Study On The Regulation Of The Expression Of The Serotype Transforming Gene RfbT Of Vibrio Cholerae

Vibrio cholerae O1 serogroup includes two major serotypes, Inaba and Ogawa. The rfbT gene, which encodes a transferase responsible for the expression of the Ogawa-specific, determines the serotype variation between Inaba and Ogawa. Under the pressures of environment or human immunity, the mutations, such as site mutation, or base missing,happened in the ORF of rfbT can cause a frame shift,thus producing a truncated RfbT protein which were previously detected in Inaba strains. Most of the researches were based on the function of rfbT, the different type of sequence of serotype conversion, the possibility of conversion in vivo and in vitro, and the conversion phenomenon during Cholera pandemic, but the influencing factor and regulation pathway of rfbT expression has seldom been reported.The aim of our project is to investigate the regulation mechanism and environmental factors that may control the expression of rfbT. Our previous gene-chip study showed that CRP mutant has a lower mRNA level of rfbT, suggesting CRP may play a positive role in rfbT expression. After analyzing the promoter region of rfbT, we predicted there is a CRP-cAMP complex binding sequence in the rfbT promoter region. CRP-cAMP is capable to bind the specific site of the promoter region in target genes and to interact with RNA polymerase to regulate the level of gene transcription. In This study, we proved that rfbT is regulated by CRP-cAMP complex. Firstly, we constructed the mutation strains with the deletion of CRP-cAMP complex formation genes: ?crp, ?cya and ?cpdA. Then,the total RNA was extracted and reverse transcribed. After that, the differences of rfbT transcription level were detected between each deletion mutant with wild-type strain. And finally, the findings were verified through gene complement. The deletions of cya and crp caused the decrease of rfbT mRNA. After manually added cAMP to complement cya, the rfbT mRNA expression was up regulated. Because the expression of cpdA could decrease cAMP, after the deletion of cpdA, the rfbT mRNA expression was increased. The results showed that CRP-cAMP complex can positively regulate rfbT mRNA level.The function of CRP-cAMP complex was further demonstrated by transcriptional fusions test. We integrated the lacZ reporter connected with rfbT promoter to the single copy on the chromosome into strains of different genetic background to analyze the gene activity. By gel shift assay (EMSA), we demonstrated that CRP-cAMP complex can bind to the promoter region of rfbT directly. However,different from the classic 6 bases interval, the CRP-cAMP complex predicting region of rfbT promoter was 7 bases, which may lead to an unstable binding,then causing a smear as a result. After the transformation of the original probe by removing a base from the interval, no matter from 5' or 3', the stability of binding was improved. In addition, the rfbT transcription start sites were identified through 5'RACE. Multiple transcription start sites were discovered, including one previous reported in the literature.Moreover, in addition to the CRP-cAMP complex, we try to find other possible regulation factors that impact rfbT expression. Considering the promoter of rfbT is an AT rich region,we tried to detect the influences of H-NS,Fis or other global regulators.Firstly, we compared the difference of rfbT mRNA level between wild type strains and the relevant gene mutant strains .The rfbT mRNA level was up regulated in the hns mutant strains, which suggested that hns gene may repress the expression of rfbT. The EMSA test showed H-NS can interact with rfbT gene promoter region by binding to the promoter region directly. Fis didn't involve in regulating the expression of rfbT in This study. In addition, we also explore other environmental factors that may affect the expression of rfbT gene such as temperature, pH, salt concentration, or bile extract and intestinal mucin.In This study, we explored the mechanism regulating the expression of Vibrio cholerae Ogawa and Inaba serotype conversion gene: rfbT. Two global regulators, CRP and H-NS,were involved in the regulation of rfbT expression. CRP-cAMP complex is a positive regulatory factor, while H-NS is a negative regulatory factor. Meanwhile, rfbT has multiple transcription start sites. These results suggested that the regulation of rfbT gene should be complex and precise. Whether there are interactions between regulators and the significance of physiological factors still need further studies.