Carbon Utilzation Of CRP And Transcription Level Of Mannitol Fermentation In Vibrio Cholerae

Objectives To study the cAMP receptor protein (CRP) in the regulation to homoserinedehydrogenase isozymes (HDH) in V. cholerae. CRP receptor protein plays an importantrole in the regulation of multiple pathways, especially in the metabolism of carbon source.Mannitol mtl operon in V. cholerae is regulated by CRP-cAMP. In V. cholerae of O1El Torbiotype, there is a significant difference in the transcription level of mannitol fermentationmtA and mtlD between cholera toxin produced strains (mainly slow fermentation strains)and non cholera toxin produced strains (mostly fast fermentation strains). To study thedifference, comparing the universality difference in transcription capacity and diversity ofprotein expression of mtlCBA gene in those two strains in mannitol fermentationexperiment, it’s necessary to explore the mechanism about mannitol fermentation speed ofthese two strains.Methods Using overlap PCR, the possible CRP binding site in the promoter of HDHgene was replaced by consensus sequence. The transcriptional reporter plasmids containingthe original promoter or changed promoter were constructed, and these plasmids wereintroduced into the V. cholerae wild type strain C7258and CRP mutant strain WL7258byconjugation. The transcription level was compared by monitoring luminescence value inC7258and WL7258. Pick up3time points (0.5h,2h,4h) in the process of mannitolfermentation in24fast fermentation of strains and24slow fermentation of strains fromdifferent years, using real-time PCR to compare the transcription level of mtlCBA gene inthose two strains. Moreover, exact of total protein to detect the protein expression level ofmtlD in those two strains by western blot.Results Our data shows that luminescence value of plasmid which containing theoriginal promoter was higher in C7258than in WL7258. After the promoter was changed,luminescence values of plasmid were raised in two strains, especially in C7258.We foundthe transcription level of mtlCBA in early stage of fast fermentation of strain is higher thanslow fermentation of during mannitol fermentation in the randomly picked24fastfermentation of strains and24slow fermentation of strains, and the protein expressionlevel of mtlD as well.Conclusions The result demonstrated that CRP regulates the transcription of HDH in V. cholerae, but not the only regulation factor. The PTS transcriptional differences andpromoter activities in mannitol fermentation are the reason of the fermentation differenceof epidemic strain and no epidemic strain.