Bacillus anthracis tagO Is Required for Vegetative Growth and Secondary Cell Wall Polysaccharide Synthesis J. Mark Lunderberg,a,b Megan Liszewski Zilla,a,b Dominique Missiakas,a,b Olaf Schneewinda,b Howard Taylor Ricketts Laboratory, Argonne National Laboratory, Lemont, Illinois, USAa; Department of Microbiology, University of Chicago, Chicago, Illinois, USAb

ABSTRACT

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. IMPORTANCE

Bacillus 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 C6-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 vegetative growth. Earlier work characterized the structure of B. anthracis SCWP as repeating units of [¡4)-␤-ManNAc-(1¡4)-␤-GlcNAc(1¡6)-␣-GlcNAc-(1¡]n, where ␣-Gal and ␤-Gal are substituted for ␣-GlcNAc at O-3 and O-4, respectively, and ␣-Gal is substituted for ␤-GlcNAc at O-3 (1). 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

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bacterial envelope (9). The enzymes that synthesize the trisaccharide repeats and galactosyl modifications of the SCWP are not yet known (10, 11). Comparative genome analysis identified the surface polysaccharide synthesis locus sps (BAS5116-BAS5127) (12). The gneZ gene (BAS5117), a member of this locus, encodes a UDP-GlcNAc 2-epimerase and catalyzes the conversion of GlcNAc into ManNAc (13). gneZ is essential for B. anthracis growth (14), and its homologue, gneY (BAS5048), is not expressed under vegetative growth conditions (15). However, the replacement of the endogenous gneY promoter with an isopropyl-␤-D-thiogalactopyranoside (IPTG)-inducible spac promoter supports growth of the B. anthracis gneZ mutant (14). Removal of the gneY inducer causes defects in S-layer assembly, suggesting that a UDP-GlcNAc 2-epimerase indeed supplies ManNAc for SCWP synthesis (14). gneY (BAS5048) is located in the same gene cluster as tagO (BAS5050). In B. subtilis and Staphylococcus aureus, TagO (TarO) links UDP-GlcNAc and undecaprenyl-phosphate (C55-P), gener-

Received 19 June 2015 Accepted 17 August 2015 Accepted manuscript posted online 31 August 2015 Citation 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. doi:10.1128/JB.00494-15. Editor: P. J. Christie Address correspondence to Olaf Schneewind, [email protected]. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

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ating C55-PP-GlcNAc and initiating WTA synthesis (16–18). C55PP-GlcNAc subsequently is modified by the TagA-catalyzed addition of ManNAc to generate C55-PP-GlcNAc-ManNAc, the precursor of the murein linkage unit (7, 19). Glycerol moieties are polymerized onto this lipid precursor, and LCP enzymes subsequently transfer the polymer onto peptidoglycan (20, 21). Although B. anthracis lacks WTA, the expression of tagO (BAS5050) restores WTA synthesis in the S. aureus tagO mutant (4). Previous attempts to delete tagO in B. anthracis were unsuccessful (4). Earlier work also suggested that BAS5051 (BA5436), which encodes a putative polysaccharide deacetylase, is required for SCWP synthesis (22); however, a specific contribution of this enzyme has not yet been revealed. Here, we use phage transduction to demonstrate that a marked deletion of tagO can be obtained only in B. anthracis strains merodiploid for tagO, and we generate a B. anthracis variant with conditional expression of tagO. The depletion of tagO leads to a loss of SCWP synthesis and S-layer assembly, as well as cell rounding and death, indicating that tagO is indeed essential for B. anthracis growth. We also show that BAS5051 (polysaccharide deacetylase) is not required for SCWP synthesis and S-layer assembly. MATERIALS AND METHODS Bacterial growth and reagents. B. anthracis wild-type (WT) strain Sterne 34F2 (23) and its variants were grown in brain heart infusion (BHI) broth or agar at 30 to 40°C. Escherichia coli was grown in Luria-Bertani (LB) broth or agar at 37°C. Where necessary, 100 ␮g/ml ampicillin, 10 ␮g/ml chloramphenicol, 200 ␮g spectinomycin/ml, or 20 ␮g/ml kanamycin was added to cultures. B. anthracis strains were sporulated in modified G medium (24). Spore preparations were heat treated at 68°C for 2 h to kill vegetative bacilli and then stored at 4°C. To monitor bacterial growth, spores were germinated at time zero by inoculation into BHI at the indicated temperatures and with appropriate antibiotics. Bacterial growth was monitored at timed intervals by recording the absorbance at 600 nm (A600). B. anthracis strains and plasmids. The temperature-sensitive vector pLM4 (25) was used to generate a clone where the tagO gene coding sequence was replaced with the aad9 gene. Briefly, 1-kbp flanking DNA sequences upstream and downstream of the tagO gene were amplified with primer pairs bearing sequences TTTCCCGGGCCTCCCGTAA AAATTCAGGATT, AAACTCGAGATAAATCACTTGTGAGTCCATTA ATACC, AAACTCGAGAAAAAACGCGAAGACTAATTGTAG, and TT TGAATTCCAAAAATAGGAAGGGTGGGAAG and ligated with the intervening spectinomycin resistance cassette aad9 as described previously (14). The resulting DNA fragment was ligated into pLM4 using K1077 (26). The recombinant plasmid was electroporated into the wild type or several tagO merodiploid variants. Allelic replacement was induced via changes in incubation temperature (25, 27) and verified by PCR using chromosomal DNA as a template and PI and PII primers (TTAAAAAAA ATTACGTTAGACTATAAAAGG and AATATTTCATAATAGTAAAGG CTATACTAC) flanking the cloning sites. Three complementing plasmids, ptagOC, ptagOi, and pts-tagOi, were used. Plasmid ptagOC (where C indicates constitutive expression) carries the minimal coding sequence of tagO amplified with primer pairs bearing the sequences AAACATATGG ACTCACAAGTGATTTATGCC and AAAGGATCCTTAGTCTTCGCG TTTTTTCAC and cloned under the constitutive hprK promoter of plasmid pWWW412 (28). Plasmid ptagOi carries the minimal coding sequence of tagO under the IPTG-inducible spac promoter of pJK4 (4, 29). Even in the absence of IPTG, expression from the spac promoter is leaky; therefore, the tagO gene was cloned into pLM5, a variant of pJK4 with a thermosensitive replicon (25), to generate plasmid pts-tagOi. Primers with sequences TTTGGTACCTTAGTCTTCGCGTTTTTTCACTT and TTTTCTAGAATGGACTCACAAGTGATTTATGC were used to

