Studys On The Different Regulation Of T6SS4 And ZnuABC By Zur In Yersinia Pseudotuberculosis

Zinc is essential for maintaining biological life,and the intracellular zinc homeostasis plays an important role in microbial growth.Zinc responsive regulator Zur?Zinc Uptake Regulator?is a major regulator for zinc uptake system.In this study,we revealed the regulation of Zur on Znu ABC?the classic zinc uptake system?and a new zinc obtained system type ? secretion system?T6SS?.And further explored its physiological function preliminarily.The results are as follows:1.The results showed that Zur can promote T6SS4 and inhibited znuA expression on both transcriptional level and post-transcriptional level through ?-galactosidase activity assay and Real-Time fluorescence quantitative PCR?qRT-PCR?experiments.The electrophoretic mobility shift assay?EMSA?showed that Zur can directly and specificly bind to T6SS4 and znuA promoter,and the DNase I footprinting confirmed the Zur binding sequence in T6SS4 promoter.Then predicted the conserved sites in znuA promoter region,the conserved binding region Zur-Box3.By comparing the expression of T6SS4 and znuA along with growth phase in,we found that T6SS4 expression increased significantly with growth phase in wild-type strain.On the contrary,the expression of znuA increased slowly in wild-type strain.4.By comparing the expression of T6SS4 and znuA under different culture conditions,we found that the expression of T6SS4 in M9 medium was significantly higher than in YLB medium,and the expression of znuA in YLB medium was higher than that in M9 medium,but the difference was not significant.5.By comparing the expression of T6SS4 and znuA at different concentrations of zinc ions,the results showed that the T6SS4 and znuA expression was induced by low concentration of zinc,whereas,was inhibited by high concentration of zinc.Zur positively regulate T6SS4 expression under all detected conditions.mutant strain,but the expression of znuA was almost not regulated by Zur under low zinc conditions.EMSA assay showed that Zur could successfully bind to T6SS4 promoter in a certain range of low zinc concentration.However,the binding ability of Zur to znuA promoter became weaker.7.By comparing the expression of T6SS4 and znuA in wild-type strain and zur mutant at different concentrations of H₂O₂,the results showed that the T6SS4 expression was induced by H₂O₂,but the expression of T6SS4 was not mediated by Zur.For znuA expression,the induced level in zur mutant strain was higher than in wild-type strain,indicating that zur could inhibit the induced expression of znuA by H₂O₂.8.By analyzing the effects of T6SS4 and znuABC on bacterial survival rate under various stress conditions,we found that both T6SS4 and znuABC were contributed to stress resistance,and the role of T6SS4 was more important than znuABC.In summary,in this research,we found that Zur could differently regulate two zinc-uptake systems,which was depended on the growth phase,nutrition,H₂O₂ and the concentration of zinc.Besides,two systems contributed differently to stress resistance.The results were important to study the zinc homeostasis in Yersinia pseudotuberculosis.