Fis对伤寒沙门菌基因表达的系统调节作用

Systemic regulation of Fis on gene expression in Salmonella enterica serovar Typhi

目的:探讨伤寒沙门菌调节因子fis对基因表达的系统调节作用。方法:用自杀质粒介导的同源重组方法制备伤寒沙门菌fis基因缺陷变异株,用脉冲场凝胶电泳分析fis缺陷变异株的基因组结构。利用伤寒沙门菌全基因组DNA芯片分析技术,比较伤寒沙门菌野生株和fis基因缺陷变异株的基因表达谱差异。用重组载体pBADfis回补fis缺陷变异株,选择部分差异表达基因进行qRT-PCR验证,用半固体平板培养观察野生株、fis缺陷变异株和回补株的动力。结果:fis基因缺陷变异株含有二相鞭毛素编码基因的线性质粒缺失,且动力消失;基因表达谱比较分析结果表明,伤寒沙门菌fis基因缺陷变异株在对数生长期94个基因出现表达差异;qRT-PCR分析所选基因表达的结果与芯片分析结果相符;fis缺陷变异株在回补fis基因后,动力及差异表达基因都获得明显恢复。结论:Fis在伤寒沙门菌中对动力、侵袭及多种代谢相关基因的表达发挥重要调节作用。

Objective： To explore the systemic regulation of factor for inversion stimulation（fis） on gene expression in Salmonella enterica serovar Typhi. Methods： The fis deleted mutant of S. enterica serovar Typhi was prepared by homologous recombination mediated by suicide plasmid; Pulse-field gel electrophoresis was performed to investigate the genome structure of thefts mutant strain. The gene expression profiles of wild-type and fis deleted mutant of S. enterica serovar Typhi incubated at log phase were investigated by S. enterica serovar Typhi genomic DNA microarray analysis. A recombinant plasmid, pBADfis, containing the fis gene with its own promoter was constructed, qRT-PCR was performed to validate the results of microarray in some selected genes. The effect of Fis on the motility phenotype was established by tests on semi-solid agar plates. Results： In the fis deleted mutant, the linear plasmid containing phase 2 flagellin gene fljBA was absent and the motility was also lost; gene expression profiles analysis revealed that expression of 94 genes were induced or decreased in thefts mutant at log phase; expression of the selected genes investigated by qRT-PCR was similar to the result of microarray assay; after complementing fis gene in the fis deleted mutant, the motility and expression of selected genes were obviously restored. Conclusion： Fis may play the central role in coordinating the expression of genes involved in motile, and in invasion or substance metabolism in S. enterica serovar Typhi.