the conversion of anthranilate to catechol was also sandwiched between two IS6100 elements as IS6100-antRAcAdAbAa-IS6100. Anthranilate 1,2-dioxygenase (ANTDO) was composed of a reductase (AntAa), a ferredoxin (AntAb), and a two-subunit terminal oxygenase (AntAcAd). Reverse transcription-PCR results suggested that carAaBaBbCAc gene cluster, fdr, and antRAcAdAbAa gene cluster were induced when strain XLDN2-5 was exposed to carbazole. Expression of both CARDO and ANTDO in Escherichia coli required the presence of the natural reductases for full enzymatic activity.

Conclusions/significance: We predict that IS6100 might play an important role in the establishment of carbazole-degrading pathway, which endows the host to adapt to novel compounds in the environment. The organization of the car and ant genes in strain XLDN2-5 was unique, which showed strong evolutionary trail of gene recruitment mediated by IS6100 and presented a remarkable example of rearrangements and pathway establishments.

Figure 1. Biodegradation of carbazole via the angular pathway by Sphingomonas sp. XLDN2-5 and other carbazole-utilizing bacteria.

The product in dashed square is unstable and has not been detected. Enzyme names: carbazole 1,9a-dioxygenase (carAaAcfdr); meta-cleavage enzyme (carBaBb); hydrolase (carC); anthranilate 1,2-dioxygenase (antAcAdAbAa). Compound I: carbazole; compound II: 2′-aminobiphenyl-2,3-diol; compound III: 2-hydroxy-6-oxo-6-(2′-aminobiphenyl)-hexa-2,4-dienoic acid; compound IV: 2-hydroxypenta-2,4-dienoate; compound V: anthranilic acid; VI: catechol.

Figure 2. Physical maps of car and ant loci.

(A) Physical map of car locus, which is delimited by IS6100 elements. The upstream IS6100 is interrupted by a novel insert element ISSsp1, and was designated IS6100::ISSsp1. (B) Schematic representation of the main features of the novel ISSsp1 sequence in Sphingomonas sp. XLDN2-5. The orientation of the ISSsp1 is shown by an arrow. The red and blue boxes represent the positions of two direct repeats (DR) and two imperfect, 32 bp, terminal inverted repeats (the left inverted repeat [IRL], and the right inverted repeat [IRR]) with one mismatch, which is indicated by lowercase letters. The nucleotide sequences of DRs and IRs are also given. (C) Physical map of ant cluster which is franked by IS6100 elements along a base pair scale.

Figure 3. Electrophoresis results of RT-PCR.

carAaBaBb (lane 1), carBbC (lane 2), carCAc (lane 3), fdr (lane 4), antAa (lane 5) and antAcAdAb (lane 6). Samples containing no reverse transcriptase (No RT) are also shown.

Figure 4. Biotransformation of substrates and accumulation of products.

(A) Biotransformation of carbazole (-▪-) and accumulation of 2′-aminobiphenyl-2,3-diol (-▾-) by E. coli DH5α harboring pUcarAaAcfdr. (B) Biotransformation of anthranilate (-♦-) and accumulation of catechol (-▴-) by E. coli DH5α harboring pUantAcdba. E. coli DH5α harboring pUC19 (-□- and -◊-) served as controls. The initial concentrations of carbazole and anthranilate were 2 mM and 1 mM, respectively. Values are means of three replicates ± SD.

Figure 5. Comparative analyses of car and ant genes from Sphingomonas sp. XLDN2-5 and other strains.

(A) Comparative analysis of car gene cluster from Sphingomonas sp. XLDN2-5 and related strains. The car genes in the three sphingomonads are more than 99% identity to each other; however, only show 60% identity to that from Pseudomonas sp. CA10. (B) Comparative analysis of ant gene cluster from Sphingomonas sp. XLDN2-5 and related strains. The ant genes in strain XLDN2-5 and KA1 are more than 99% identity to each other; however, only show 40–76% identity to that from Burkholderia cepacia DPO1.