Bacillus anthracis is the causative agent of anthrax and a biodefense category A priority pathogen. Wild type B. anthracis carries two plasmids, pXO1 and pXO2, both of which are absolutely required for production of disease. pXO1 encodes the components of the anthrax toxin and pXO2 encodes the genes for production of the poly-D-glutamic acid capsule, both of which are essential virulence factors. Most of the research on these plasmids has focused on the toxin and capsule genes and their regulation, but little is known about the biology of the plasmids themselves. The experiments described in this proposal are designed to identify and characterize small RNAs that may be involved in regulation of pXO1 plasmid-encoded functions. Emphasis will initially focus on plasmid maintenance functions, including those required for replication and stable inheritance, since small RNAs frequently regulate these functions in other systems. However, work in other bacteria has clearly shown that RNA-mediated regulation has been underappreciated in prokaryotic systems and probably plays a much more important role in regulatory circuits than previously imagined. Because of this, procedures developed to examine the role of small RNAs in controlling plasmid maintenance will be expanded to screen the entire plasmid for RNAs that may mediate other regulatory aspects of plasmid function, including toxin production. After potential regulatory RNAs have been located, their targets will be identified and their role in regulating those targets will be determined. Detailed studies will be conducted to determine the molecular mechanisms of regulation of these small RNAs. It is hoped that by identifying RNA controlled targets of plasmid function, compounds can be developed to interfere with these functions and thereby "detoxify" the organism. Furthermore, determination of the rules of RNA regulation in this system will pave the way for further study of regulatory RNAs that may be encoded on pXO2 or the B. anthracis chromsome.