AphB influences acid tolerance of Vibrio vulnificus by activating expression of the positive regulator CadC.
A mutant of Vibrio vulnificus that was more sensitive to low pH was screened from a library of mutants constructed by random transposon mutagenesis. By use of a transposon-tagging method, an open reading frame encoding a LysR homologue, AphB, was identified and cloned from V. vulnificus. The deduced amino acid sequence of AphB from V. vulnificus was 80% identical to that reported from V. cholerae. A mutational analysis demonstrated that the gene product of aphB contributes to acid tolerance of V. vulnificus. The lysine decarboxylase activity and cellular level of the cadA transcript were decreased in the aphB mutant, indicating that AphB exerts its effect on the acid tolerance of V. vulnificus by enhancing the expression of cadBA. Western blot analyses demonstrated that the cellular level of CadC, a transcription activator of the cadBA operon, was significantly reduced by aphB mutation, and a primer extension analysis revealed that the cadC promoter (P(cadC)) activity was under the positive control of AphB. A direct interaction between AphB and the P(cadC) DNA was demonstrated by gel mobility shift assays. The AphB binding site mapped by deletion analyses of the P(cadC) regulatory region and confirmed by a DNase I protection assay was centered at the 61.5 bp upstream of the transcription start site. Accordingly, these results demonstrate that AphB and CadC function sequentially in a regulatory cascade to activate cadBA expression and that AphB activates the expression of cadC by directly binding to an upstream region of P(cadC).
Rhee
Jee Eun
JE
Department of Food Science and Technology, School of Agricultural Biotechnology, Center for Agricultural Biomaterials, Seoul National University, Seoul, 151-742, South Korea.
Jeong
Hee Gon
HG
Lee
Jeong Hyun
JH
Choi
Sang Ho
SH
D000222:Adaptation, Physiological / Q000235:genetics; D000900:Anti-Bacterial Agents / Q000494:pharmacology*; D001426:Bacterial Proteins / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D001665:Binding Sites; D015152:Blotting, Northern; D015153:Blotting, Western; D002262:Carboxy-Lyases / Q000096:biosynthesis; D003001:Cloning, Molecular; D018983:DNA Footprinting; D004252:DNA Mutational Analysis; D004251:DNA Transposable Elements / Q000235:genetics; D004269:DNA, Bacterial / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000096:biosynthesis / Q000378:metabolism; D024202:Electrophoretic Mobility Shift Assay; D005798:Genes, Bacterial; D006863:Hydrogen-Ion Concentration; D016254:Mutagenesis, Insertional; D009154:Mutation; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D012329:RNA, Bacterial / Q000032:analysis; D012333:RNA, Messenger / Q000032:analysis; D017386:Sequence Homology, Amino Acid; D015534:Trans-Activators / Q000235:genetics / Q000502:physiology*; D014157:Transcription Factors / Q000235:genetics; D041261:Vibrio vulnificus / Q000187:drug effects* / Q000235:genetics
D000900:Anti-Bacterial Agents; C120327:AphB protein, Vibrio cholerae; D001426:Bacterial Proteins; C080050:CadC protein, Bacteria; D004251:DNA Transposable Elements; D004269:DNA, Bacterial; D004268:DNA-Binding Proteins; D012329:RNA, Bacterial; D012333:RNA, Messenger; D015534:Trans-Activators; D014157:Transcription Factors; C073470:LysR protein, Bacteria; D002262:Carboxy-Lyases; C021520:lysine decarboxylase