RESEARCH ARTICLE

crossm Genes Required for Bacillus anthracis Secondary Cell Wall Polysaccharide Synthesis So-Young Oh, J. Mark Lunderberg, Alice Chateau, Olaf Schneewind, Dominique Missiakas Howard Taylor Ricketts Laboratory, Argonne National Laboratory, Lemont, Illinois, USA, and Department of Microbiology, University of Chicago, Chicago, Illinois, USA

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.

Received 11 August 2016 Accepted 7 October 2016 Accepted manuscript posted online 24 October 2016 Citation Oh S-Y, Lunderberg JM, Chateau A, Schneewind O, Missiakas D. 2017. Genes required for Bacillus anthracis secondary cell wall polysaccharide synthesis. J Bacteriol 199:e00613-16. https://doi.org/10.1128/ JB.00613-16. Editor Piet A. J. de Boer, Case Western Reserve University School of Medicine Copyright © 2016 American Society for Microbiology. All Rights Reserved. Address correspondence to Dominique Missiakas, [email protected].

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. January 2017 Volume 199 Issue 1 e00613-16

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Bacillus anthracis, a member of the B. cereus group and the causative agent of anthrax, elaborates an SCWP with the trisaccharide repeat structure (¡4)-␤-ManNAc(1¡4)-␤-GlcNAc-(1¡6)-␣-GlcNAc-(1¡), where ␣-GlcNAc is replaced with ␣-Gal and ␤-Gal at O3 and O4, respectively, and ␤-GlcNAc is replaced with ␣-Gal at O3 (11). 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). Murein linkage units, formed via TagO- and TagA-mediated assembly of undecaprenol-(PO4)2GlcNAc-ManNAc, function as an assembly platform for WTA precursors that, once translocated across the membrane, are tethered by LytR-CpsA-Psr (LCP) enzymes via a phosphodiester linkage to the C-6 hydroxyl of N-acetylmuramic acid (MurNAc) in the glycan strands of peptidoglycan (5, 10, 15). B. anthracis expression of tagO is essential for vegetative growth and contributes to SCWP assembly, which also involves the lcpB1 to -4 (lcpB1– 4), lcpC, and lcpD genes of this microbe (13, 16, 17). The SCWP promotes envelope attachment of murein hydrolases that specifically bind to the carbohydrate structure (18–21). The ketal-pyruvyl-modified SCWP is a ligand for the S-layer homology (SLH) domains of native B. anthracis proteins. These include the S-layer proteins Sap and EA1, which assemble into a paracrystalline S-layer (22–24), as well as B. anthracis S-layer-associated proteins (BSLs) that fulfill the specific functions of host cell adhesion (25), nutrient transport (26), and cell separation and chain length determination (27). To identify the genetic determinants for SCWP synthesis, we combined experimental and bioinformatic approaches and identified two factors, WpaA and WpaB, that belong to the heretofore-uncharacterized PF13425 family; PF13425 is one of five protein families in CL0499, a clan that also includes bacterial membrane proteins involved in the assembly of O-antigen lipopolysaccharide (LPS). We present a model whereby WpaA and WpaB contribute to the assembly of lipid-linked precursors of SCWP in B. anthracis. RESULTS PF13425 family proteins and cell wall polysaccharide assembly (Wpa) of B. anthracis. 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). A preliminary analysis suggested that, similar to mutations in genes of the S-layer cluster, a deletion of bas0847 caused mutant bacilli to exhibit an increased chain length phenotype, whereby the mutant forms longer chains of cells than do wild-type bacilli owing to a cell wall separation defect (see below) (14, 23). Using BLAST searches with the predicted protein sequence of BAS0847, we identified bas5274 in the B. anthracis genome (48% identity; 66% similarity) (Fig. 1A). Analyzing microbial genomes via BLAST, we identified close homologues of bas0847 and bas5274 in B. cereus and Bacillus thuringiensis, as well as in one B. subtilis isolate, JRS10, extracted from the rhizosphere of the desert plant Rhazya stricta (accession number CUB52033.1; E value of 0). However, no functional or family domain had been assigned for these proteins. When B. anthracis, B. cereus, and B. thuringiensis were excluded from BLAST searches, a new group of proteins was identified, with the closest homologues in the genomes of several Streptococcus pneumoniae isolates, including the proteins deposited under accession numbers COE85730.1 and CKE53299.1 (E value of 0). This group of proteins was not encoded in the genomes of the better characterized pneumococcal strains otherwise endowed with genetic loci for Wzy- and synthase-dependent synthesis of S. pneumoniae serotype 2 and serotype 3 capsular polysaccharide, respectively (29, 30). More distant homologues of BAS0847 and BAS5274 were also identified in Gram-negative bacteria, such as Bacteroides fragilis (accession number EXY84983.1; E value of 2e⫺08). None of these proteins have been studied thus far; however, we noted that some candidates in S. pneumoniae and B. January 2017 Volume 199 Issue 1 e00613-16

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FIG 1 Wpa proteins of Bacillus anthracis. (A) Illustration of gene clusters associated with secondary cell wall polysaccharide (SCWP) synthesis in B. anthracis, including the S-layer cluster with csaB (encoding the ketal-pyruvyl transferase of the SCWP), sap and eag (encoding S-layer proteins), patA1-patB1-patA2-patB2 (encoding O-acetyl transferases of the SCWP), and wpaA (bas0847; encoding a PF13425 [O-antigen_lig] family member). bslS, encoding S-layer-associated murein hydrolase S, is located in the vicinity of wpaA. bslT, encoding S-layer-associated murein hydrolase T, is located adjacent to bslO. Finally, wpaB (bas5274) is located in an operon for SCWP synthesis together with tagA1. (B) PFAM-predicted transmembrane topology of PF13425 family members from Streptococcus pneumoniae (O-antigen ligase-like protein; accession number CKE53299) and B. anthracis (WpaA [BAS0847] and WpaB [BAS5274]). (C) Experimentally determined topologies of Wzy and WaaL from P. aeruginosa (34). The numbers at top and bottom indicate positions of amino acid residues in the sequences of the respective proteins. Numbering of transmembrane segments is indicated in the middle as 1 through 12 (WaaL and PF13425) and 1 through 14 (Wzy). PL, periplasmic loop.

fragilis were designated “Lipid A core-O-antigen ligase and related enzymes.” The S. pneumoniae protein deposited under accession number CKE53299.1 was found to bear a small domain between amino acids 355 and 423 with a hit (E value ⫽ 6.62e⫺03) toward pfam13425 (PF13425), an O-antigen ligase-like membrane family protein of the TIGR04370 family (oligosaccharide repeat unit polymerase) (Fig. 2, red box). According to the shotgun sequence deposited in the GenBank data bank (www.ncbi.nlm.nih.gov/ nuccore/CIDK01000005.1), the S. pneumoniae gene with accession number CKE53299.1 is immediately upstream from predicted cps4H (rfaB) and tagA genes, an arrangement identical to that of bas5274, bas5273 (rafB), and bas5272 (tagA1) (Fig. 1A). The PF13425 family is a member of a PFAM clan designated “O-anti_assembly” (CL0499), a superfamily characterized by bacterial proteins that lie in the membrane and comprise enzymes involved in the assembly of O-antigen lipopolysaccharide in Gram-negative bacteria. In addition to PF13425 (O-antigen_ lig), clan CL0499 comprises the following 4 additional members: (i) the PF14296 (O-ag_pol_Wzy) family of established O-antigen polymerases with the well-defined Wzy protein (31), (ii) the PF01901 family of putative O-antigen polymerases found in archaebacteria, (iii) the PF04932 (Wzy_C) family of O-antigen ligases, such as Escherichia coli RfaL (32), and (iv) the PF06899 (WzyE) family January 2017 Volume 199 Issue 1 e00613-16

