The Mechanism Study Of Regulation Of Natural Competence By CAMP Receptor Protein CRP In Vibrio Cholerae And Vibrio Fluvialis

The Bacterial species can take up environmental DNA and incorporate the exogenous genetic elements into chromosomes by homologous recombination under natural conditions. This phenomenon, referred to as natural transformation, is a prime example of horizontal gene transfer. By natural transformation, bacteria are able to acquire new characters that can enhance their fitness in different environments. Numerous studies have revealed that natural transformation is prevalent among members of the Vibrionaceae family, which plays an important role in Vibrio genetic diversity and evolutional process.Competence is a prerequisite for natural transformation in Bacteria. TfoX (also known as SXY), coded by VC1153, is an integral regulator of natural transformation in V. cholerae. TfoX induces the chitin-dependent natural transformation in V. cholerae. In the present of chitin, TfoX was shown to positively regulate the expression of four transformation machinery genes including pilQ, pilA, comEA and ComEC, whose gene products played essential roles in the uptake and transport of exogenous DNA. The adenosine 3',5'cyclic phosphate (cAMP) receptor protein (CRP) is an important regulator in global gene control in prokaryotes, and plays a key role in forming natural competence in V. cholerae. Literature reports that CRP can promote the expression of pilA, comEA and chiA-1, but the exact mechanism remains unclear. In this study, We explored the regulatory mechanism of CRP on Vibrionaceae natural competence by using V. cholerae and V.fluvialis as model organisms.Based on the sequence information of identified promoter region of tfoXVC, we predicted a possible CRP binding site in the tfoXVC promoter region by Virtual Foot print and Regulatory Sequence Analysis Tools (RSAT). By comparing tfoXVC mRNA levels and LacZ activity of tfoXVC-lacZ transcriptional reporter system under widetype and CRP deletion background of V. cholerae, we showed that CRP transcriptionally regulates competence regulator, TfoXVC. Electrophoretic mobility shift assays (EMSA) experimentally demonstrated that this regulation is achieved through the direct binding of cAMP-CRP complex to the predicted CRP binding site of tfoXVC promoter region. Results of natural transformation experiments show that transformation frequencies of ?CRP and ?TfoXVC were significantly lower than that of wildtype strain. Consistently, the mRNA expression levels of competence genes, including chiA-1, comEA, pilB and pilM were down-regulated in ?CRP, ?TfoXVC and CRPTfoXVC double mutant compared with the wildtype strain of V. choleraeThe conservative of the regulation mechanism of cAMP-CRP complex on the tfoX expression, we conducted verification in new emerging foodborne pathogen Vibrio fluvialis. Sequence alignments have showed that the identities of the nucleotides and amino acids between TfoXVF and TfoXVC were 75.21% and 80.69%, respectively. Firstly, we identified the transcriptional start site (TSS) of tfoXVF by 5'RACE-PCR, and the TSS is located 106bp upstream of the tfoXVF open reading frame. According to TSS, we speculated the-10 region (TATGAT),-35 region (TCCGGA) and SD sequence (GGGA) of tfoXVF promoter. Sequence analysis of the promoter region also indicates the presence of CRP binding sites. tfoXVF mRNA levels, reporter gene activity of tfoXVF-lux promoter and gel shift experiment confirmed that cAMP-CRP complex transcriptionally regulates the tfoXVF expression through directly binding to the tfoXVF promoter region, as same as in V. cholerae. Further sequence alignment and bioinformatics analysis showed that this regulatory mechanism was conserved among the members of Vibrio, such as V. vulnificus, V. Vibrio furnissii, V. anguillarum,etc. Similar cAMP-CRP binding sites were found in the promoter region of each TfoX homolog.In addition, we conducted pilot investigation of the function of VC1152. VC1152 and tfoXVC are diversely transcribed and share the common intergenic region/promoter sequences on the chromosome, as well as the predicted CRP binding sites. Domain analysis showed that VC1152 contains HD-domain/PDEase-1. HD-domain is a conserved protein domain and found in a superfamily of enzymes with a predicted or known phosphohydrolase activity. These proteins appear to be involved in bacterial nucleic acid metabolism, signal transduction or other functions regulation. In this study, we investigated the possibility of VC1152 participating in natural competence and the regulating relationship between VC1152 and CRP. Firstly, we identified the TSS 8 bp upstream of the putative TTG start codon of VC1152 ORF by 5'RACE. Analysis of VC1152 mRNA level and activity of promoter reporter gene indicate that CRP has a positive regulatory role on VC1152. In subsequent natural transformation experiments, transformation frequency of ?VC1152 was significantly lower than that of wild type strain. Consistent to the impaired transformation phenotype, the mRNA expression levels of competence related genes, including chiA-1, pilB and comEA were downregulated in VC1152 mutant compared to the wild-type. In summary, our results provides preliminary tips that VC1152 may be involved in natural competence of V. cholerae, the exact mechanism remains to be determined.