The research to be performed in this proposal will define a molecular mechanism for capsule expression in Bacillus anthracis. Production of the poly-D-glutamic acid capsule within the host allows the bacterium to evade detection by the immune system facilitating anthrax disease progression. Environmental and culture conditions that promote capsule expression have been defined, however the mechanism leading to expression of the capsule biosynthetic operon are not well understood. The capsule biosynthetic enzymes are encoded by an operon, capBCADE, on plasmid pXO2. Transcription of the operon is induced during growth in elevated atmospheric CO2 and medium containing bicarbonate. The capsule biosynthetic operon is flanked by two genes, acpA and acpB, that encode proteins that positively regulate transcription of the operon. Transcriptional read-through of capBCADE results in expression of acpB, resulting in a positive feedback loop. AtxA, the master virulence regulator in B. anthracis, positively regulates capsule gene transcription via control of acpA. When expressed in high copy or in growth conditions that enhance AtxA activity, AtxA can control capsule operon expression in the absence of acpA and acpB. The activity of AcpA, AcpB, and AtxA on transcription of the capsule operon demonstrates functional similarity between these proteins. In addition to influencing expression of the capsule biosynthetic operon, AtxA has a broad regulon consisting of genes on both virulence plasmids and the chromosome. The regulons of AcpA and AcpB are less well-defined, but are thought to contain only a few genes. The three regulators have similar putative DNA-binding domains at their amino termini, but the basis for their target specificities is unknown. Structure-function studies have identified that AtxA activity is influenced by dimerization and phosphorylation. Aside from phenotypic studies designed to assess capsule expression in acpA- and acpB-null mutants, nothing is known regarding the mechanism of AcpA and AcpB activity. The research proposed here will (1) determine AtxA, AcpA, and AcpB domains associated with target gene specificity and (2) assess multimerization states and phosphorylation sites of AcpA and AcpB and the effects on regulator activity. This study is designed to define discrete roles for three proteins responsible for expression of capsule synthesis genes in B. anthracis. Identifying the role of discrete functional domains and protein-protein interactions required for activity of AtxA, AcpA, and AcpB will highlight novel targets for the inhibition of capsule expression and anthrax pathogenesis.