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FIG 2 Amino acid sequence alignment of the prototypic O-antigen ligase WaaL of P. aeruginosa (Pa) (accession number CTQ39963.1), the S. pneumoniae (Sp) protein with accession number CKE53299.1, and B. anthracis BAS0847 (YP_027123.1) and BAS5274 (YP_031512.1) using ClustalO (http://www.ebi.ac.uk/Tools/msa/clustalo/). The large periplasmic loop PL5 of WaaL (34) that defines the prototypic PF04932 (Wzy_C) domain is shown with a line above the amino acid sequence. The conserved RX3V motif and catalytic H303 are highlighted in blue in the WaaL sequence. The red box marks the position of the PF13425 (O-antigen_lig) domain. Residues highlighted in yellow are shared by at least two of the three S. pneumoniae and B. anthracis PF13425 proteins. Residues identical between all four proteins are highlighted in red and marked with an asterisk, whereas double and single dots mark residues that are highly and weakly conserved, respectively, in all four sequences.

that is specific to enterobacteria and necessary for the assembly of O-antigen lipopolysaccharides (http://pfam.xfam.org/family/PF13425#tabview⫽tab2). Gram-positive bacteria, such as B. anthracis, do not synthesize LPS. Nevertheless, the PF13425 domains borne by BAS0847 and BAS5274 are most closely related to the Wzy_C domain found in the O-antigen ligase RfaL (also known as WaaL), for which functional domains and architectures have been well characterized in Vibrio cholerae January 2017 Volume 199 Issue 1 e00613-16

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and Pseudomonas aeruginosa (33, 34). The topology of P. aeruginosa WaaL has been experimentally determined with translational hybrids, revealing 12 transmembrane (TM) and 5 periplasmic loops (PL1 to -5) (Fig. 1B) (34). According to PFAM prediction, the PF13425 family S. pneumoniae protein with accession number CKE53299.1 and PF13425 proteins B. anthracis BAS0847 and BAS5274 are predicted to encompass 12 TM segments with one large extracytoplasmic loop between TM9 and TM10, reminiscent of WaaL PL5 (Fig. 1B). In P. aeruginosa WaaL, residues 209 to 322 spanning TM8 through TM10 match the PF04932 (Wzy_C) domain with an E value of 1.98e⫺06 (Fig. 2). Based on Clustal sequence alignment of WaaL and the S. pneumoniae protein with accession number CKE53299.1, we propose that residues 328 to 390 and residues 335 to 397 define the PF13425 (O-antigen_lig) domain of BAS0847 and BAS5274, respectively (Fig. 2, red box). We therefore designated BAS0847 as WpaA and BAS5274 as WpaB, for wall polymer assembly factors A and B. B. anthracis wpaA mutants display increased chain length. To study wpaA, we generated a deletion mutant (ΔwpaA). In a chain length analysis of vegetative forms (27), the average length of cell chains for the ΔwpaA mutant increased by twofold compared to that of wild-type B. anthracis (Fig. 3A). The chain length phenotype was restored to wild-type levels when the ΔwpaA mutant was transformed with a plasmid providing for the constitutive expression of wpaA (pPhrpK-wpaA) but not by a plasmid providing for the constitutive expression of wpaB (pPhrpK-wpaB) (Fig. 3A). Earlier work showed that B. anthracis mutants with phenotypic defects in chain length control fall into two categories: variants that cannot properly assemble the S-layer protein Sap (23, 35), and mutants with defects in murein hydrolase deposition at the cell septa of vegetative chains (27). To analyze the chain length phenotype of the ΔwpaA mutant, B. anthracis cultures were fractionated into medium, S-layer, and cell lysate fractions and then analyzed by immunoblotting. The subcellular distribution of the S-layer protein Sap was not perturbed by the deletion of wpaA (Fig. 3B). However, the N-acetylglucosaminidase BslO (27) and two predicted peptidoglycan hydrolases, BslS and BslT, class 3 muramidases that heretofore had not been studied, were found in the extracellular medium of cultures derived from the wpaA deletion mutant but not in cultures from wild-type B. anthracis (Fig. 3B and C). As a control, the subcellular localization of PrsA1, a lipoprotein with peptidyl-prolyl isomerase activity (36), was not affected by the deletion of wpaA. The deletion of bslS, whose structural gene (bas0851) is located in close proximity to wpaA (bas0847), but not deletion of bslT (bas1682), which is located immediately adjacent to bslO (bas1683), caused vegetative forms to assemble elongated chains (Fig. 3A; also data not shown). Taken together, these data indicate that WpaA contributes to the assembly of BslO, BslS, and BslT in the B. anthracis envelope and promotes physiological control of the length of B. anthracis cell chains. Furthermore, the phenotypic defects of the wpaA mutant were restored to wild-type levels by plasmids expressing wpaA but not by wpaB (Fig. 3A and B). B. anthracis wild-type and the ΔwpaA mutant (with or without complementing plasmids) and ΔbslS mutant were analyzed by immunofluorescence microscopy for the assembly of the S-layer protein Sap, using antibodies against Sap. As expected, wild-type B. anthracis assembled Sap in S-layers along the cylindrical envelope of vegetative cells (data not shown). S-layer Sap assembly was not perturbed by the wpaA deletion and complementing plasmids (data not shown). Deletion of wpaB impedes vegetative growth of B. anthracis. Using allelic replacement, we generated ΔwpaB, a deletion of wpaB marked with a spectinomycin resistance cassette (aad9) in the presence of pPhrpK-wpaB. The ΔwpaB allele was transduced with bacteriophage CP51 into wild-type B. anthracis with and without the complementing plasmid pwpaA or pwpaB. The resulting B. anthracis variant carrying the wpaB::aad9 mutation, ΔwpaB, formed small colonies on agar plates (Fig. 4A). The growth phenotype of the ΔwpaB mutant was complemented by pwpaB and, to a lesser extent, also by pwpaA; growth could be enhanced by the addition of isopropyl-␤-DJanuary 2017 Volume 199 Issue 1 e00613-16

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FIG 3 Mislocalization of murein hydrolases causes the chain length phenotype in B. anthracis ΔwpaA mutants. Spores (1 ⫻ 106/ml) derived from various B. anthracis strains were germinated and grown in BHI at 37°C for 4 h prior to fixation for microscopy or cell fractionation and immunoblot analysis. (A) The lengths of 150 chains of vegetative cells were measured and graphed with a box-and-whiskers plot, where the box denotes the 25th and 75th percentiles, the whiskers denote the minimum and maximum measured chain length, and the center line indicates the median value. Data were analyzed by unpaired Student’s t tests for significant differences compared to the wild type. *, P ⬍ 0.001. (B) Subcellular localization of S-layer and S-layer-associated murein hydrolases in the ΔwpaA mutant. Cultures from germinated spores were fractionated into medium (M), S-layer (S), and cellular (C) fractions. Samples were analyzed by 12% SDS-PAGE and Coomassie staining. (C) Samples obtained in the experiment whose results are shown in panel B were subjected to immunoblot analysis utilizing polyclonal antisera raised against S-layer protein (Sap), S-layer-associated murein hydrolases (BslO, BslS, and BslT), or a membrane lipoprotein (PrsA1). Numbers to the left in panels B and C indicate the migratory positions of molecular mass markers in kilodaltons.

thiogalactopyranoside (IPTG), the inducer for Pspac promoter-driven expression of wpaA and wpaB (Fig. 4A). To evaluate the contributions of WpaA and WpaB to vegetative growth, spores derived from B. anthracis Sterne and the ΔwpaA and ΔwpaB mutants were inoculated in brain heart infusion (BHI) broth and vegetative growth was monitored as the increase in absorbance at 600 nm (A600). Compared to that of the wild-type and the ΔwpaA variant, the vegetative growth of the ΔwpaB mutant occurred at a lower rate until cultures reached late exponential phase; thereafter, the ΔwpaB cultures expanded at a rate similar to those of wild-type and ΔwpaA mutant bacilli. The growth defect of the ΔwpaB mutant was ameliorated by transformation with plasmid pwpaA or pwpaB (data not shown). When subjected to chain length analysis of vegetative forms, the average chain length of the ΔwpaB mutant increased by eightfold compared to that of wild-type B. anthracis (Fig. 4B). The chain length phenotype was restored to wild-type levels when the ΔwpaB mutant was complemented with pwpaA or pwpaB (Fig. 4B). To analyze S-layer assembly, B. anthracis cultures were fractionated into medium, S-layer, and cell lysates and then analyzed by immunoblotting. The subcellular distribution of Sap and January 2017 Volume 199 Issue 1 e00613-16

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FIG 4 Mislocalization of BslO and BslS, chain length, and growth phenotypes in B. anthracis ΔwpaB mutants. (A) Small-colony phenotype of the ΔwpaB mutant. Spores (1 ⫻ 103) were inoculated on BHI agar, and vegetative forms grown with or without 1 mM IPTG at 37°C for 24 h. (B) Box-and-whisker plot representing measured vegetative-form chain lengths of wild-type B. anthracis and the ΔwpaB mutant. Samples were prepared and analyzed as described in the legend to Fig. 3A. (C) B. anthracis cultures were fractionated into medium (M), S-layer (S), and cellular (C) fractions, which were analyzed by immunoblotting as described in the legend to Fig. 3B. Numbers to the left indicate the migratory positions of molecular mass markers in kilodaltons.