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amplify tagO. The ftsZ gene, BAS3757, was amplified by PCR without its stop codon from the genomic DNA of B. anthracis using primer pair AA AGGATCCTTCTAGGGGGATTTCGAC and AAAGGTACCTCGTCTA CGGCGATTAC. The fragment was digested with KpnI and ligated into the pMutin-green fluorescent protein (GFP) plasmid (30). The inserts for pgfp and pftsZ-gfp were amplified using primer pairs with sequences AA AACTAGTGGTACCAGCGCTATCGATC and CGCACTAGTGGTACC ATTATTTGTAGAG for pgfp and AAACATATGACTAGTTTAGAGTTT GATACTACTCAAGATC and CGCACTAGTGGTACCATTATTTGTA GAGCTC for pftsZ-gfp. The PCR products were cloned into pWH1520 using the SpeI cloning site (31). The ⌬BAS5051 mutant was obtained by allelic replacement using plasmid pLM4 (25, 27). Here, 1-kbp flanking DNA sequences upstream and downstream of BAS5051 were amplified with two primer pairs bearing sequences AAAGAATTCTCGGTATTT TAGATGATATGTACGAG and AAACTCGAGTCGCTTGATCATAAC ATTCACC as well as AAACTCGAGACAAGATTGTAACAGCTTTAG TACC and AAACCCGGGGACAGAGTCACGTTTCTTTGTC. The amplification products were ligated without intervening sequences into pLM4. The plasmid was transformed into B. anthracis for allelic replacement (25, 27). The BAS5051::bursa aurealis mutant allele was obtained by transposon mutagenesis with bursa aurealis (32–34). The mutant allele was transduced into wild-type B. anthracis with bacteriophage CP-51 (35, 36), and the position of transposon insertion was verified by DNA sequencing (34). B. anthracis strains with mutational lesions in sap, eag, bslO, csaB, and bslR have been described (4, 6, 32, 37). Bacteriophage CP-51 was used to perform transduction experiments (35, 36). mCherry and mCherry hybrids. Plasmids expressing mCherry or its hybrids from the T7 promoter of pET16b included pJK81 (mCherry alone) as well as pJK87 and pJK88, which express the mCherry coding sequence fused to the 3= end of the coding sequence for the Sap or EA1 SLH domain, i.e., residues 30 to 210 (pSapSLH-mCherry and pEA1SLHmCherry) (4, 9). Plasmid pJK81 was digested with XhoI and ligated with the XhoI-restricted, PCR-amplified portion of plyG encoding the carbohydrate binding domain (amino acids 166 to 233 of PlyG [38]) to create pPlyGCBD-mCherry. All cloning steps were performed in E. coli DH5␣. E. coli BL21(DE3) cultures harboring expression plasmids were induced for T7 polymerase expression with 0.1 mM IPTG overnight at 26°C. Cells were sedimented by centrifugation, lysed in a French press, and purified by affinity chromatography on Ni-nitrilotriacetic acid (NTA)-Sepharose as described previously (4). Analysis of SCWP. For the analysis of SCWP from the tagO variant, spores were derived from wild-type B. anthracis(pLM5) and the tagO:: aad9(pts-tagOi) mutant, each grown on lawns on six agar plates supplemented with 200 ␮g/ml spectinomycin, scraped off, and suspended in modified G medium. Spores were germinated at 40°C in 1 liter of BHI broth and grown with rotation for 5 h. Bacilli were sedimented by centrifugation (10,000 ⫻ g for 10 min) and washed in water. The remainder of the purification of SCWP from wild-type and tagO::aad9(pts-tagOi) bacilli, as well as that of wild-type and BAS5051 mutant strains, which were propagated on BHI agar plates, was performed as described earlier (4, 37). Washed bacilli were boiled for 30 min in a 4% SDS solution, washed, and suspended in water. The bacteria were mechanically lysed with 0.1-mm glass beads. The resulting murein sacculi were sedimented by centrifugation at 17,000 ⫻g for 15 min, suspended in 100 mM Tris-HCl (pH 7.5), and incubated for 4 h at 37°C with 10 ␮g/ml DNase and 10 ␮g/ml RNase supplemented with 20 mM MgSO4. Samples then were incubated for 16 h at 37°C with 10 ␮M trypsin supplemented with 10 mM CaCl2. Enzymes were inactivated by boiling for 30 min in a water bath in 1% SDS. The SDS was removed by repeated centrifugation and water-washing cycles. The murein sacculi then were washed with water, 100 mM Tris-HCl (pH 8.0), water, 0.1 M EDTA (pH 8.0), water, acetone, and finally twice with water. Murein sacculi were suspended in 5 to 7 ml water, and 25 ml of 48% hydrofluoric acid (HF) was added. Samples were incubated overnight on ice with shaking. The acid-extracted murein sacculi were sedimented by centrifugation at 17,000 ⫻ g for 15 min. SCWP-containing supernatant

