鼠伤寒沙门氏菌群体感应复苏oxyR基因缺失引起的VBNC状态

VBNC Status Caused by Group Sensing Resuscitation of oxyR Gene Deletion in Salmonella typhimurium

探讨鼠伤寒沙门氏菌oxyR基因缺失株引起的VBNC状态及其与群体感应的关系。运用同源重组的方法构建oxyR基因无痕缺失的鼠伤寒沙门氏菌并检测该菌株对H_2O_2的敏感性;将oxyR基因缺失株和亲本株(WT)涂布或滴于LB固体培养基,观察其是否生长及其浓度依赖性生长情况;用swimming和swarming平板检测oxyR基因缺失株和WT的运动能力;检测固体培养基和液体培养基中沙门氏菌分解H_2O_2的能力。成功构建了oxyR无痕缺失菌株;oxyR基因缺失株在0.1 mmol/L H_2O_2的LB平板上形成的菌苔发生了变形,在1 mmol/L H_2O_2的LB平板上不能生长,而WT均能生长;6×10~6和6×10~5 cfu/mL的WT涂布于LB平板上能长满菌苔,而等量的oxyR缺失株不能生长菌落;不同浓度的WT滴于LB平板均能形成菌苔,而oxyR缺失株仅在OD_(600)≥10^(-1)浓度时才能形成菌苔;oxyR缺失株泳动距离无显著性变化,而群集运动距离显著性大于WT;固体培养的沙门氏菌比液体培养的沙门氏菌有更强的分解H_2O_2的能力。鼠伤寒沙门氏菌的群体感应系统通过调控其群集运动和H_2O_2分解能力来复苏由oxyR基因缺失引起的VBNC状态。

VBNC status caused by oxy R deletion and the relationship with group sensing in Salmonella typhimurium was investigated. Homogenetic combination method was used to construct oxy R gene traceless deletion S. typhimurium strain and check its hydrogen peroxide sensibility. The oxy R deletion strain and parent spread or dropped on LB solid media was observed if their growth depend on their concentration. Swimming and swarming plate were used to check moving ability of oxy R deletion strain and parent strain, and determined the ability of Salmonella to decompose H 2O 2 solid and liquid media. The results oxy R traceless deletion strain was successfully constructed. The bacterial lawn of oxy R deletion strain formed on LB plate with 0.1 mmol/L H 2O 2 was distorted, and cannot grow on LB plate with 1 mmol/L H 2O 2, but the parent strain can grow,while the WT had growth under the same condition. 6×10 6 or 6×10 5 cfu/mL oxy R mutant strain were inoculated onto LB plates and could not grow. Only if its concentration great than or equal to OD 600 0.1, the oxy R deletion strain could form bacterial lawn on LB plates. For the parent strain, it can grow in both cases. The swimming distance of the oxy R deletion strain had no significant change while its swarming distance increased obviously. The H 2O 2 decomposing ability of the strain collected from plate has significant increase than that of the strain collected from broth. Group sensing systems up-regulate the swarming ability and H 2O 2 decomposing ability to resuscitate VBNC caused by oxy R deletion.