BslT was not perturbed by the deletion of wpaB (Fig. 4C). However, the S-layerassociated proteins BslO and BslS were found in the extracellular medium of cultures derived from the ΔwpaB deletion mutant but not in cultures from wild-type B. anthracis (Fig. 4C). The subcellular distribution of murein hydrolases in the ΔwpaB mutant was restored to the wild-type levels by IPTG-induced expression of plasmid-borne wpaA or wpaB (Fig. 4C). Immunofluorescence microscopy and staining with anti-BslS antibody revealed that the murein hydrolase was more abundantly distributed in the envelope of the ΔwpaB mutant than in wild-type B. anthracis (Fig. 5). This phenotype was restored to the wild type when the ΔwpaB mutant harbored pwpaA or pwpaB (Fig. 5B). Of note, S-layer Sap assembly was not affected by the ΔwpaB mutation (Fig. 5A). wpaA and wpaB are together indispensable for B. anthracis growth. The B. anthracis ΔwpaA variant was transformed with empty vector (pJK4 or pLM5) or with pwpaA, pwpaAts, pwpaB, or pwpaBts. The resulting strains were transduced with CP51 bacteriophage lysates derived from B. anthracis ΔwpaB (wpaB::aad9) or B. anthracis bslA::aad9. The latter strain carries a mutation in bslA, a gene that encodes an S-layerassociated adhesin (Fig. 6A) (27). Transduction events were enumerated by CFU counts on agar plates supplemented with spectinomycin and 1 mM IPTG. Transductants with the bslA::aad9 mutation arose with similar frequencies in all B. anthracis strains analyzed. In contrast, transductants with the ΔwpaB mutation could be isolated in wild-type B. anthracis Sterne(pJK4) and in the ΔwpaA(pwpaA), ΔwpaA(pwpaAts), ΔwpaA(pwpaB), January 2017 Volume 199 Issue 1 e00613-16

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FIG 5 Deposition and distribution of S-layer and S-layer-associated proteins in the envelope of wpaB mutant B. anthracis. Spores derived from wild-type and ΔwpaB mutant B. anthracis strains were germinated and grown in BHI at 37°C for 4 h prior to fixation. Samples were stained with BODIPY-vancomycin (B-vancomycin) or rabbit antiserum raised against purified Sap (␣Sap) (A) or BslS (␣BslS) (B) and viewed via differential interference contrast (DIC) or fluorescence microscopy, and images were acquired and merged. Scale bars denote 5 ␮m.

and ΔwpaA(pwpaBts) mutants but not in the ΔwpaA(pJK4) or ΔwpaA(pLM5) mutant. These data suggest that B. anthracis cannot grow when both genes, wpaA and wpaB, have been deleted (Fig. 6A). B. anthracis ⌬wpaAB(pwpaAts) and ⌬wpaAB(pwpaBts) strains cannot grow under conditions nonpermissive to plasmid replication. To investigate the effect of wpaAB deletion on B. anthracis vegetative growth, we took advantage of wpaA or wpaB

FIG 6 B. anthracis wpaA and wpaB are indispensable for vegetative growth. (A) CP-51 phage lysate was prepared from B. anthracis strains carrying spectinomycin-resistance determinants in a ΔbslA(bslA::aad9) or ΔwpaB(wpaB::aad9) background and incubated with wild-type or ΔwpaA mutant bacilli harboring plasmid vector pJK4 or pLM5 (empty vectors), pwpaA, pwpaAts, pwpaB, or pwpaBts. Samples were plated and incubated for 30 h at 30°C, and colonies were enumerated. The mean numbers of colonies and associated standard errors from 3 independent experimental determinations were plotted. (B) Spores (1 ⫻ 103) were inoculated on BHI agar with or without 1 mM IPTG and grown at 30°C or 42°C for 24 h. (C) B. anthracis strains were assessed for growth by inoculating spores (1 ⫻ 106/ml) into BHI with 1 mM IPTG at 30°C or into BHI without IPTG at 42°C. Vegetative growth was monitored by recording optical density (A600) over time. January 2017 Volume 199 Issue 1 e00613-16

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merodiploid strains (ΔwpaAB strain with pwpaA, pwpaAts, pwpaB, or pwpaBts) carrying wpaA or wpaB on plasmids whose replicons were stable or temperature sensitive. When either the wpaA or wpaB gene is placed under the control of the IPTG-inducible spac promoter, leaky transcription occurs in B. anthracis; the strains do not require the addition of IPTG for vegetative growth. Additionally, the presence of wpaB allowed ΔwpaAB(pwpaB) and ΔwpaAB(pwpaBts) merodiploids to form larger colonies than strains expressing wpaA on a plasmid, i.e., ΔwpaAB(pwpaA) and ΔwpaAB(pwpaAts) strains (Fig. 6B). Furthermore, IPTG-inducible expression of wpaA or wpaB enabled merodiploid strains to form colonies of equal or larger size than the ΔwpaB mutant (Fig. 6B). When wpaA or wpaB is placed on pLM5, the plasmids with temperature-sensitive replicons, pwpaAts and pwpaBts, cannot replicate at above 32°C and the ΔwpaAB(pwpaAts) and ΔwpaAB(pwpaBts) merodiploid strains are unable to form colonies at 42°C, even in the presence of IPTG (Fig. 6B). When propagated in liquid broth at 30°C, the ΔwpaAB(pwpaA), ΔwpaAB(pwpaAts), ΔwpaAB(pwpaB), and ΔwpaAB(pwpaBts) mutants grew at rates similar to that of the ΔwpaB mutant (Fig. 6C). Incubation of merodiploid strains at 42°C revealed that, in contrast to the ΔwpaAB(pwpaA) and ΔwpaAB(pwpaB) mutants, the ΔwpaAB(pwpaAts) and ΔwpaAB(pwpaBts) mutants are unable to grow beyond an initial small increase in A600 (Fig. 6C). Cell shape defects in B. anthracis ⌬wpaAB(pwpaAts) and ⌬wpaAB(pwpaBts) mutants incubated under conditions nonpermissive to plasmid replication. Differential interference contrast (DIC) microscopy of bacilli grown at 42°C revealed cell shape distortions in ΔwpaAB(pwpaAts) and ΔwpaAB(pwpaBts) mutants (Fig. 7A). When incubated at 42°C for 2 h, both the ΔwpaAB(pwpaAts) and the ΔwpaAB(pwpaBts) variant formed elongated chains of vegetative forms (Fig. 7A). The variants subsequently increased their cell diameters and formed aberrant, spherical shapes (Fig. 7A). To monitor the viability of bacterial cells as a function of membrane integrity, bacilli were stained with Syto9 (green) and propidium iodide (red) and visualized by fluorescence microscopy at timed intervals. Following 6 h of incubation at 42°C, many cells of the ΔwpaAB(pwpaAts) and ΔwpaAB(pwpaBts) variants stained red, indicating membrane permeability and cell death. In contrast, wild-type B. anthracis did not stain with propidium iodide (Fig. 7A). Depletion of wpaAB affects B. anthracis S-layer assembly. To determine the effect of wpaAB deletion on S-layer assembly, spores derived from B. anthracis Sterne and ΔwpaAB(pwpaAts) and ΔwpaAB(pwpaBts) variants were incubated at 42°C for 6 h. Cultures were fractionated into medium, S-layer, and cell lysates and were analyzed by Coomassie-stained SDS-PAGE or immunoblotting. Sap and EA1 were detected in the S-layer fraction of the ΔwpaAB(pwpaAts) and ΔwpaAB(pwpaBts) mutants, albeit the abundance of S-layer proteins was diminished compared to their abundance in wildtype B. anthracis (Fig. 7B and C). The membrane protein PrsA1 was detected in the extracellular medium of cultures derived from both the ΔwpaAB(pwpaAts) and the ΔwpaAB(pwpaBts) mutant, indicating bacterial lysis in cells depleted of wpaAB. Furthermore, the subcellular distribution of BslO and BslT was perturbed following depletion of wpaAB in the ΔwpaAB(pwpaBts) strain but not in the ΔwpaAB(pwpaAts) strain (Fig. 7B and C). Immunofluorescence microscopy was used to visualize the deposition and localization of S-layer and S-layer-associated proteins on the surface of the wpaAB depleted cells. Unlike wild-type B. anthracis, which assembles homogeneous deposits of Sap along its cylindrical axis, the ΔwpaAB(pwpaAts) and ΔwpaAB(pwpaBts) variants displayed irregular deposits of Sap. Increased cell deformity, manifested as a spherical cell shape, was associated with diminished assembly of Sap in the bacterial envelope (Fig. 8A). In contrast to that of Sap, BslS assembly on the surface of wpaAB-depleted cells exhibited differences between the ΔwpaAB(pwpaAts) and ΔwpaAB(pwpaBts) variants (Fig. 8B). Cells from the ΔwpaAB(pwpaAts) mutant assembled BslS in irregular patches, whereas ΔwpaAB(pwpaBts) mutant cells were generally devoid of BslS (Fig. 8B). When stained with BODIPY-vancomycin, which binds to lipid II, i.e., the peptidoglycan synthesis January 2017 Volume 199 Issue 1 e00613-16

