| Summary: | Bacillus anthracis is the causative agent of anthrax, a disease which can affect most mammals, including humans. The bacterium is transmitted as infectious spores which, upon germination into vegetative forms and massive expansion of these cells, precipitate disease. The envelope of the B. anthracis vegetative cell mediates its interactions with the host environment. Therefore, understanding the assembly of the B. anthracis envelope is important in the effort to develop new strategies for the prevention and treatment of anthrax disease. The assembly of two structures within the B. anthracis envelope are evaluated within this thesis. First, the secondary cell wall polysaccharide (SCWP) is a polymer linked to the peptidoglycan. Like other Gram positive peptidoglycan-linked polymers, its assembly impacts cellular division and cell morphology. The SCWP also serves as an anchoring point, through a noncovalent interaction, for twenty four proteins in the Surface layer (S layer); these proteins have previously been implicated in playing a role in anthrax pathogenesis. Requirements for S-layer protein interaction with the SCWP are first, having a protein with three repeating S-layer homology domains, and second, having a CsaB-mediated modification of the polysaccharide. Prior to the studies presented in this thesis, no direct evidence was shown for any other genes involved in B. anthracis SCWP synthesis or modification. This thesis will establish molecular mechanisms for SCWP assembly and modification as well as define the effects of the implicated genes upon S-layer assembly. Chapter II identifies a cluster of four genes, patA1/patB1 and patA2/patB2 as O-acetyl transferases of the SCWP. O-acetylation of the SCWP affects the retention of some, but not all proteins in the S-layer. From this work, a model could be developed in which the selective O-acetylation of the polysaccharide allows the localization of specific proteins in the S-layer to discrete locations. Chapter III explores the function of the murein linkage unit in SCWP synthesis. The murein linkage unit, comprised of N-acetyl mannosamine and N-acetyl glucosamine linked to the peptidoglycan through a phosphodiester bond, is necessary for the synthesis of other Gram positive secondary wall polymers, such as wall teichoic acid of Staphylococcus aureus. Here, we provide evidence that B. anthracis synthesis of the murein linkage unit is essential for bacterial cell division, S-layer assembly and viability. Chapter IV identifies Wzy-like polysaccharide repeat polymerase A (wppA) and B (wppB) as effectors of SCWP synthesis, presumably polymerizing the repeating units of the polysaccharide. Strains with defects in wppA or wppB are defective in SCWP synthesis and S-layer protein localization and B. anthracis requires at least one wpp gene for viability. Homology with other bacterial organisms suggests that wpp genes may be a common means for the synthesis of some extracellular polymers.
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