Role of BvgA phosphorylation and DNA binding affinity in control of Bvg-mediated phenotypic phase transition in Bordetella pertussis.
To investigate the mechanism by which the Bordetella BvgAS phosphorelay controls expression of at least three distinct phenotypic phases, we isolated and characterized two B. pertussis mutants that were able to express Bvg- and Bvg(i) phase phenotypes but not Bvg+ phase phenotypes. In both cases, the mutant phenotype was due to a single nucleotide change in bvgA resulting in a single amino acid substitution in BvgA. In vitro phosphorylation assays showed that BvgA containing the T194M substitution was significantly impaired in its ability to use either BvgS or acetyl phosphate as a substrate for phosphorylation. Binding studies indicated that this mutant protein was able to bind an oligonucleotide containing a high-affinity BvgA binding site in a manner similar to wild-type BvgA, but was defective for binding the fhaB promoter in the absence of RNA polymerase (RNAP). By contrast, BvgA containing the R152H substitution had wild-type phosphorylation properties but was severely defective in its ability to bind either the high-affinity BvgA binding site-containing oligonucleotide or the fhaB promoter by itself. Both mutant BvgA proteins were able to bind the fhaB promoter in the presence of RNAP however, demonstrating the profound effect that RNAP has on stabilizing the ternary complexes between promoter DNA, BvgA and RNAP. Our results are consistent with the hypothesis that BvgAS controls expression of multiple phenotypic phases by adjusting the intracellular concentration of BvgA-P and they demonstrate the additive nature of BvgA binding site affinity and protein-protein interactions at different Bvg-regulated promoters.
Jones
Allison M
AM
Department of Molecular, Cellular and Developmental Biology, University of California, Santa Barbara, CA 93109-9610, USA.
Boucher
Philip E
PE
Williams
Corinne L
CL
Stibitz
Scott
S
Cotter
Peggy A
PA
D019943:Amino Acid Substitution; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001886:Bordetella pertussis / Q000235:genetics / Q000502:physiology*; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism; D015964:Gene Expression Regulation, Bacterial; D046911:Macromolecular Substances; D008958:Models, Molecular; D010641:Phenotype; D010766:Phosphorylation; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D020836:Protein Structure, Quaternary; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction / Q000502:physiology; D014157:Transcription Factors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D001426:Bacterial Proteins; C065601:BvgA protein, Bacteria; D004268:DNA-Binding Proteins; D046911:Macromolecular Substances; D011993:Recombinant Fusion Proteins; D014157:Transcription Factors; D012321:DNA-Directed RNA Polymerases