Bacillus anthracis elaborates a linear secondary cell wall polysaccharide (SCWP) that retains surface (S)-layer and associated proteins via their S-layer homology (SLH) domains. The SCWP is comprised of trisaccharide repeats [¡4)--ManNAc-(1¡4)--GlcNAc-(1¡6)-␣-GlcNAc-(1¡]and tethered via acid-labile phosphodiester bonds to peptidoglycan. Earlier work identified UDP-GlcNAc 2-epimerases GneY (BAS5048) and GneZ (BAS5117), which act as catalysts of ManNAc synthesis, as well as a polysaccharide deacetylase (BAS5051), as factors contributing to SCWP synthesis. Here, we show that tagO (BAS5050), which encodes a UDP-N-acetylglucosamine:undecaprenyl-P N-acetylglucosaminyl 1-P transferase, the enzyme that initiates the synthesis of murein linkage units, is required for B. anthracis SCWP synthesis and S-layer assembly. Similar to gneY-gneZ mutants, B. anthracis strains lacking tagO cannot maintain cell shape or support vegetative growth. In contrast, mutations in BAS5051 do not affect B. anthracis cell shape, vegetative growth, SCWP synthesis, or S-layer assembly. These data suggest that TagO-mediated murein linkage unit assembly supports SCWP synthesis and attachment to the peptidoglycan via acid-labile phosphodiester bonds. Further, B. anthracis variants unable to synthesize SCWP trisaccharide repeats cannot sustain cell shape and vegetative growth.
IMPORTANCEBacillus anthracis elaborates an SCWP to support vegetative growth and envelope assembly. Here, we show that some, but not all, SCWP synthesis is dependent on tagO-derived murein linkage units and subsequent attachment of SCWP to peptidoglycan. The data implicate secondary polymer modifications of peptidoglycan and subcellular distributions as a key feature of the cell cycle in Gram-positive bacteria and establish foundations for work on the molecular functions of the SCWP and on inhibitors with antibiotic attributes.T he cell wall envelope of Bacillus anthracis, the causative agent of anthrax, is comprised of peptidoglycan and its attached secondary cell wall polysaccharide (SCWP) (1). The SCWP retains two surface (S)-layer proteins, surface array protein (Sap) and extractable antigen 1 (EA1) (2), as well as 22 S-layer-associated proteins whose S-layer homology (SLH) domains associate with ketal-pyruvylated SCWP (3, 4). Unlike nonpathogenic Bacillus spp., for example, Bacillus subtilis and Bacillus cereus AHU1030, B. anthracis does not synthesize wall teichoic acid (WTA) (5, 6). Nevertheless, B. anthracis expresses functional tagO and tagA genes, whose products provide for the synthesis of murein linkage units [P-GlcNAc-(4¡1)--ManNAc-R] that are phosphodiester linked to the C 6 -hydroxyl of N-acetylmuramic acid in the repeating disaccharide of peptidoglycan [MurNAc(P-GlcNAc-ManNAc-R)-GlcNAc] (4). In B. subtilis, -R represents wall teichoic acid, i.e., either polyglycerol-phosphate or polyribitol-phosphate (7). Here, we test the hypothesis that in B. anthracis the SCWP is attached via the murine linkage unit and that the tagO gene is required for vegetati...