Abstract
BackgroundCarbazole 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.FindingsThe 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/SignificanceWe 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.
Highlights
Carbazole is an N-heterocyclic compound that is known to possess mutagenic and toxic activities [1]
A closer look at the left region suggested that an IS6100, exhibiting 100% identity to that of Mycobacterium fortuitum [16], was interrupted by a novel insertion element ISSsp1
E. coli DH5a completely transformed 1 mM anthranilate to catechol in 24 h (Figure 4B), while E. coli DH5a encoding only ferredoxin and oxygenase transformed only 5% of the anthranilate to catechol. These results suggested that the expression of CARDO and ANTDO in E. coli requires the presence of the ancillary reductases for full enzymatic activity
Summary
Carbazole is an N-heterocyclic compound that is known to possess mutagenic and toxic activities [1]. The degradation of carbazole starts with angular dioxygenation in all studied strains so far [2,3,4,5,6,7,8] In this pathway, carbazole is initially attacked at the 1 and 9a positions by carbazole 1,9adioxygenase (CARDO), resulting in the formation of a highly unstable hemiaminal, which gives rise to 29-aminobiphenyl-2,3diol after spontaneous cleavage and rearomatization. The resulting metabolite, anthranilate, is converted to catechol in a single step by anthranilate 1,2-dioxygenase (ANTDO) (Figure 1) [9,10] In this upper carbazole degradation pathway, CARDO is considered as the key enzyme. 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 This process is mediated by mobile genetic elements such as plasmids, transposons, and insertion sequences
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