Bacillus anthracis is a gram-positive bacterium that causes Anthrax. Two megaplasmids pXO1 and pXO2 in B. anthracis have been shown to encode for the virulence factors of this bacteria. Biosynthesis of the anthrax toxin requires the presence of the pXO1 plasmid (110 Mda), since it contains the genes encoding for the proteins protective antigen, lethal factor and edema factor. The other plasmid pXO2 (60 Mda) contains the genes capB, capC, capA and dep that are required for the synthesis of poly-D-glutamic acid capsule. Both of these plasmids are required for the full virulence of B. anthracis. In spite of the importance of these two plasmids for the virulence, many of the genes in these plasmids are yet to be studied in detail. Recently, the sequence and organization of the plasmid pXO1 has been reported by Okinaka et al and has been shown to contain a total of 143 ORFs. Functions for the proteins encoded by 35 of these ORFs have been assigned based on their similarities to proteins from other organisms. All the toxin genes in the pXO1 plasmid are contained in a 44.8-kb region designated as pathogenecity island (PAI). Interestingly, ORFs 93, 94 and 95 which encode for proteins with sequence similarity to hyaluronic acid synthetase (hasA), UDP-glucose-pyrophosphorylase (hasC) and UDP-glucose-dehydrogenase (hasB) of Streptococcus are present immediately adjacent to the PAI. Hyaluronic acid is capsular polysaccharide and a virulence factor for Streptococcus pyogenes and Pasteurella multocida. There is no precedence for the presence of a polysaccharide capsule in B. anthracis. Therefore, it is not known if these genes (ORFs 93, 94 and 95) are even functional and even if they express to form the corresponding proteins, their importance in terms of virulence is unknown. The objectives of this project are: 1. To determine if the hyaluronic acid synthesis genes (ORFs 93, 94 and 95) in the virulence plasmid pXO1 of Bacillus anthracis are functional genes. 2. To characterize the hyaluronic acid synthetase encoded by ORF 93 in the plasmid pXO1. 3. To determine whether hyaluronic acid synthetase is expressed in B. anthracis. If indeed these genes are found to be functional, these results will be the first evidence for the possibility of presence of a virulent capsular polysaccharide in B. anthracis.
