| Vibrio cholerae is the causative agent of cholera that inhabits marine and estuarine environments. When V. cholerae cells are subjected to stringent conditions they may form viable but not culturable (VBNC) state cells. These bacteria could be recovered from the VBNC state and may cause danger to human. The cholera surveillance system can not detect these bacteria because the V.cholerae in VBNC state fail to grow on the routine bacteriological media.To study the process by which V. cholerae enters the VBNC state cultured in artificial seawater at4℃, we get6bacterial samples of different time points during entering the state together with the reference sample. The RNA intergrety of the seven samples were assessed. The resuts show that small RNA fragment increased with the time, this indicate that protein synthesis was reduced in the VBNC state bacteria. We performed comparative transcriptome analysis of different stage versus control sample. Four-fold changes are used as criteria adjudging whether the gene expression varied or not. We found that the number of down-regulated and silenced genes increasd with the time. Analysis of Gene Ontology (GO), KEGG pathways showed that up-regulated genes are involved major in amino acid metabolism, fatty acid metabolism, Amino sugar and nucleotide sugar metabolism, down-regulated genes are involved in bacterial chemotaxis, oxidative phosphorylation, and ABC transporters.This study also investigate proteomic profiles of V.cholerae between the normal state and VBNC state. Differentially expressed proteins were analyzed for significant down-or up-regulation, which was calculated by twice folds, a total of1523unique proteins were identified, of which there were87up-regulated and239down-regulated proteins. Analysis of GO, KEGG pathways showed that up-regulated proteins are involved in amino acid metabolism, phosphotransferase system (PTS),Amino sugar and nucleotide sugar metabolism, down-regulated proteins are involved in ribonucleoprotein complex, glycolysis/gluconeogenesis, pyrimidine metabolism, purine metabolism. There are50down-regulated proteins was enriched in ribonucleoprotein complex, which is in accordance with transcriptome research.The transcriptom result showed that there are ten contiguous genes from VC0611to VC0620were up-regulated in all the stage. Our results demonstrate that low temperature is the reason for these gene activated.We delete some genes of this operon and comparied the difference between mutant strian and wild type strian entering the VBNC state using plate count method. The result shows that there are no difference between the wild type and mutant strian.Carbohydrate metabolosm involved in V.cholerae entering VBNC state, the cyclic AMP receptor protein (CRP) is an important regulator in carbohydrate sources metabolosm.We found that CRP plays an important role in V. cholera entering VBNC state. To elucidate the CRP regulation cascade and the bacterial response to environmental stimuli,we performed a high-throughput strategy that compared the carbon sourse metabolic differences between the wild-type and CRP gene mutant strains of V. cholerae01El Tor. Twenty four carbon sources metabolism were subgected to CRP regulation, and11of these carbon sources were not previously reported in any bacteria. The genes known to be involved in the metabolism of four of these carbon sources were validated by quantitative RT-PCR. Gel shift experiments confirmed that the utilizations of inosine, adenosine and L-aspartic acid were directly regulated by CRP. This comprehensive analysis of CRP-mediated catabolite control in V. cholerae identifies new candidate carbon sources for future experimental studies.In this study, we used transcriptomic and proteomic approach investigated the gene expression of V.cholerae during entering VBNC state. These research provide significant information in the biology of the V. cholerae, it also provide the basic data for the further study of the VBNC state. |
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