The Acid Survival Mechanism Study Of Arginine-dependent Acid Resistant System In Yersinia Pseudotuberculosis

The arginine-dependent acid resistance system(AR3) is important for pathogenic enterobacteria, which is composed of arginine decarboxylase(AdiA) and antiporter(AdiC). This system depends on AdiA utilizing arginine to consume intracellular proton and AdiC delivering arginine in and removing decarboxylation product agmatine out of cells for next reaction, which is resulting a pH homeostasis in cytoplasm while the cells are suffering in acid environment.Yersinia pseudotuberculosis(YP) is one of the three major pathogens in Yersinia. As a common pathogenic enterobacteria, YP must pass through the extremely acidity of gastrointestinal tract in mammal to establish successful symptoms. It will cause mesenteric lymphadenitis, chronic diarrhea or sometimes sepsis once YP infects in human tissues. This study focused on the acid resistance mechanism of Yersinia pseudotuberculosis type III(YPIII) that have completed the whole genome sequence, providing the theoretical basis for the control of infection and spread. This thesis mainly includes the following several aspects:1. Through a brief survey to compare the pH3.5 acid survival in YPIII wild type, ΔrovM and ΔrovM-com cells, we find an increased acid survival rate with presence of arginine in ΔrovM comparing with wild type, which confirmed that the LysR-family regulator RovM is related to the acid resistance of YPIII.2. Through comparing the pH3.5 acid survival in YPIII wild type, ΔadiA and ΔadiA-com cells, we find an increased acid survival rate with additional arginine in wild type but not in ΔadiA; through colorimetric Adi A activity assay, we find a higher activity in wild type comparing with ΔadiA, which confirmed YPIII depends on AR3 system orchestrating a process of internal proton consumption to maintain a pH suitable for cell survival.3. In YPIII wild type, ΔrovM and ΔrovM-com cells,through colorimetric AdiA activity assay, we find a higher activity in ΔrovM comparing with wild type; through constructing lacZ fusions and a qRT-PCR analysis of adiC gene which is critically located in AR3 system chromosomes in YPIII, we find the AR3 expression dramatically increased in ΔrovM, confiming that RovM negatively regulated AR3 system in YPIII.4. Through EMSA between His6-RovM protein and the promoter DNA fragment of AR3 system, we find RovM could directely binds to the AR3 promoter region; through a deletion analysis of the AR3 promoter region with six different length promoter regions fused with a promoterless lacZ reporter in YPIII wild type and ΔrovM, we find the RovM binding site in AR3 system promoter region located in approximately upstream-300 bp region of AR3 system; Through DNase I footprint assay, we finally confimed the specific hindering region of RovM in the AR3 promoter as upstream-236 bp to-309 bp region of AR3 system.In conclusion, our study have revealed that YPIII depends on AR3 system pumping the extra proton out of cell membrane to keep pH homeostasis in cytoplasm responding to acid stress, and the expression of this system is repressed by the lysR-family regulator RovM.