title: Bacillus anthracis virulence regulator AtxA: oligomeric state, function and CO(2) -signalling.
abstract: AtxA, a unique regulatory protein of unknown molecular function, positively controls expression of the major virulence genes of Bacillus anthracis. The 475 amino acid sequence of AtxA reveals DNA binding motifs and regions similar to proteins associated with the phosphoenolpyruvate: carbohydrate phosphotransferase system (PTS). We used strains producing native and functional epitope-tagged AtxA proteins to examine protein-protein interactions in cell lysates and in solutions of purified protein. Co-affinity purification, non-denaturing polyacrylamide gel electrophoresis and bis(maleimido)hexane (BMH) cross-linking experiments revealed AtxA homo-multimers. Dimers were the most abundant species. BMH cross-links available cysteines within 13 Å. To localize interaction sites, six AtxA mutants containing distinct Cys→Ser substitutions were tested for multimerization and cross-linking. All mutants multimerized, but one mutation, C402S, prevented cross-linking. Thus, BMH uses C402 to make the inter-molecular bond between AtxA proteins, but C402 is not required for protein-protein interaction. C402 is in a region bearing amino acid similarity to Enzyme IIB proteins of the PTS. The AtxA EIIB motif may function in protein oligomerization. Finally, cultures grown with elevated CO(2) /bicarbonate exhibited increased AtxA dimer/monomer ratios and increased AtxA activity, relative to cultures grown without added CO(2) /bicarbonate, suggesting that this host-associated signal enhances AtxA function by shifting the dimer/monomer equilibrium towards the dimeric state.
authors.affiliation: Department of Microbiology and Molecular Genetics, The University of Texas - Houston Health Science Center, Medical School, Houston, TX, USA.
authors.forename: Troy G
authors.initials: TG
authors.lastname: Hammerstrom
authors.forename: Jung Hyeob
authors.initials: JH
authors.lastname: Roh
authors.forename: Edward P
authors.initials: EP
authors.lastname: Nikonowicz
authors.forename: Theresa M
authors.initials: TM
authors.lastname: Koehler
mesh_terms: D019943:Amino Acid Substitution; D001408:Bacillus anthracis / Q000472:pathogenicity* / Q000502:physiology; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D002245:Carbon Dioxide / Q000378:metabolism*; D002846:Chromatography, Affinity; D003432:Cross-Linking Reagents / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D015964:Gene Expression Regulation, Bacterial; D006801:Humans; D016297:Mutagenesis, Site-Directed; D050505:Mutant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D025941:Protein Interaction Mapping; D055503:Protein Multimerization; D015534:Trans-Activators / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D014774:Virulence
chemical_list: C082932:ATXA protein, Bacillus anthracis; D001426:Bacterial Proteins; D003432:Cross-Linking Reagents; D050505:Mutant Proteins; D015534:Trans-Activators; D002245:Carbon Dioxide