Study On Antioxidant Function Of T6SS Regulated By Reut_A2805 In Cupriavidus Necator JMP134

Bacteria type ? secretion system(T6SS)has been widely found in Gram-negative bacteria.It is a newly found secretion system with a variety of functions in recent years.At present,it has been found that T6 SS was involved in a series of life activities such as host invasion,biofilm formation,interaction between bacteria,environmental stress response and quorum sensing.T6 SS with complex functions helps bacteria adapt to changing living conditions.Cupriavidus necator JMP134,as an environmental friendly microbes,was initially regarded as the degradation bacteria of benzene contaminants and PHB synthesis.There are two sets of T6 SS in the Cupriavidus necator JMP134,and there is no report of T6 SS related functions in Cupriavidus necator JMP134 so far.In this study,the first set of type ? secretion system(T6SS1)in Cupriavidus necator JMP134 was used as the research object to study its function and regulation.The specific research results were as follows:1.T6SS1 core conserved gene clpV(reut_A1727)defective mutant in Cupriavidus necator JMP134 was constructed.Comparison differences of survival rates under the stress of acid,oxidant and salt were determined.It was found that the survival rate of wild type under oxidant stress was significantly higher than that of mutant strains.It showed that the type ? secretion system was associated with antioxidant function.2.We found the heterogeneous gene of reut_A2805 in Cupriavidus necator JMP134 by protein sequence alignment.We constructed the mutant of reut_A2805 gene,and compared the survival rate of mutant with wild strain under oxidative stress.The result showed that the survival rate of wild strain was significant higher than the mutant.So,it was speculated that this gene may be involved in the regulation of T6SS1.3.The T6SS1 promoter was analyzed and the possible binding sites of Reut_A2805 was found.The results of EMSA showed that the Reut_A2805 protein in Cupriavidus necator JMP134 could directly bound to the T6SS1 promoter.Promoter activity determination,RT-PCR experiment and Hcp1 secretion test showed that Reut_A2805 positively regulated T6SS1.In summary,the resistance ability of T6SS1 to oxidative stress in Cupriavidus necator JMP134 was studied.It suggested when the Cupriavidus necator JMP134 was subjected to oxidative stress,the regulator of T6SS1,Oxy R,could directly binding to the T6SS1 promoter region,and up-regulated the expression of T6SS1,therefore reduced the damage of oxidation.