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FIG 7 Depletion of wpaA and wpaB affects B. anthracis cell shape, survival, and S-layer assembly. (A) Fluorescence micrographs of B. anthracis cells incubated for 2, 4, or 6 h in BHI without IPTG at 42°C. Bacterial cells were derived from germinated wild-type B. anthracis Sterne or ΔwpaAB(pwpaAts) or ΔwpaAB(pwpaBts) strain spores. At each time point, bacilli were stained with Syto9 (green) and propidium iodide (red) and visualized by fluorescence microscopy to monitor the viability of bacterial populations as a function of the membrane integrity of the cell. Cells with compromised membranes stain red, whereas cells with intact membranes stain green. Merged fluorescence microscopy images are presented. (B) Subcellular fractionation of B. anthracis strains grown for 6 h into medium (M), S-layer (S), and cellular (C) fractions. Proteins were separated on 12% SDS-PAGE and stained with Coomassie blue. (C) Samples obtained in the experiment whose results are shown in panel B were analyzed by immunoblotting with antibodies raised against purified Sap (␣Sap), EA1 (␣EA1), BslO (␣BslO), BslS (␣BslS), BslT (␣BslT), and PrsA1 (␣PrsA). Numbers to the left in panels B and C indicate the migratory positions of molecular mass markers in kilodaltons.

precursor, as well as cell wall pentapeptides with D-Ala–D-Ala (37), wpaAB-depleted cells displayed increased vancomycin staining, which is indicative of a severe defect in peptidoglycan synthesis (Fig. 8A and B). Spores that had germinated and were incubated as vegetative bacilli for 6 h at 42°C were fixed, thin sectioned, stained with uranyl acetate, and analyzed by transmission electron microscopy (Fig. 8C). As expected, images of wild-type B. anthracis revealed smooth cell surfaces and cylindrical-shaped peptidoglycan, indicative of physiological January 2017 Volume 199 Issue 1 e00613-16

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FIG 8 Localization of Sap and BslS in the envelope of wpaAB-depleted B. anthracis strains. Immunofluorescence micrographs stained with BODIPY-vancomycin (B-vancomycin) and anti-Sap antibody (␣Sap) (A) or anti-BslS antibody (␣BslS) (B) and transmission electron micrographs (C) of strains after 6 h of incubation in BHI without IPTG at 42°C following the germination of spores derived from B. anthracis Sterne or the ΔwpaAB(pwpaAts) or ΔwpaAB(pwpaBts) mutant. Scale bars denote 5 ␮m (A and B) and 1 ␮m (C).

cell wall synthesis and assembly (Fig. 8C). In contrast, mutants depleted of wpaAB displayed either very small cells or greatly expanded envelopes with a spherical shape and rough, irregular cell surfaces, as well as unevenly thickened peptidoglycan (Fig. 8C). Depletion of wpaAB diminishes B. anthracis SCWP synthesis. Spores of wild-type B. anthracis or the ΔwpaAB(pwpaAts) and ΔwpaAB(pwpaBts) variants were germinated and incubated at 42°C for 6 h. Murein sacculi of vegetative bacilli were normalized to an A206 of 20 and incubated with hydrofluoric acid (HF). The SCWP released was analyzed by size exclusion high-performance liquid chromatography (SEC-HPLC). The SCWP peaks of ΔwpaAB(pwpaAts) and ΔwpaAB(pwpaBts) mutant cells were much smaller than that of wild-type B. anthracis and eluted with increased retention time (Fig. 9). SCWP extracted from wild-type cells eluted in a broader and larger peak centered at 7.5 min (⬃15 kDa) (8), whereas SCWP of ΔwpaAB(pwpaAts) and ΔwpaAB(pwpaBts) cells January 2017 Volume 199 Issue 1 e00613-16

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FIG 9 SCWP synthesis defects of wpaAB-depleted B. anthracis. Size exclusion high-performance liquid chromatography (SEC-HPLC) chromatographs of SCWP isolated from B. anthracis Sterne or the ΔwpaAB(pwpaAts) or ΔwpaAB(pwpaBts) mutant are shown. Spores of the three strains were germinated and incubated in BHI without IPTG at 42°C for 6 h. Murein sacculi were isolated and treated with hydrofluoric acid. Released SCWP was separated by SEC-HPLC, and absorbance was recorded at 206 nm (mAu, milli-absorbance units). The SEC-HPLC data are representative of two independent experiments.

eluted at 8.12 (6 kDa) and 8.4 (6 kDa) min, respectively (Fig. 9). Together, these data indicate that the ΔwpaAB(pwpaAts) and ΔwpaAB(pwpaBts) mutants produced less SCWP than wild-type B. anthracis and the polymer size was aberrant. DISCUSSION The SCWP of B. anthracis and closely related B. cereus species is comprised of the trisaccharide repeat (¡4)-␤-ManNAc-(1¡4)-␤-GlcNAc-(1¡6)-␣-GlcNAc-(1¡) (8, 11). Earlier work proposed that the genes for SCWP synthesis were located in the surface polysaccharide synthesis gene cluster (sps; bas5116 to bas5127) (18). In agreement with this model, gneZ (bas5117), which encodes a UDP-ManNAc epimerase, is essential for B. anthracis growth and SCWP synthesis (38). Nevertheless, gneY (bas5048), a homologue of gneZ, can substitute for loss of gneZ function to restore B. anthracis growth and SCWP synthesis (38). gneY is a member of another gene cluster that is involved in SCWP synthesis; this cluster includes lcpD (bas5047) and tagO (bas5050). LCP enzymes promote the phosphodiester linkage of secondary wall polymers to the peptidoglycan of Gram-positive bacteria (5). TagO, the UDP-N-acetylglucosamine:undecaprenyl-P N-acetylglucosaminyl 1-P transferase of bacteria, initiates the synthesis of murein linkage units (39, 40). Although the B. anthracis chromosome harbors only one tagO gene, the genome does contain two tagA genes, bas5272 (tagA1) and bas2675 (tagA2). Thus, with the exception of tagO, the B. anthracis genome harbors duplicate genes for the enzymes involved in the synthesis of ManNAc and murein linkage units, as well as the anchoring of SCWP. Therefore, we would anticipate the existence of homologous genes involved in the synthesis and assembly of the SCWP. Here, we identify B. anthracis wpaA (bas0847) mutants that display increased lengths of cell chains, a phenotype that could be attributed to mislocalization of the S-layerassociated murein hydrolases BslO, BslS, and BslT. Bioinformatic analysis identified a homologue, bas5274, which prompted us to examine the requirement of this gene for similar phenotypes. We found that a wpaB (bas5274) mutant is defective in vegetative replication and, importantly, the deletion of both wpaA and wpaB did not yield viable bacilli. Further bioinformatic searches identified homologues in other bacteria, some of which were curated in the PFAM data bank under the protein family PF13425; this family belongs to CL0499, a clan that also encompasses Wzy and RfaL/WaaL proteins January 2017 Volume 199 Issue 1 e00613-16

