Objective The complex of the cyclic AMP receptor protein （CRP） and cAMP is an important transcriptional regulator of numerous genes in prokaryotes. The transport of mannitol through the phosphotransferase systems （...Objective The complex of the cyclic AMP receptor protein （CRP） and cAMP is an important transcriptional regulator of numerous genes in prokaryotes. The transport of mannitol through the phosphotransferase systems （PTS） is regulated by the CRP-cAMP complex. The aim of the study is to investigate how the CRP-cAMP complex acting on the mannitol PTS operon mtl of the Vibrio cholerae El Tot biotype. Methods The crp mutant strain was generated by homologous recombination to assess the need of CRP to activate the mannitol PTS operon of V. choleroe El Tor. Electrophoretic mobility shift assays （EMSA） and the reporter plasmid pBBRlux were used to confirm the role that the CRP-cAMP complex playing on the mannitol PTS operon intl. Results In this study, we confirmed that CRP is strictly needed for the activation of the mtl operon. We further experimentally identified five CRP binding sites within the promoter region upstream of the mannitol PTS operon mtl of the Vibrio cholerae El Tor biotype and found that these sites display different affinities for CRP and provide different contributions to the activation of the operon. Conclusion The five binding sites collectively confer the strong activation of mannitol transfer by CRP in V. choleroe, indicating an elaborate and subtle CRP activation mechanism.展开更多
基金supported by NSFC key project grants 30830008 and 81171640
文摘Objective The complex of the cyclic AMP receptor protein （CRP） and cAMP is an important transcriptional regulator of numerous genes in prokaryotes. The transport of mannitol through the phosphotransferase systems （PTS） is regulated by the CRP-cAMP complex. The aim of the study is to investigate how the CRP-cAMP complex acting on the mannitol PTS operon mtl of the Vibrio cholerae El Tot biotype. Methods The crp mutant strain was generated by homologous recombination to assess the need of CRP to activate the mannitol PTS operon of V. choleroe El Tor. Electrophoretic mobility shift assays （EMSA） and the reporter plasmid pBBRlux were used to confirm the role that the CRP-cAMP complex playing on the mannitol PTS operon intl. Results In this study, we confirmed that CRP is strictly needed for the activation of the mtl operon. We further experimentally identified five CRP binding sites within the promoter region upstream of the mannitol PTS operon mtl of the Vibrio cholerae El Tor biotype and found that these sites display different affinities for CRP and provide different contributions to the activation of the operon. Conclusion The five binding sites collectively confer the strong activation of mannitol transfer by CRP in V. choleroe, indicating an elaborate and subtle CRP activation mechanism.
