鸡白痢沙门氏菌生物被膜形成相关基因rpoE的鉴定

Identification of rpoE gene associated with biofilm formation of Salmonella pullorum

摘要:

摘要:【目的】通过鸡白痢沙门氏菌基因表达和缺失株生物特性的测定，鉴定其生物被膜形成的相关σ因子。【方法】利用结晶紫染色定量法测定沙门氏菌生物被膜形成能力;通过触酶试验测定rpoS活性，确定rpoS基因依赖性和非依赖性生物被膜形成株;利用建立的荧光定量PCR方法比较rpoS基因非依赖株在指数期和生物被膜形成期6个σ因子的基因表达差异;运用Red同源重组系统构建所鉴定σ因子基因缺失株，并测定野生株和基因缺失株对于环境应激的抵抗力差异。【结果】鸡白痢沙门氏菌S6702能够形成生物被膜，触酶试验阴性，确定S6702为rpoS基因非依赖性生物被膜形成株;荧光定量PCR检测显示，培养4－24 h后S6702中rpoE基因表达量最高;与野生株相比，ΔrpoS缺失株保留了生物被膜形成能力，而ΔrpoE缺失株不能形成生物被膜。rpoS和rpoE基因缺失株对于环境应激的抵抗力均显著降低。【结论】在rpoS基因非依赖性生物被膜形成株中，rpoE基因为参与生物被膜形成调控的σ因子之一，这一发现可用于进一步研究沙门氏菌生物被膜形成的调控机制。

Abstract:

Abstract:［Objective］In order to identify Sigma factor associated with biofilm formation of Salmonella pullorum，we determined the gene expression in the wild type strain and biological characteristics of deletion mutants.［Methods］Biofilm forming ability of S． pullorum strain was detected by crystal violet assay．The rpoS gene-dependent or -independent strain for biofilm formation was determined by catalase test．Real-time PCR was established to compare the expression of six different Sigma factors during biofilm formation．Deletion mutants were constructed using the Red recombination system，and their resistance to environmental stress was determined.［Results］ S. pullorum strain S6702 had strong ability of biofilm formation．The result of catalase test was negative，indicating that S6702 was an rpoS-independent strain for biofilm formation．The expression of rpoE gene was the highest during 4 h and 24 h post-incubation．Compared to wildtype strains，ΔrpoS kept the biofilm-forming ability，whereas ΔrpoE mutant could not produce biofilm．Both mutants with deletion of the rpoS and rpoE genes had reduced resistance to environmental stress.［Conclusion］The rpoE gene was identified as one of biofilm formation associated genes in a S． pullorum rpoS-independent strain．The finding may help to elucidate the regulatory mechanism of Salmonella biofilm formation.