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(31, 32). Clustal alignment with a PF13425 member, the S. pneumoniae protein with accession number CKE53299.1, suggested that WpaA and WpaB belong to the same family. Thus far, members of this protein family have not been experimentally investigated. In Gram-negative bacteria, Wzy proteins are involved in the polymerization of the LPS O-antigen on the trans side of the plasma membrane by ligating together newly flipped undecaprenol-linked O-antigen repeat units (41, 42). Chain extension occurs at the reducing terminus, whereby the nascent chain is transferred from the lipid carrier to the nonreducing terminus of another undecaprenol-linked subunit (41, 42). The extent of Wzy-mediated polymerization and, thus, chain-length modality is determined by Wzz (41, 42). Wzx acts upstream from Wzy by flipping undecaprenol-Oantigen repeat units across the membrane. RfaL/WaaL acts downstream from Wzy by transferring the polymerized O-antigen from the undecaprenol carrier to the lipid A-core oligosaccharide, thereby effectively completing the synthesis of LPS (41, 42). The synthesis of several exopolysaccharides of both Gram-negative and Gram-positive organisms is also supported by the Wzy-dependent mechanism (reviewed in references 43, 44, and 45). In Gram-positive organisms, capsular gene clusters have been extensively examined in S. pneumoniae serotypes that elaborate distinct exopolysaccharides (45, 46). For example, the biosynthesis of the S. pneumoniae serotype 2 capsule involves the canonical Wzx (CpsJ) and Wzy (CpsH) proteins, along with modulators of chain extension (CpsBCD), suggesting that polysaccharide elongation occurs in a manner analogous to O-antigen assembly (44–46). However, S. pneumoniae CPS clusters do not encompass RfaL/WaaL-like factors (44–46). During LPS assembly, Wzy and RfaL/WaaL share the same substrate, undecaprenol-O-antigen polymer, which is subsequently transferred onto the sugar acceptor of the lipid A-core intermediate. Both Wzy and RfaL/WaaL proteins are highly hydrophobic; however, topology and biochemical studies point to distinct features (33, 34, 47). Wzy proteins encompass 14 TM segments with 2 large periplasmic loops, PL3 and PL5 (Fig. 1B). PL3 and PL5 share the motif RX10G that has been postulated to bind O-antigen repeat units: one loop binds incoming units that are then transferred to the second loop, which retains the elongating chain. In contrast, RfaL/WaaL proteins encompass 12 TM segments with only 1 large periplasmic loop, PL5 (Fig. 1B). Clustal alignments and domain and topology predictions suggest that WpaA and WpaB are more closely related to RfaL/WaaL proteins. The amino acid motif RX3V/L and His 303 in P. aeruginosa or His 311 in V. cholerae (Fig. 2, residues highlighted in blue) are required for the catalytic activity of WaaL (33, 34). The RX3V/L motif is missing in the sequences of WpaA and WpaB, and a lysine is found in place of the histidine within the PF13425 (O-antigen_lig) domain. Genome analysis of B. anthracis did not identify canonical Wzx, Wzy, and WaaL proteins; however, the bas5279 gene downstream from wpaB is predicted to encode a multidrug and toxin extrusion (MATE)-like protein that could be responsible for flipping the undecaprenol-bound GlcNAc-GlcNAc-ManNAc precursor repeat of SCWP across the plasma membrane. In this model, polymerization of repeat units occurs on the trans side of the plasma membrane (polymerization reaction). Next, the undecaprenol-bound polymer is ligated onto murein linkage units presumably assembled by TagO and TagA1/-2 (ligation reaction). The final polymer is then attached to peptidoglycan by LCP proteins to complete the envelope assembly (attachment reaction). It is conceivable that WpaA and WpaB may carry out both polymerization and ligation reactions. However, based on sequence and topology relatedness of PF13425 members to the RfaL/WaaL family of proteins (PF04932), we favor a model whereby both WpaA and WpaB carry out the ligation reaction of SCWP to murein linkage units. Irrespective of which reaction may be catalyzed by WpaA/WpaB, the loss of either polymerase or ligase activity would lead to a reduction of HF-extractable SCWP, and the resulting accumulation of precursor polysaccharides in the membrane would result in the depletion of undecaprenol-P. While we have not examined the lipid content, we observe that depletion of both wpaA and wpa results in a loss of viability and reduction of peptidoglycan-bound polysaccharide. The genome of B. anthracis may not encode a Wzz-like factor that elsewhere is required to regulate the chain length of January 2017 Volume 199 Issue 1 e00613-16

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O-antigen and capsular polymers in Gram-negative bacteria (41, 42, 44). The conspicuous lack of a Wzz-like factor for SCWP synthesis may be explained by a unique mechanism of trisaccharide repeat modification: the terminal ManNacGlcNAc-GlcNAc repeat of SCWP is modified by CsaB with ketal-pyruvyl at the hydroxyl moiety necessary for SCWP polymerization. If both WpaA and WpaB acted as ligases, why would B. anthracis require two WaaL-like catalysts for the assembly of SCWP in the envelope? As suggested by earlier work (16), B. anthracis envelope assembly appears to involve SCWP molecules tethered to TagO/TagA1-derived murein linkage units (undecaprenol-GlcNAcManNAc) and to linkage units with unknown structure that are presumably assembled by TagA2. This model is also corroborated by data for B. anthracis lcpB1– 4, lcpC, or lcpD mutants, which display discrete positional and biological defects in the assembly of SCWP that may explain the need for murein linkage unit (tagO)dependent and independent SCWP synthesis (16). Thus, the need for two WaaL-like ligases may be based on substrate specificity and subcellular localization so that SCWP can be immobilized at multiple locations in the bacterial envelope to support the physiological synthesis of peptidoglycan and S-layer assembly. MATERIALS AND METHODS Bacterial strains and growth conditions. B. anthracis strains were routinely grown in brain heart infusion (BHI) broth or propagated on BHI agar at 30 and 37°C. To deplete wpaAB, spores were germinated and grown at 42°C for 6 h. Plasmid maintenance and transduction of marked mutations among B. anthracis strains were achieved by supplementing the medium with spectinomycin (200 ␮g/ml), kanamycin (20 ␮g/ml), or chloramphenicol (10 ␮g/ml). B. anthracis strains were sporulated in modified G medium (ModG) (48). Plasmid maintenance in Escherichia coli was accomplished by supplementing the medium with spectinomycin (200 ␮g/ml), kanamycin (50 ␮g/ml), or ampicillin (100 ␮g/ml). Allelic replacement using pLM4-derived vectors was performed as previously described (49). B. anthracis mutants and plasmids. Plasmids for electroporation into B. anthracis strains were isolated from E. coli K1077 (dam dcm) (50). Allelic replacement, using the temperature-sensitive pLM4 shuttle vector and derived constructs, was used to generate unmarked deletions in bslS, bslT, and wpaA. To generate the pLM4-ΔbslS, pLM4-ΔbslT, and pLM4-ΔwpaA plasmids, 1-kb fragments upstream and downstream from the genes of interest were amplified from B. anthracis genomic DNA. Upstream and downstream PCR products were then cloned into pLM4 using the EcoRI/XhoI and XhoI/XmaI restriction enzyme sites, respectively. The plasmids pPhrpK-wpaA and pPhrpK-wpaB were generated by inserting the coding sequences for wpaA and wpaB into pWWW412 using the NdeI/BamHI sites (51). The wpaB::aad9 lesion in wpaB, marked by the spectinomycin resistance cassette aad9, was created in B. anthracis Sterne (52) containing pPhrpK-wpaB via allelic replacement with pLM4-wpaB::aad9. To construct pLM4-wpaB:: aad9, 1-kb regions flanking wpaB were amplified, and the PCR products were digested with EcoRI/XhoI and XhoI/XmaI, respectively, and inserted into pLM4. The resulting plasmid was digested with XhoI and ligated with an XhoI-digested aad9 PCR fragment. The aad9-marked lesion in wpaB was transferred using the transducing phage CP51 to B. anthracis strains harboring empty vector pJK4 (13) or pLM5 (temperature sensitive for replication) (49) or plasmid pwpaA (pSY194), pwpaAts (pSY195), pwpaB (pSY196), or pwpaBts (pSY197) (Table 1). Briefly, the donor strain containing the wpaB::aad9 mutation was incubated with CP51 phage (53, 54). The resulting lysate was incubated with the recipient strains, and bacteria were then spread on plates containing spectinomycin. Colonies were isolated after 36 to 48 h of growth at 30°C and analyzed by PCR amplification and DNA sequencing of regions of interest. To construct pwpaA, pwpaAts, pwpaB, and pwpaBts, the coding sequences for wpaA and wpaB were cloned into XbaI/KpnI restriction enzyme sites of pJK4 or pLM5. The list of plasmids used in this work is summarized in Table 1. Cell fractionation and immunoblotting. Spores (1 ⫻ 106/ml) derived from various strains were germinated and incubated in BHI with or without IPTG at 37°C for 4 h or 42°C for 6 h. Next, cultures were normalized to the same optical density at 600 nm (A600) and bacilli were sedimented by centrifugation at 16,000 ⫻ g for 1 min. Culture medium was separated from the sedimented cells. The cells were washed twice with phosphate-buffered saline (PBS) and suspended in a 3 M urea–PBS solution. This mixture was heated to 95°C for 10 min to extract S-layer and S-layer-associated proteins (35). S-layer-stripped cells were sedimented by centrifugation (16,000 ⫻ g for 1 min), and the supernatant fraction was removed as the S-layer fraction. The remaining cells were washed twice with PBS and then mechanically lysed by silica bead beating for 3 min (Fastprep-24; MP Biomedical); this fraction was designated the cellular fraction. Proteins in each of the three fractions, medium, S-layer, and cellular, were precipitated with trichloroacetic acid (TCA), and TCA precipitates were washed in acetone, dried, and solubilized in sample buffer. Proteins were separated by SDS-PAGE, transferred onto a polyvinylidene difluoride (PVDF) membrane, and probed with rabbit antibodies specific for Sap, EA1, BslO, BslS, BslT, and PrsA1. Immunoreactive signals were detected via chemiluminescence. Microscopy and chain length measurements. Spores (1 ⫻ 106/ml) of various B. anthracis strains were germinated in BHI broth, and vegetative cells were fixed with 4% formalin and washed with PBS. Bacilli were visualized by differential interference contrast (DIC) microscopy and images acquired with a January 2017 Volume 199 Issue 1 e00613-16

