Study Of Mechanism Of Mannitol And Sorbitol Metabolism In Vibrio Cholerae El Tor

Cholera is caused by V. cholerae and it is generally accepted that at least seven distinct pandemics have occured since 1871. In 1961 the seventh and present pandemic began, caused by the O1 E1 Tor biotype. In China, the Phage-biotyping Scheme has been developed to distinguish the O1 E1 Tor biptype strains as "epidemic strains" and "non-epidemic strains", Further studies revealed that all "epidemic strains" were toxigenic and almost "non-epidemic strains" were nontoxigenic. The scheme of Phage-biotype has been used for many years in China and until now, it is also a useful tool for the identification of V. cholerae toxigenic and nontoxigenic strains. Sorbitol fermentation test, one of important tests in the Phage-biotyping scheme, shows different fermentation rate between "epidemic strains" and "non-epidemic strains", therefore are designated slow-fermenting strain and fast-fermenting strain respectively.Firstly, the result obtained from the expression profiles of genes of N16961 and 93097 in 0.2% mannitol fermentation medium revealed that the expression level of the components of the PTS in 93097 was higher than that in N16961, which may cause the different fermentation rate in the two classes of strains. In addition, the result showed that more mannitol was metabolized to produce acid in fast-ferrmenting strain, but more energy was produced by TCA in slow-fermenting strain.We make further analysis of the mannitol-specific PTS in fast-fermenting and slow-fermenting strains. Our previous study has confirmed that mtlA,mtlR and mtlD were the components of mtl operon. The transcription level of mtlA and mtlD in 93097 was higher than that in N16961. In our study, we analyzed the mtl peomoter, and we found that the gene VCA1044 located in the upper part of mtl operon was very important to the activity of mtl promoter. And the activity of mtl promoter from fast-fermenting strain was higher than that in slow-fermenting strain. What's more, the more mannitol was utilized in toxigenic and nontoxigenic strains in which the expression level of mtlA was upregulated. These results could confirm that transcript level of mtl operon was related to the ability of mannitol in fast-fermenting and slow-fermenting strains, which may cause different fermentation rate.As the important regulator in bacteria, cAMP-CRP complex play a key role in metabolism of carbohydrate. So we analyzed the role of cAMP-CRP complex in regulation of manitole-specific PTS. The results of the gene crp and cya deletion mutants fermenting mannitol confirmed that cAMP-CRP complex could regulate positively the metabolism of mannitol. The transcription level of cya in fast-fermenting strain was higher that that in slow-fermrnting strain. What's more, the color in mannitol fermentation medium of fast-fermnting and slow-fermenting strains changed to yellow more earlier after the expression level of cya was upregulated. These results suggested that the regulation of cAMP-CPP complex was different in the two classes of strains. Higher activity of cAMP-CRP complex caused higher transcription level of mtl operon in fast-fermentation strain. We predicted the conservative site in mtl promoter, that was "TGTGA.....TCACA", the result of EMSA confirmend that cAMP-CRP complex could bind to the region.Our previous study indicated that the results of mannitol fermentation were similar to those of sorbitol fermentation of V. cholerae E1 Tor. But there was no sorbitol-specific PTS in V. cholerae, which was different from those other bacteria. Sorbitol was PTS sugar in many other bacteria. The ptsI deletion mutant was constructed and the result of sorbitol fermenting indicated that the mutant could not metabolize sorbitol, which confirmed that sorbitol was also transported by PTS in V. cholerae. What's more, sorbitol could induce the high expression level of mtl operon. Therefore theses results indicated that sorbitol was transported by mannitol-specific PTS in V. cholerae.The study make further analysis of mechanism of mannitol and sorbitol metabolism in V. cholerae, and make clear both that cAMP-CRP complex play a key role in regulation of mannitol and sorbitol metabolism, and sorbitol is transported by manniol-specific PTS as the inducer of mannitol-specific PTS. The characteristic was different from other bacteria. And the study help understand the mechanism of difference of mannitol and sorbitol fermentation rate and metabolism between fast-fermentig and slow-fermenating strains. We also found the different metabolism pathway of mannitol and sorbitol in the two classes of strains. Whether these differences are related to the need of growth and regulation of metabolism? In addition, whether growth of V. cholerae is affected by the level of cAMP? Further study is necessary.