Monitoring analysis of VBNC state of Escherichia coli O157 in soil

许多致病或非致病细菌进入“活的但不可培养的(VBNC)状态”。近年来，大量的研究揭示了细胞在进入和存在这种状态时在生理和形态上发生了什么。本研究的目的是主要通过蛋白质组分析来鉴定与大肠杆菌O157的VBNC状态相关的基因。首次建立了进入VBNC状态的实验条件，即在无菌水中孵育4℃。经56天培养后，可培养细胞数由10^7个/ml降至10^2个/ml，但活细胞数稳定在10~7个/ml，用SDS-PAGE和双向凝胶电泳法比较了培养0d和56d的细胞蛋白质谱，但两者差异无统计学意义。用LC/MS/MS进行蛋白质组分析，共检测到约1000个基因，但未发现它们与VBNC状态的关系。利用“KO集合法”，即在大肠杆菌中对所有基因敲除突变体、VBNC基因进行鉴定。对3800株突变菌株中的480株进行了VBNC状态检测。目前还没有检测到这些基因。

Many pathogenic or non pathogenic bacteria enter the "viable but nonculturable (VBNC) state". In recent years, a great amount of research has revealed what occurs in cells as they enter and exist in this state, physiologically and morphologically. An aim of this study is to identify genes involved in the VBNC state of Escherichia coli O157 by mainly proteome analysis. At the first time, the experimental condition to enter the VBNC state was constructed, that is the incubation in sterile water at 4℃. The culturable cells decreased from 10^7 cells/ml to 10^2 cells/ml during 56 days incubation, but viable cells were constant at 10^7 cells/ml. The protein profiles of cells between VBNC 0 day and 56 days incubation were compared using SDS-PAGE and two dimensional gel electrophoresis, but both profiles were not observed significant difference. From proteome analysis with LC/MS/MS, about 1000 genes were detected, but they could not be found out the relation to VBNC state. Using "KO collection", that is all genes knock-out mutants in E.coli, VBNC genes were tried to identify. For 480 out of 3800 mutant strains, their VBNC states were examined. The genes have not been detected yet.