Genes Required for Bacillus anthracis Secondary Cell Wall Polysaccharide Synthesis

Abstract

The secondary cell wall polysaccharide (SCWP) is thought to be essential for vegetative growth and surface (S)-layer assembly in Bacillus anthracis; however, the genetic determinants for the assembly of its trisaccharide repeat structure are not known. Here, we report that WpaA (BAS0847) and WpaB (BAS5274) share features with membrane proteins involved in the assembly of O-antigen lipopolysaccharide in Gram-negative bacteria and propose that WpaA and WpaB contribute to the assembly of the SCWP in B. anthracis. Vegetative forms of the B. anthracis wpaA mutant displayed increased lengths of cell chains, a cell separation defect that was attributed to mislocalization of the S-layer-associated murein hydrolases BslO, BslS, and BslT. The wpaB mutant was defective in vegetative replication during early logarithmic growth and formed smaller colonies. Deletion of both genes, wpaA and wpaB, did not yield viable bacilli, and when depleted of both wpaA and wpaB, B. anthracis could not maintain cell shape, support vegetative growth, or assemble SCWP. We propose that WpaA and WpaB fulfill overlapping glycosyltransferase functions of either polymerizing repeat units or transferring SCWP polymers to linkage units prior to LCP-mediated anchoring of the polysaccharide to peptidoglycan. IMPORTANCE The secondary cell wall polysaccharide (SCWP) is essential for Bacillus anthracis growth, cell shape, and division. SCWP is comprised of trisaccharide repeats (¡4)-␤-ManNAc-(1¡4)-␤-GlcNAc-(1¡6)-␣-GlcNAc-(1¡) with ␣-Gal and ␤-Gal substitutions; however, the genetic determinants and enzymes for SCWP synthesis are not known. Here, we identify WpaA and WpaB and report that depletion of these factors affects vegetative growth, cell shape, and S-layer assembly. We hypothesize that WpaA and WpaB are involved in the assembly of SCWP prior to transfer of this polymer onto peptidoglycan.KEYWORDS Bacillus anthracis, S-layers, Wzy repeat polymerase, cell wall polysaccharide, envelope assembly T he peptidoglycan cell wall of Gram-positive microbes is a determinant of bacterial shape and integrity (1, 2). Peptidoglycan also serves as a scaffold for the attachment of secondary polymers, which support specific cell cycle functions of bacteria (3, 4). Some Gram-positive bacteria, for example, Bacillus subtilis and Staphylococcus aureus, attach wall teichoic acid (WTA) to peptidoglycan, thereby positioning peptidoglycan synthesis enzymes, division septa, and murein hydrolases for cell growth and division (5-7). Other microbes, including Streptococcus species and Bacillus cereus, attach a secondary cell wall polysaccharide (SCWP) to peptidoglycan, and these polymers presumably fulfill functions similar to that of WTA during the bacterial cell cycle (8, 9). While WTA synthesis has been studied in detail (10), little is known about the genetic determinants and assembly reactions for SCWP synthesis.

Assembly and Function of the Bacillus anthracis S-Layer

“…BslO localization is dependent on S-layer assembly (64). Further, patA1 and patA2 (encoding two acetyltransferases that modify the SCWP) and wpaA [whose product belongs to the family of Wzy-/WaaL (also known as O-antigen ligase)-like enzymes] are also required for proper localization of BslO (83, 105) (Figure 2). Mutations in patA1 and patA2 affect the deposition of EA1 in the vicinity of cell septa, suggesting that SLP distribution is influenced by chemical modifications to the ligand of SLH domains (83).…”

“…Computational simulation suggests that BslO effects a nonrandom distribution of B. anthracis chain lengths, implying that all septa are not equal candidates for BslO-mediated cell separation (5). BslS is an N -acetylmuramoyl-L-alanine amidase; its structural gene is located downstream of the wpaA gene and the S-layer locus (105). Deposition of BslS and BslT, a homolog of BslS, in the S-layer of B. anthracis is perturbed in wpaA mutant bacilli (105).…”

“…BslS is an N -acetylmuramoyl-L-alanine amidase; its structural gene is located downstream of the wpaA gene and the S-layer locus (105). Deposition of BslS and BslT, a homolog of BslS, in the S-layer of B. anthracis is perturbed in wpaA mutant bacilli (105). …”

“…We presume that some of these genes are responsible for the galactosylation patterns of B. anthracis and B. cereus SCWP (Figure 3 b ). Some of the genes predicted to be involved in SCWP synthesis are present in duplicate; their products often display functional redundancy (105, 106). …”

“…WpaA ( slc encoded) and WpaB ( scwp2 encoded) belong to the same clan as members of the Wzy polymerase/WaaL family of proteins. Both genes are involved in the synthesis of the SCWP of B. anthracis (105). B. anthracis mutants blocking the expression of a single O-antigen ligase–like gene, wpaA or wpaB , cannot synthesize SCWP and form misshapen cells without S-layers (105).…”