The Molecular Mechanism Of Carbazole Degradation By A Sphingobium Sp. Strain

The genus Sphingomonas was established by Yabuuchi et a1. in 1990. Due to its unique ecological distribution and metabolic features, it has caused environmental microbial scholars' attention. These properties make Sphingomonas in environmental pollution control and biological technology has wide application prospects. But because of scabbard Sphingomonas understanding of late, the research also stays in the primary stage. In this study, we identified a carbzole-degragrading bacterium and investigated its degradation characteristics. An alignment of the DNA sequences of the 16S rDNA of the bacterium with DNA sequences, available in GenBank database indicates that strain XLDN2-5 is most closely related to Sphingobium yanoikuyae DSM 7462 (99% homology). Whole cell fatty acid analysis also indicates that closest relative of XLDN2-5 is Sphingobium yanoikuyae DSM 7462. Based on these results, strain XLDN2-5 was identified as Sphingobium yanoikuyae?Benzothiophenes is a toxic and relatively recalcitrant fraction of coal tar creosote. We investigated cometabolic transformation of benzothiophenes by a carbazole-degrader strain XLDN2-5, which was not able to grow on benzothiophenes as the sole carbon source. Among the benzothiophenes tested, benzothiophene, 2-methylbenzothiophene and 5-methylbenzothiophene could be cometaboliclly converted. For 3-methylbenzothiophene, a total inhibition of growth on carbazole was observed. The common transformation products for benzothiophene, 2-methylbenzothiophene, and 5-methylbenzothiophene are the corresponding sulfoxides and sulfones. For benzothiophene, several high-molecular-weight sulfur-containing aromatic compounds, including benzo[b]naphtho[1,2-d]thiophene, benzo[b]naphtho[1,2-d]thiophene-7-oxide, 6a,11b-dihydrobenzo[b]naphtho[1,2-d]thiophene, 6a,11b-dihydrobenzo[b]naphtho[1,2-d]thiophene-7-oxide, and a new product 6,12-epithiobenzo[b]naphtho[1,2-d]thiophene, were also detected by gas chromatography-mass spectrometry. The data suggested that it concerned a Diels-Alder reaction. GC-FID investigations showed that about 17% of benzothiophene was transformed to benzo[b]naphtho[1,2-d]thiophene. Aerobic transformation of benzothiophenes to sulfoxides and sulfones can reduce the toxicity and facilitate the biodegradation of benzothiophenes. However, the more persistent products such as benzo[b]naphtho[1,2-d]thiophene in the environment might have profound implications for the regulation of these compounds.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. The genes encoding the enzymes responsible for the degradation of carbazole to catechol via anthranilate were cloned, sequenced, and characterized from a carbazole-degrading 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. 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.The microbial degradation of carbazole is of interest for catabolism of N-heterocyclic aromatic compounds. We have characterized a gene cluster from Sphingobium yanoikuyae XLDN2-5 responsible for the aerobic catechol degradation via the meta-cleavage pathway. This gene cluster, organized as carLM-catS-carJK-catR-carDEFGHIYX, is composed by 14 genes, i.e., 12 enzyme-encoding genes distributed in three putative operons, carDEFGHIYX, carJK, and carLM, which were separated by two oppositely oriented regulator genes, carR and carS. The CatR and CatS proteins are members of the GntR and IclR families, respectively, of transcriptional regulators. The amino acids of the meta-pathway enzymes of strain XLDN2-5 show significant but limited homology to that of pseudomonads origin, but share much lower levels of sequence similarity with that of sphingomonads origin. The order of the genes in meta-pathway of strain XLDN2-5 is remarkably different from that of plasmid-borne xyl, dmp, nah, and bph pathways, and surprisingly from that of sphingomonads. Thus, the sequencing and characterization of genes from strain XLDN2-5 show a novel type organization of the catechol meta-pathway genes.Finally, whole genome of XLDN2-5 was sequenced. After annotation, we found that the strains contain a lot of insert sequences movable components. Carbazole biodegradation pathway of genes is in a plasmid, which was named pCar4. In addition, through comparing proteinomics, we found that carbazole biodegradation related proteins expressed when carbazole existed, but not glucose.