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TABLE 1 Plasmids used in this study Plasmid pWWW412 pLM4 pLM5 pJK4 pPhrpK-wpaA pPhrpK-wpaB pLM4-ΔbslS pLM4-ΔbslT pLM4-ΔwpaA pLM4-wpaB::aad9 pwpaA pwpaAts pwpaB pwpaBts

Description hrpK promoter for constitutive expression in pOS1 Thermosensitive pET194ts-based vector with aphA3a IPTG-inducible spac promoter expression in pLM4 Thermostable variant of pLM5 wpaA in pWWW412 wpaB in pWWW412 One-kilobase flanking regions of bslS in pLM4 to delete bslS One-kilobase flanking regions of bslT in pLM4 to delete bslT One-kilobase flanking regions of wpaA in pLM4 to delete wpaA One-kilobase flanking regions of wpaB and aad9b in pLM4 to delete wpaB wpaA in pJK4; pSY194 wpaA in pLM5; pSY195 wpaB in pJK4; pSY196 wpaB in pLM5; pSY197

Reference or source 51 49 49 13 This study This study This study This study This study This study This This This This

study study study study

aaphA3 baad9

is a kanamycin resistance determinant. is a spectinomycin resistance determinant.

charge-coupled device (CCD) camera on an Olympus IX81 microscope. The lengths of 150 chains of vegetative cells were measured from the acquired microscopy images using ImageJ and graphed with a box-and-whiskers plot, where the box denotes the 25th and 75th percentiles, the whiskers denote the minimum and maximum measured chain lengths, and the center line indicates the median value. An unpaired, two-tailed Student’s t test was used to analyze data sets comparing the chain lengths of wild-type and mutant bacilli. For immunofluorescence microscopy, fixed bacilli were incubated with polyclonal rabbit sera raised against purified recombinant B. anthracis S-layer (Sap and EA1) or BSL proteins (BslO, BslS, and BslT) (27, 35). The bacilli were then washed with PBS and incubated with Alexa Fluor 594-conjugated goat anti-rabbit polyclonal antibody (Fisher) and 1-␮g/ml vancomycin (4,4-difluoro-4bora-3a,4a-diaza-s-indacene [BODIPY]-vancomycin) (Invitrogen Molecular Probes). Excess staining agent was removed by washing with PBS, and digital micrographs of the bacilli were acquired on a Leica SP5 II STED-CW superresolution laser scanning confocal microscope. Fluorescence microscopy images were merged using the ImageJ software. Electron microscopy. Spores derived from B. anthracis Sterne or the ΔwpaAB(pwpaAts) or ΔwpaAB(pwpaBts) variant were grown at 42°C for 6 h. Bacilli were washed twice with PBS, incubated in fixative (2% glutaraldehyde, 4% paraformaldehyde, 0.1 M sodium cacodylate buffer) overnight at 4°C, and postfixed with 1% OsO4 in 0.1 M sodium cacodylate buffer for 1 h. Fixed samples were stained in 1% uranyl acetate in maleate buffer for 1 h, serially dehydrated with increasing concentrations of ethanol, embedded in spurr resin for 48 h at 60°C, thin sectioned (90 nm) using a Reichert-Jung Ultracut device, and poststained in uranyl acetate and lead citrate. The samples were imaged on a Tecnai F30 (Philips/FEI) transmission electron microscope with a CCD camera and Gatan digital microscope software. Analysis of SCWP. Purification of SCWP was performed as previously described (14). Briefly, spores derived from B. anthracis Sterne or the ΔwpaAB(pwpaAts) or ΔwpaAB(pwpaBts) variant were germinated and grown in BHI broth at 42°C for 6 h. Bacilli were sedimented by centrifugation (8,000 ⫻ g for 15 min), boiled in a 4% SDS solution, and mechanically lysed with 0.1-mm glass beads. The resulting murein sacculi were incubated for 4 h with 10-␮g/ml DNase and 10-␮g/ml RNase supplemented with 20 mM MgSO4, followed by 12 h of 10 ␮M trypsin treatment supplemented with 10 mM CaCl2. Enzymes were inactivated by boiling in 1% SDS. The murein sacculi were first washed extensively with water, followed by washes with 100 mM Tris–HCl (pH 8.0), water, 0.1 M EDTA (pH 8.0), acetone, and water. Murein sacculi were normalized by measuring the absorbance at 206 nm (A206). Next, 5 ml of each suspension was incubated with 25 ml 48% hydrofluoric acid (HF) at 4°C for 16 h. Acid-extracted murein sacculi were sedimented by centrifugation (17,000 ⫻ g for 15 min), and the SCWP-containing supernatant was precipitated with ice-cold ethanol. The SCWP was sedimented by centrifugation (17,000 ⫻ g for 15 min), washed with ice-cold ethanol, suspended in water, and subjected to size exclusion high-performance liquid chromatography (SEC-HPLC) on a 300-mm by 7.8-mm BioBasic SEC300 (Thermos) column equilibrated with 50 mM sodium phosphate buffer (pH 7.5) and 1-ml/min flow rate. The absorbance at 206 nm was monitored to assess the retention time of SCWP.

