MOE Key Laboratory of Microbial Metabolism and School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai, People's Republic of China.
PLoS One. 2010 Apr 2;5(4):e10018. doi: 10.1371/journal.pone.0010018.
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.
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.
咔唑是一种具有二恶英结构的顽固化合物，具有致突变性和毒性。细菌通过招募外源基因来建立一条降解外来化合物的途径来应对外来化合物，这对于它们的适应和生存是必要的。通常，这个过程是由移动遗传元件介导的，如质粒、转座子和插入序列。
从一种咔唑降解的鞘氨醇单胞菌菌株 XLDN2-5 中，克隆、测序并表征了负责将咔唑通过邻氨基苯甲酸降解为儿茶酚的酶的基因。car 基因簇（carRAaBaBbCAc）和 fdr 基因两侧各有两个 IS6100 元件，组织为 IS6100::ISSsp1-ORF1-carRAaBaBbCAc-ORF8-IS6100-fdr-IS6100。咔唑由咔唑 1,9a-双加氧酶（CARDO，CarAaAcFdr）、间裂解酶（CarBaBb）和水解酶（CarC）转化为邻氨基苯甲酸和 2-羟戊二烯二酸。fdr 基因编码一种新的铁氧还蛋白还原酶，其缺失导致 CarAa 和 CarAc 对咔唑的转化活性降低。参与将邻氨基苯甲酸转化为儿茶酚的 ant 基因簇（antRAcAdAbAa）也被夹在两个 IS6100 元件之间，形成 IS6100-antRAcAdAbAa-IS6100。邻氨基苯甲酸 1,2-双加氧酶（ANTDO）由一个还原酶（AntAa）、一个铁氧还蛋白（AntAb）和一个双亚基末端加氧酶（AntAcAd）组成。当 XLDN2-5 菌株暴露于咔唑时，carAaBaBbCAc 基因簇、fdr 和 antRAcAdAbAa 基因簇被诱导。在大肠杆菌中表达 CARDO 和 ANTDO 需要存在天然还原酶才能获得完全的酶活性。
结论/意义：我们预测，IS6100 可能在建立咔唑降解途径中发挥重要作用，使宿主能够适应环境中的新化合物。XLDN2-5 菌株中 car 和 ant 基因的组织是独特的，这显示了基因招募的强烈进化轨迹，由 IS6100 介导，并呈现了一个显著的重排和途径建立的例子。
