高渗条件下RpoE与RpoS调节因子共同影响伤寒沙门菌生长代谢

Metabolism of Salmonella enterica serovar typhi influenced by RpoE and RpoS under hyperosmosis

目的了解RpoE和RpoS调节因子在高渗应激下是否共同调节伤寒沙门菌的代谢生长。方法运用自杀质粒介导的同源重组方法制备伤寒沙门菌rpoS／rpoE双缺失突变株，用PCR观察重组现象；以吸光度值（A。值）为纵坐标，培养时间为横坐标绘制生长曲线，观察细菌在高渗条件下的生存能力，分析其在对数生长早期（4h）和对数生长晚期（12h）的生长差异；利用伤寒沙门菌全基因组芯片分析技术，比较伤寒沙门菌野生株和各基因缺失突变株代谢相关基因在高渗应激下的表达差异，选择部分差异表达基因利用实时定量RT—PCR进一步验证。结果成功制备伤寒沙门菌rpoS／rpoE双缺失突变株。生长曲线分析发现，rpoS缺失突变株在生长4h和12h的A600值分别为0．503±0．018和2．060±0．112，rpoE缺失突变株分别为0．293±0．053和1．933±0．115，rpoS／rpoE双缺失突变株分别为0．051±0．007和0．9634-0．111，均低于野生株的0．725±0．097和2．496±0．171，差异具有统计学意义（P〈0．05）。全基因组芯片筛选到42个受RpoE、RpoS调节的涉及代谢的基因。实时定量PCR结果发现，rpsE、rbsK、nusG、etuB在rpoS缺失突变株中的表达分别为（1．86±0．14）×10^6、（1．37±0．11）×10^6、（2．72±0．58）×10^6、（8．27±1．01）×10^6拷贝／μg，在rpoE缺失突变株中的表达分别为（2．19±0．17）×10^6、（1．51±0．12）×10^6、（2．73-4-0．57）×10^6、（9．63±1．42）×10^6拷贝／μg，与野生株中相应基因表达比较[分别为（1．94±0．10）×10^6、（1．52±0．11）×10^6、（2．39±0．52）×10^6、（10．83±1．52）×10^6拷贝／μg]，差异无统计学意义（P〉0．05）；与rpoS／rpoE双缺失突变株比较[分别为（5．64±0．59）×10^6、（4．17±0．40）×10^6、（9．44±1．22）×10^6、（2．95±0．88）×10^6拷贝／μg]，差异均有统计学意义（P〈0．05）。结论RpoE与RpoS能够影响大量代谢相关基因的表达，共同调控伤寒沙门菌在高渗应激下的代谢生长。

Objective To study the role of RpoE and RpoS on the influence of the metabolism and growth of bacterials under byperosmotic stress. Methods The rpoS/rpoE double deletion mutant of Salmonella enterica serovar typhi （S. typhi） was prepared by homologous recombination through the suicide plasmid mediated. The recombination was visualized by PCR. Growth curves were drawn by using photometric value A600 as the ordinate and cultivation time as abscissa. The survival abilities of bacterials were compared under hyperosmotic stress. Statistical differences of early logarithmic growth stage （4 h ） and laters logarithmic growth stage （ 12 h） were analyzed by one-way ANOVA. The expression difference of metabolism related genes of wild-type and mutant strains of S. Typhi incubated under hyperosmotic stress were investigated by Salmonella genomic DNA microarray. Real-time quantitative PCR （qRT-PCR） was performed to validate the results of microarray assay in some selected genes. Results The rpoS/rpoE double deletionmutant of S. Typhi was successfully generated. The analysis of growth curve showed that the 4-hour and 12-hour A600 values were separately 0. 503 ± 0. 018 and 2. 060 ±0. 112 in rpoS deletion mutant strains, 0. 293±0. 053 and 1. 933 ±0. 115 in rpoE deletion mutant strains,and 0. 051 ±0. 007 and 0. 963 ±0. 111 in rpoS/rpoE double deletion mutant strains; all of which were lower than the values of wild-type strains, who were 0. 725± 0. 097 and 2. 496± 0. 171, respectively. The difference were statistically significant （ P 〈 0. 05 ）. The genomie DNA mieroarray revealed that 42 genes relevant with bacterial metabolism were influenced by RpoE and RpoS. Results of qRT-PCR showed that the expression values of rpsE, rbsK, nusG and etuB in rpoS deletion mutant strains were （ 1.86±0. 14） ×10^6, （ 1. 37 ±0. 11 ） ×10^6, （2. 72 ±0. 58） ×10^6 and （8.27 ± 1.01 ） ×10^6 copies/μg, respectively; while those in rpoE deletion mutant strains were （2. 19 ± 0. 17 ） ×10^6, （ 1.51 ± 0. 12 ） ×10^6, （ 2. 73± 0. 57 ） ×10^6 and （ 9. 63 ± 1.42 ）×10^6 copies/μg, respectively. Compared with the values in wild-type strains, which were separately （ 1.94 ± 0. 10 ） ×10^6 , （ 1.52±0. 11 ） ×10^6, （2. 39 ± 0. 52） ×10^6 and （ 10. 83 ± 1.52） ×10^6 copies/μg, the differences was not statistical significance （ P 〉 0. 05 ）. However, compared with the values in rpoS/rpoE double mutant strains, which were separately （ 5.64 ± 0. 59 ）×10^6, （ 4. 17 ± 0.40 ） ×10^6, （ 9. 44 ±1.22 ） ×10^6 and （ 2. 95 ±0. 88 ） ×10^6 copies/μg, the difference was significant （ P 〈 0. 05 ）. Conclusion RpoE and RpoS could influence the expression of lots of metabolism genes. Together, they regulated the metabolism and growth of S. Typhi under hyperosmotic stress.