ACKNOWLEDGMENTS We thank Yimei Chen (Electron Microscopy Facility at the University of Chicago) for experimental advice on microscopy and members of our laboratory and Joseph S. Lam for discussions. We are grateful to the reviewers of this work for their insightful comments. January 2017 Volume 199 Issue 1 e00613-16

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J.M.L. is a trainee of the Medical Scientist Training Program at the University of Chicago, which is supported by a National Institutes of Health (NIH) training grant (grant GM07281), and a recipient of the NIH Ruth L. Kirschstein National Research Service award (number 1F30AI110036). This research was supported by grant AI069227 from the National Institute of Allergy and Infectious Diseases, Infectious Disease Branch (to O.S. and D.M.).

REFERENCES 1. Salton MRJ. 1964. The bacterial cell wall. Elsevier, Amsterdam, Netherlands. 2. Shockman GD, Barrett JF. 1983. Structure, function, and assembly of cell walls of gram-positive bacteria. Annu Rev Microbiol 37:501–527. https:// doi.org/10.1146/annurev.mi.37.100183.002441. 3. Kojima N, Araki Y, Ito E. 1983. Structure of linkage region between ribitol teichoic acid and peptidoglycan in cell walls of Staphylococcus aureus H. J Biol Chem 258:9043–9045. 4. D’Elia MA, Millar KE, Beveridge TJ, Brown ED. 2006. Wall teichoic acid polymers are dispensable for cell viability in Bacillus subtilis. J Bacteriol 188:8313– 8316. https://doi.org/10.1128/JB.01336-06. 5. Kawai Y, Marles-Wright J, Cleverley RM, Emmins R, Ishikawa S, Kuwano M, Heinz N, Bui NK, Hoyland CN, Ogasawara N, Lewis RJ, Vollmer W, Daniel RA, Errington J. 2011. A widespread family of bacterial cell wall assembly proteins. EMBO J 30:4931– 4941. https://doi.org/10.1038/ emboj.2011.358. 6. D’Elia MA, Pereira MP, Chung YS, Zhao W, Chau A, Kenney TJ, Sulavik MC, Black TA, Brown ED. 2006. Lesions in teichoic acid biosynthesis in Staphylococcus aureus lead to a lethal gain of function in the otherwise dispensable pathway. J Bacteriol 188:4183– 4189. https://doi.org/ 10.1128/JB.00197-06. 7. Atilano ML, Pereira PM, Yates J, Reed P, Veiga H, Pinho M, Filipe SR. 2010. Teichoic acids are temporal and spatial regulators of peptidoglycan cross-linking in Staphylococcus aureus. Proc Natl Acad Sci U S A 107: 18991–18996. https://doi.org/10.1073/pnas.1004304107. 8. Forsberg LS, Choudhury B, Leoff C, Marston CK, Hoffmaster AR, Saile E, Quinn CP, Kannenberg EL, Carlson RW. 2011. Secondary cell wall polysaccharides from Bacillus cereus strains G9241, 03BB87 and 03BB102 causing fatal pneumonia share similar glycosyl structures with the polysaccharides from Bacillus anthracis. Glycobiology 21:934 –948. https:// doi.org/10.1093/glycob/cwr026. 9. van Sorge NM, Cole JN, Kuipers K, Henningham A, Aziz RK, Kasirer-Friede A, Lin L, Berends ET, Davies MR, Dougan G, Zhang F, Dahesh S, Shaw L, Gin J, Cunningham M, Merriman JA, Hütter J, Lepenies B, Rooijakkers SH, Malley R, Walker MJ, Shattil SJ, Schlievert PM, Choudhury B, Nizet V. 2014. The classical Lancefield antigen of group A Streptococcus is a virulence determinant with implications for vaccine design. Cell Host Microbe 15:729 –740. https://doi.org/10.1016/j.chom.2014.05.009. 10. Xia G, Peschel A. 2008. Toward the pathway of S. aureus WTA biosynthesis. Chem Biol 15:95–96. https://doi.org/10.1016/j.chembiol.2008 .02.005. 11. Choudhury B, Leoff C, Saile E, Wilkins P, Quinn CP, Kannenberg EL, Carlson RW. 2006. The structure of the major cell wall polysaccharide of Bacillus anthracis is species specific. J Biol Chem 281:27932–27941. https://doi.org/10.1074/jbc.M605768200. 12. Forsberg LS, Abshire TG, Friedlander A, Quinn CP, Kannenberg EL, Carlson RW. 2012. 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 CDC684. Glycobiology 22:1103–1117. https:// doi.org/10.1093/glycob/cws080. 13. Kern J, Ryan C, Faull K, Schneewind O. 2010. Bacillus anthracis surfacelayer proteins assemble by binding to the secondary cell wall polysaccharide in a manner that requires csaB and tagO. J Mol Biol 401:757–775. https://doi.org/10.1016/j.jmb.2010.06.059. 14. Lunderberg JM, Nguyen-Mau SM, Richter GS, Wang YT, Dworkin J, Missiakas DM, Schneewind O. 2013. Bacillus anthracis acetyltransferases PatA1 and PatA2 modify the secondary cell wall polysaccharide and affect the assembly of S-layer proteins. J Bacteriol 195:977–989. https:// doi.org/10.1128/JB.01274-12. 15. Chan YGY, Frankel MB, Dengler V, Schneewind O, Missiakas DM. 2013. Staphylococcus aureus mutants lacking the LytR-CpsA-Psr (LCP) family of January 2017 Volume 199 Issue 1 e00613-16

16.

17.

18.

19.

20.

21.

22.

23.

24.

25.

26.

27.

28.

29. 30.

31.

enzymes release wall teichoic acids into the extracellular medium. J Bacteriol 195:4650 – 4659. https://doi.org/10.1128/JB.00544-13. Liszewski Zilla M, Chan YG, Lunderberg JM, Schneewind O, Missiakas D. 2015. LytR-CpsA-Psr enzymes as determinants of Bacillus anthracis secondary cell wall polysaccharide assembly. J Bacteriol 197:343–353. https://doi.org/10.1128/JB.02364-14. Lunderberg JM, Liszewski Zilla M, Missiakas D, Schneewind O. 2015. Bacillus anthracis tagO is required for vegetative growth and secondary cell wall polysaccharide synthesis. J Bacteriol 197:3511–3520. https:// doi.org/10.1128/JB.00494-15. Schuch R, Pelzek AJ, Raz A, Euler CW, Ryan PA, Winer BY, Farnsworth A, Bhaskaran SS, Stebbins CE, Xu Y, Clifford A, Bearss DJ, Vankayalapati H, Goldberg AR, Fischetti VA. 2013. Use of a bacteriophage lysin to identify a novel target for antimicrobial development. PLoS One 8:e60754. https://doi.org/10.1371/journal.pone.0060754. Low LY, Yang C, Perego M, Osterman A, Liddington RC. 2005. Structure and lytic activity of a Bacillus anthracis prophage endolysin. J Biol Chem 280:35433–35439. https://doi.org/10.1074/jbc.M502723200. Mo KF, Li X, Li H, Low LY, Quinn CP, Boons GJ. 2012. Endolysins of Bacillus anthracis bacteriophages recognize unique carbohydrate epitopes of vegetative cell wall polysaccharides with high affinity and selectivity. J Am Chem Soc 134:15556 –15562. https://doi.org/10.1021/ja3069962. Kern JW, Wilton R, Zhang R, Binkowski A, Joachimiak A, Schneewind O. 2011. Structure of the SLH domains from Bacillus anthracis surface array protein. J Biol Chem 286:26042–26049. https://doi.org/10.1074/ jbc.M111.248070. Mesnage S, Fontaine T, Mignot T, Delepierre M, Mock M, Fouet A. 2000. Bacterial SLH domain proteins are non-covalently anchored to the cell surface via a conserved mechanism involving wall polysaccharide pyruvylation. EMBO J 19:4473– 4484. https://doi.org/10.1093/ emboj/19.17.4473. Kern VJ, Kern JW, Theriot JA, Schneewind O, Missiakas DM. 2012. Surface (S)-layer proteins Sap and EA1 govern the binding of the S-layer associated protein BslO at the cell septa of Bacillus anthracis. J Bacteriol 194:3833–3840. https://doi.org/10.1128/JB.00402-12. Nguyen-Mau SM, Oh SY, Schneewind DI, Missiakas D, Schneewind O. 2015. Bacillus anthracis SlaQ promotes S-layer protein assembly. J Bacteriol 197:3216 –3217. https://doi.org/10.1128/JB.00492-15. Kern JW, Schneewind O. 2010. BslA, the S-layer adhesin of Bacillus anthracis, is a virulence factor for anthrax pathogenesis. Mol Microbiol 75:324 –332. https://doi.org/10.1111/j.1365-2958.2009.06958.x. Tarlovsky Y, Fabian M, Solomaha E, Honsa E, Olson JS, Maresso AW. 2010. A Bacillus anthracis S-layer homology protein that binds heme and mediates heme delivery to IsdC. J Bacteriol 192:3503–3511. https:// doi.org/10.1128/JB.00054-10. Anderson VJ, Kern JW, McCool JW, Schneewind O, Missiakas DM. 2011. The SLH domain protein BslO is a determinant of Bacillus anthracis chain length. Mol Microbiol 81:192–205. https://doi.org/10.1111/j.1365 -2958.2011.07688.x. Mesnage S, Tosi-Couture E, Mock M, Gounon P, Fouet A. 1997. Molecular characterization of the Bacillus anthracis main S-layer component: evidence that it is the major cell-associated antigen. Mol Microbiol 23: 1147–1155. https://doi.org/10.1046/j.1365-2958.1997.2941659.x. Iannelli F, Pearce BJ, Pozzi G. 1999. The type 2 capsule locus of Streptococcus pneumoniae. J Bacteriol 181:2652–2654. Yother J, Ambrose KD, Caimano MJ. 1997. Association of a partial H-rpt element with the type 3 capsule locus of Streptococcus pneumoniae. Mol Microbiol 25:201–203. https://doi.org/10.1046/j.1365-2958 .1997.4361798.x. Samuel G, Reeves P. 2003. Biosynthesis of O-antigens: genes and pathways involved in nucleotide sugar precursor synthesis and O-antigen jb.asm.org 16

