| Objective: To determine the function of mannitol transportation of Phosphoenolpyruvate- dependent phosphotransferase systems (PTS) in O1 El Tor Vibrio cholerae (V. cholerae) and compare the generality of the mtlCBA genes transcription difference between epidemic (rapid-ferment) and non-epidemic (slow-ferment) strains which have been defined as two-kind strains and showed different fermatation rates in both sorbitol and mannitol ferment tests. To identify the role of postutulated regulating gene mtlR in transcription of mtl operon, and explore why epidemic and non-epidemic strains of V. cholerae showed different fermenting-speed in mannitol-containing broth.Methods: We analyzed the basic structure of PTS gene cluster and constructed mutants with in-frame deletion and revertants in mtlCBA and mtlR, and determined the functions of PTS genes in V. cholerae. Growth curves of 10 slow-ferment (epidemic) and 10 rapid-ferment (non-epidemic) strains were also detected by mannitol fermentation test, and mtlCBA transcription levels of these strains were determined by quantitative real-time reverse-transcriptional PCR Moreover, Rluc report gene system was established to measure the activities of different predicted PTS promoters. In addition, transcription regulator His-MtlR was expressed and purified.Results: Although no influence on growth velocity in 0.2% mannitol-containing broth was found, the growth of both mtlCBA deletion mutants of N16961 (slow-ferment strain) and 93097 (rapid-ferment strain) in M9 medium supplemented with 0.2% mannitol as the sole carbon source were restrained, and the mannitol ferment tests indicated no positive reaction. The mtlR deletion mutants of N16961 (slow-ferment strain) and 93097 (rapid-ferment strain) cultured in M9 medium and mannitol-containing broth containing 0.2% mannitol showed a more quick increase in cell numbers and a faster positive reaction compared with wild strains respectively. The data of transcription levels of mtlCBA genes determined by quantitative realtime reverse-transcriptional PCR suggested that the transcription levels of mtlCBA of the rapid-ferment strains were much more higher at the 1st and 2nd hour and lower at the 4th and 8th hour than that of the slow-ferment strains. After 4 hours, the non-epidemic strains became positive in mannitol ferment test, the growth density of most fast-ferment strains were higher than of slow-ferment strains, except that some were lower all the time, however, the transcriptional level of mtlCBA mRNA of rapid-ferment strains remained higher compared with slow-ferment strains. In contrast, when slow-ferment strains got positive (at the 8th hour in the test), the growth density of them were higher than of fast-ferment strains. Meanwhile, the results refered to the activity of predicted mtlCBA promoters between two-kind strains in the test by using the Rluc as a report gene, recommended that the activity of promoter of fast-ferment strains always much higher than of slow-ferment strains. In order to further study the mechanism of transcription regulation, we expressed and purified MtlR tagged with His6 at the N terminus.Conclusion: The mtlCBA in mannitol PTS encoded a protein mtlEII, participating in the translocation-phosphorylation of substrate. The transcription repressor gene, mtlR, of mannitol PTS operon in V. cholerae is not the immediate cause of the ferment difference between the rapid-ferment and slow-ferment strains. The difference of mannitol PTS operon transcription level and the activity of promoter between two-kind strains resulted in the different mannitol fermentation rate in the test. This study also indicated that there were probably other regulational factors effect on the mtl promoter, in which sequence were identical. The different effect on transcription resulted in the difference of mannitol ferment between the toxigenic and non-toxigenic V. cholerae strains. |
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