The Mechanism Of Oxygen Sensitivity Of Aphb In Vibrio Cholerae

The disease cholera is caused by Vibrio cholerae which is a member of Gram-negative bacterium. This causative agent has caused seven cholera global pandemics in the history. There are an array of virulence factors in V.cholerae that the primary factors are cholera toxin (CT) and the toxin co-regulated pilus (TCP).The LTTRs comprise one of the largest families of transcriptional regulator in prokaryotes. Members of this family have a similarly sized molecule and a conserved structure, which one with an N-terminal helix-turn-helix domain for DNA-binding and a C-terminal co-inducer-binding domain. The LysR family transcriptional regulators control the expression of an extremely diverse set of genes, and there are many signals affect their activity, which include O2. Many of the regulators involved in response to oxidative stress are basal activators that lose their activity due to oxidative modification of cysteine residues.AphB is a member of the LysR family protein, which plays most important role in virulence regulating. As diverse bacterial species, V.cholerae uses thiol-based regulatory modifications to respond to oxidative stress. We choose Vibrio cholerae AphB regulator as our research object, and find out other mechanism which is important for oxygen sensitivity.We used E.coli mutator strain XL1Red to introduce mutations to the pACYC-aphb plasmid. Then the mutated plasmids were transformed into BW27784strain, which contained a pBBRLux-PtcpP plasmid as the reporter.Approximately7,800colonies were screened, of which had point mutations in the aphB gene. Compared to wild-type AphB, there are four AphB mutants (S267L, M1131, M244I and E213K) had ability to activate the expression of tcpP in the presence of oxygen, and these mutations all located in the C-terminal domain.We used bacterial two-hybrid system to detect protein-protein interaction in vivo to analysis the mechanism that the mutations’sensitivity to oxygen. As a result of this assay, E213K had the ability to form a viable dimer, while the other mutants are not able to. Then we studied the crystal structure of AphB C-terminal domain to detect whether mutant residues triggered a conformational change that changed that stimulates transcription. In this research, we have got the crystal of WT AphB protein, and ones of mutations need to make further research.In a word, the new four positions are important for AphB to response oxygen. In the future research, this result will be the basis for our investigation to understand V.cholerae virulence gene regulation.