Localization and structural analysis of a conserved pyruvylated epitope in Bacillus anthracis secondary cell wall polysaccharides and characterization of the galactose-deficient wall polysaccharide from avirulent B. anthracis CDC 684

Forsberg, Lennart S.; Abshire, Teresa G.; Friedlander, Arthur M.; Quinn, Conrad P.; Kannenberg, Elmar L.; Carlson, Russell W.

doi:10.1093/glycob/cws080

Cited by 41 publications

(93 citation statements)

References 43 publications

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“…Bioinformatic analysis of the S-layer gene cluster of B. anthracis identified bas0847, which is located immediately adjacent to patB1-patA1-patA2-patB2, genes whose products promote O-acetylation of SCWP (14), to sap and eag, the S-layer protein genes of B. anthracis (23,28), and to csaB, whose product catalyzes SCWP ketalpyruvylation (12,22) (Fig. 1A).…”

Section: Resultsmentioning

confidence: 99%

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Genes Required for Bacillus anthracis Secondary Cell Wall Polysaccharide Synthesis

Lunderberg

Château

et al. 2017

J Bacteriol

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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.

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Section: Resultsmentioning

confidence: 99%

“…The SCWP is modified with ketal-pyruvyl and acetyl groups, which enable assembly of the bacterial surface (S)-layer and its S-layer-associated proteins (12). The genetic determinants for SCWP modification are known; however, they are neither required for vegetative growth, nor do they contribute to the bacterial cell cycle (13,14).…”

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Genes Required for Bacillus anthracis Secondary Cell Wall Polysaccharide Synthesis

Lunderberg

Château

et al. 2017

J Bacteriol

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“…The terminal ManNAc of some SCWP molecules is ketal pyruvylated by the product of csaB, a prerequisite for the assembly of S-layer proteins (4,8). Further, SCWP is acetylated by patA1B1 and patA2B2, contributing to the assembly of proteins with SLH domains at discrete sites in the bacterial envelope (9).…”

Section: Importancementioning

confidence: 99%

Bacillus anthracis tagO Is Required for Vegetative Growth and Secondary Cell Wall Polysaccharide Synthesis

et al. 2015

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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...

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“…Insertional lesions in bslO, sap, and csaB, as well as patA1 and patA2, increase the chain lengths of B. anthracis vegetative forms without affecting the size of bacterial cells (15)(16)(17)(18). bslO encodes a secreted cell wall hydrolase with S-layer homology (SLH) domains that bind to pyruvylated secondary cell wall polysaccharide (SCWP) (15), comprised of the repeating unit [¡4)-␤-ManNAc-(1¡4)-␤-GlcNAc-(1¡6)-␣-GlcNAc-(1¡)] n (19,20). BslO functions as a murein hydrolase when deposited at the division septa of vegetative cells and promotes cell separation, thereby reducing chain length (15).…”

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confidence: 99%

“…csaB mutants display the most severe cell separation defect, and the variants are unable to deposit any S-layer or S-layer-associated proteins with SLH domains in the envelope of B. anthracis (17,21). csaB is thought to encode a pyruvyl transferase that transfers ketal-pyruvyl onto the terminal ␤-ManNAc residue at the distal end of the SCWP (20). Finally, PatA1 and PatA2 catalyze acetylation of the SCWP, thereby enabling the deposition of EA1 as well as BslO near the septal region of the B. anthracis envelope (18).…”

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Bacillus anthracis SlaQ Promotes S-Layer Protein Assembly

Nguyen-Mau

Schneewind

et al. 2015

J Bacteriol

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Bacillus anthracis vegetative forms assemble an S-layer comprised of two S-layer proteins, Sap and EA1. A hallmark of S-layer proteins are their C-terminal crystallization domains, which assemble into a crystalline lattice once these polypeptides are deposited on the bacterial surface via association between their N-terminal S-layer homology domains and the secondary cell wall polysaccharide. Here we show that slaQ, encoding a small cytoplasmic protein conserved among pathogenic bacilli elaborating S-layers, is required for the efficient secretion and assembly of Sap and EA1. S-layer protein precursors cosediment with SlaQ, and SlaQ appears to facilitate Sap assembly. Purified SlaQ polymerizes and when mixed with purified Sap promotes the in vitro formation of tubular S-layer structures. A model is discussed whereby SlaQ, in conjunction with S-layer secretion factors SecA2 and SlaP, promotes localized secretion and S-layer assembly in B. anthracis. IMPORTANCES-layer proteins are endowed with the propensity for self-assembly into crystalline arrays. Factors promoting S-layer protein assembly have heretofore not been reported. We identified Bacillus anthracis SlaQ, a small cytoplasmic protein that facilitates S-layer protein assembly in vivo and in vitro.

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Localization and structural analysis of a conserved pyruvylated epitope in Bacillus anthracis secondary cell wall polysaccharides and characterization of the galactose-deficient wall polysaccharide from avirulent B. anthracis CDC 684

Cited by 41 publications

References 43 publications

Genes Required for Bacillus anthracis Secondary Cell Wall Polysaccharide Synthesis

Genes Required for Bacillus anthracis Secondary Cell Wall Polysaccharide Synthesis

Bacillus anthracis tagO Is Required for Vegetative Growth and Secondary Cell Wall Polysaccharide Synthesis

Bacillus anthracis SlaQ Promotes S-Layer Protein Assembly

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