产甲酸草酸杆菌细胞数群体感应系统基因hdtS克隆和功能分析

Cloning and identification of hdtS gene from Oxalobacter formigenes encoding an acylhomoserine lactone synthase of quorum-sensing system

为鉴定产甲酸草酸杆菌(Oxalobacter formigenes)是否具有细胞数群体感应(QS)系统,将费氏弧菌(Vibrio fischeri)和荧光假单胞菌(Pseudomonas fluorences)QS系统相关基因与产甲酸草酸杆菌(O.formigenes)基因组序列进行比对,初步确定hdtS和AGPA为QS系统候选基因。通过PCR和T-A克隆方法分别克隆以上两个基因并将其构建成表达载体pET19-hdtS和pET19-AGPA,借助SDS-PAGE凝胶电泳证实以上两个基因均在大肠杆菌(Escherichia coli)BL21(DE3)中高效表达,采用平板菌株报告法鉴定hdtS具有酰基高丝氨酸内酯(AHL)合成酶的功能,而AGPA则不具有该功能。这是国内外首次报道产甲酸草酸杆菌(O.formigenes)中QS系统有关基因,对该菌株QS系统了解及分析影响该菌株在人肠道内定殖因素具有重要意义。

To identify the quorum-sensing system （QS） genes in Oxalobacter formigenes, QS-related genes from Vibrio fischeri and Pseudomonas fluorences were aligned with the genome sequence of O. formigenes. The genes hdtS and AGPA were chosen as the candidate QS-related genes, cloned by PCR into the vector pEASY-T3, and constructed into pET19-pp-histag. The resulting expression vectors designated pET19-hdtS and pET19-AGPA were transformed into Escherichia coli BL21 （DE3）, respectively, fol owed by the induction by IPTG. SDS-PAGE showed that the genes hdtS and AGPA were over-expressed in E. coli BL21 （DE3）. The hdtS gene, but AGPA not, was final y established to encode an acylhomoserine lactone （AHL） synthase via an AHL reporter strain, Agrobacterium tumefaciens NTIA （pZLR4） through the double-agar diffusion method. To the best of our knowledge, this is the first report on the cloning and identification of the QS-realted gene from O. formigenes, which is very helpful for better understanding of the quorum sensing system of O. formigenes and analysis of impact factors of its colonization in human intestines.