Bacillus anthracis Wall Polysaccharide Synthesis

32.

33.

34.

35.

36.

37. 38.

39.

40. 41.

42. 43.

assembly. Carbohydr Res 338:2503–2519. https://doi.org/10.1016/ j.carres.2003.07.009. Klena JD, Pradel E, Schnaitman CA. 1992. Comparison of lipopolysaccharide biosynthesis genes rfaK, rfaL, rfaY, and rfaZ of Escherichia coli K-12 and Salmonella typhimurium. J Bacteriol 174:4746 – 4752. Schild S, Lamprecht AK, Reidl J. 2005. Molecular and functional characterization of O antigen transfer in Vibrio cholerae. J Biol Chem 280: 25936 –25947. https://doi.org/10.1074/jbc.M501259200. Islam ST, Taylor VL, Qi M, Lam JS. 2010. Membrane topology mapping of the O-antigen flippase (Wzx), polymerase (Wzy), and ligase (WaaL) from Pseudomonas aeruginosa PAO1 reveals novel domain architectures. mBio 1:e00189-10. https://doi.org/10.1128/mBio.00189-10. Nguyen-Mau S-M, Oh SY, Kern V, Missiakas D, Schneewind O. 2012. Secretion genes as determinants of Bacillus anthracis chain length. J Bacteriol 194:3841–3850. https://doi.org/10.1128/JB.00384-12. Williams RC, Rees ML, Jacobs MF, Praqai Z, Thwaite JE, Baillie LW, Emmerson PT, Harwood CR. 2003. Production of Bacillus anthracis protective antigen is dependent on extracellular chaperone, PrsA. J Biol Chem 278:18056 –18062. https://doi.org/10.1074/jbc.M301244200. Walsh CT. 1993. Vancomycin resistance: decoding the molecular logic. Science 261:308 –309. https://doi.org/10.1126/science.8392747. Wang YT, Missiakas D, Schneewind O. 2014. GneZ, a UDP-GlcNAc 2-epimerase, is required for S-layer assembly and vegetative growth of Bacillus anthracis. J Bacteriol 196:2969 –2978. https://doi.org/10.1128/ JB.01829-14. Soldo B, Lazarevic V, Karamata D. 2002. tagO is involved in the synthesis of all anionic cell-wall polymers in Bacillus subtilis 168. Microbiology 148:2079 –2087. https://doi.org/10.1099/00221287-148-7-2079. Kojima N, Arakai Y, Ito E. 1985. Structure of the linkage units between ribitol teichoic acids and peptidoglycan. J Bacteriol 161:299 –306. Raetz CR, Whitfield C. 2002. Lipopolysaccharide endotoxins. Annu Rev Biochem 71:635–700. https://doi.org/10.1146/annurev.biochem.71 .110601.135414. Valvano MA. 2003. Export of O-specific lipopolysaccharide. Front Biosci 8:s452–s471. https://doi.org/10.2741/1079. Cuthbertson L, Mainprize IL, Naismith JH, Whitfield C. 2009. Pivotal roles of the outer membrane polysaccharide export and polysaccharide copolymerase protein families in export of extracellular polysaccharides in

January 2017 Volume 199 Issue 1 e00613-16

Journal of Bacteriology

44.

45.

46.

47.

48.

49.

50.

51.

52. 53. 54.

gram-negative bacteria. Microbiol Mol Biol Rev 73:155–177. https:// doi.org/10.1128/MMBR.00024-08. Taylor VL, Huszczynski SM, Lam JS. 9 February 2016. Membrane translocation and assembly of sugar polymer precursors. Curr Top Microbiol Immunol https://doi.org/10.1007/82_2015_5014. Yother J. 2011. Capsules of Streptococcus pneumoniae and other bacteria: paradigms for polysaccharide biosynthesis and regulation. Annu Rev Microbiol 65:563–581. https://doi.org/10.1146/annurev .micro.62.081307.162944. Mavroidi A, Aanensen DM, Godoy D, Skovsted IC, Kaltoft MS, Reeves PR, Bentley SD, Spratt BG. 2007. Genetic relatedness of the Streptococcus pneumoniae capsular biosynthetic loci. J Bacteriol 189:7841–7855. https://doi.org/10.1128/JB.00836-07. Abeyrathne PD, Daniels C, Poon KK, Matewish MJ, Lam JS. 2005. Functional characterization of WaaL, a ligase associated with linking O-antigen polysaccharide to the core of Pseudomonas aeruginosa lipopolysaccharide. J Bacteriol 187:3002–3012. https://doi.org/10.1128/ JB.187.9.3002-3012.2005. Kim HU, Goepfert JM. 1974. A sporulation medium for Bacillus anthracis. J Appl Bacteriol 37:265–267. https://doi.org/10.1111/j.1365-2672 .1974.tb00438.x. Marraffini LA, Schneewind O. 2006. Targeting proteins to the cell wall of sporulating Bacillus anthracis. Mol Microbiol 62:1402–1417. https:// doi.org/10.1111/j.1365-2958.2006.05469.x. Gaspar AH, Marraffini LA, Glass EM, DeBord KL, Ton-That H, Schneewind O. 2005. Bacillus anthracis sortase A (SrtA) anchors LPXTG motifcontaining surface proteins to the cell wall envelope. J Bacteriol 187: 4646 – 4655. https://doi.org/10.1128/JB.187.13.4646-4655.2005. Bubeck Wardenburg J, Williams WA, Missiakas D. 2006. Host defenses against Staphylococcus aureus infection require recognition of bacterial lipoproteins. Proc Nat Acad Sci U S A 103:13831–13836. https://doi.org/ 10.1073/pnas.0603072103. Sterne M. 1937. Avirulent anthrax vaccine. Onderstepoort J Vet Sci Anim Ind 21:41– 43. Green BD, Battisti L, Koehler TM, Thorne CB, Ivins BE. 1985. Demonstration of a capsule plasmid in Bacillus anthracis. Infect Immun 49:291–297. Ruhfel RE, Robillard NJ, Thorne CB. 1984. Interspecies transduction of plasmids among Bacillus anthracis, B. cereus, and B. thuringiensis. J Bacteriol 157:708 –711.

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Genes Required for Bacillus anthracis Secondary Cell Wall Polysaccharide Synthesis.

The secondary cell wall polysaccharide (SCWP) is thought to be essential for vegetative growth and surface (S)-layer assembly in Bacillus anthracis; h...
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