Direct interaction between quorum-sensing regulator SmcR and RNA polymerase is mediated by integration host factor to activate vvpE encoding elastase in Vibrio vulnificus.
It has been suggested that quorum sensing is an important signal transduction system regulating the expression of numerous virulence genes in bacterial pathogens. We previously revealed that SmcR, a LuxR homologue of Vibrio vulnificus, activates promoter S, an RpoS-dependent promoter of vvpE encoding a potential virulence factor elastase and binds in vitro to a binding site centered at -196.5. In this study, chromatin immunoprecipitation assays and promoter deletion analyses demonstrated that SmcR binds to the vvpE regulatory region in vivo and directly interacts with RNAP for activation of the vvpE expression. A search for regulatory genes involved in the regulation of elastase production singled out ihfA, which encodes for a subunit of integration host factor (IHF). Levels of both elastase activity and vvpE transcript decreased significantly as a result of inactivation of ihfA, and primer extension analyses demonstrated that IHF regulates the vvpE transcription by activating PS. Direct binding of IHF to the two distinct binding sites centered at -174 and -131, respectively, was determined using an electrophoretic mobility shift assay and a DNase I protection assay. Chromatin immunoprecipitation assays revealed that the interaction of SmcR with RNAP in vivo was mediated by IHF. Collectively, the results proposed a model whereby IHF positions SmcR to contact RNAP by looping the vvpE regulatory DNA, thus allowing precise control of the expression level of VvpE during the pathogenesis of V. vulnificus.
Jeong
Hye Sook
HS
National Research Laboratory of Molecular Microbiology and Toxicology, Department of Agricultural Biotechnology, Center for Agricultural Biomaterials, and Research Institute for Agriculture and Life Sciences, Seoul National University, Seoul 151-921, South Korea.
Kim
Seung Min
SM
National Research Laboratory of Molecular Microbiology and Toxicology, Department of Agricultural Biotechnology, Center for Agricultural Biomaterials, and Research Institute for Agriculture and Life Sciences, Seoul National University, Seoul 151-921, South Korea.
Lim
Moon Sub
MS
National Research Laboratory of Molecular Microbiology and Toxicology, Department of Agricultural Biotechnology, Center for Agricultural Biomaterials, and Research Institute for Agriculture and Life Sciences, Seoul National University, Seoul 151-921, South Korea.
Kim
Kun Soo
KS
Department of Life Science and Interdisciplinary Program of Integrated Biotechnology, Sogang University, Seoul 121-742, South Korea.
Choi
Sang Ho
SH
National Research Laboratory of Molecular Microbiology and Toxicology, Department of Agricultural Biotechnology, Center for Agricultural Biomaterials, and Research Institute for Agriculture and Life Sciences, Seoul National University, Seoul 151-921, South Korea. Electronic address: choish@snu.ac.kr.
D001426:Bacterial Proteins / Q000378:metabolism*; D001483:Base Sequence; D001665:Binding Sites; D047369:Chromatin Immunoprecipitation; D003001:Cloning, Molecular; D017931:DNA Primers; D012321:DNA-Directed RNA Polymerases / Q000235:genetics / Q000378:metabolism*; D034982:Integration Host Factors / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D010196:Pancreatic Elastase / Q000378:metabolism; D010957:Plasmids / Q000378:metabolism; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D015534:Trans-Activators / Q000378:metabolism*; D014158:Transcription, Genetic; D041261:Vibrio vulnificus / Q000201:enzymology*
D001426:Bacterial Proteins; D017931:DNA Primers; D034982:Integration Host Factors; D015534:Trans-Activators; C410093:smcR protein, Vibrio vulnificus; D012321:DNA-Directed RNA Polymerases; D010196:Pancreatic Elastase