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.