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was mixed with ice-cold ethanol in a 1:5 ratio, causing SCWP to precipitate. The polysaccharide was sedimented by centrifugation at 17,000 ⫻ g and 4°C for 15 min, and the sediment was washed extensively with icecold ethanol. The SCWP was 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 with a 3-␮mparticle-size guard column (Thermos) equilibrated with 50 mM sodium phosphate buffer (pH 7.5) and a 0.4 ml/min flow rate. The absorbance at 206 nm was monitored to assess the retention time of SCWP material. Analysis of S-layer assembly. For the fractionation of S-layer proteins, spores derived from wild-type B. anthracis(pLM5) and the tagO:: aad9(pts-tagOi) mutant were germinated at 40°C in BHI and grown for 2 or 5 h. For fractionation studies involving the BAS5051 variants, overnight cultures of B. anthracis wild-type, ⌬BAS5051, and BAS5051::bursa aurealis strains were diluted 1:100 into fresh BHI broth and incubated with rotation at 37°C to an A600 of 2. B. anthracis cultures then were centrifuged at 16,000 ⫻ g to separate the medium (M) from the bacterial sediment. Bacteria were washed twice with phosphate-buffered saline (PBS) and boiled at 95°C for 10 min in 3 M urea. Cells were sedimented by centrifugation at 16,000 ⫻ g, and the supernatant containing S-layer proteins (S) was removed. Urea-extracted bacteria were washed twice with PBS and mechanically lysed by silica bead beating for 3 min (Fastprep-24; MP Biomedical) to generate the cell lysate (C). Proteins in all three fractions (M, S, and C) were precipitated with 10% (vol/vol) trichloroacetic acid (TCA), separated on 10% SDS-PAGE gels, and analyzed by Coomassie staining or electrotransferred onto a polyvinylidene difluoride (PVDF) membrane for immunoblotting (39). Proteins were detected with rabbit antisera raised against purified antigens. mCherry experiments. For mCherry binding experiments, spores were germinated in BHI at 40°C for 5 h and bacilli sedimented by centrifugation, suspended in PBS containing 3 M urea, and heated at 95°C for 10 min. Bacteria were sedimented by centrifugation, washed three times with water, suspended in PBS, and normalized by optical density (A600). Similar amounts of bacterial cell suspensions were incubated with equimolar quantities of purified mCherry, SapSLH-mCherry, EA1SLH-mCherry, or PlyGCBD-mCherry overnight at 4°C. Bacteria were sedimented by centrifugation, washed two times with PBS, and either imaged by fluorescence microscopy or had their fluorescence intensity measured in 96-well format with a BioTek Synergy HT microplate reader. The excitation and emission wavelengths were set at 590 ⫾ 20 nm and 645 ⫾ 40 nm, respectively. Fluorescence measurements were converted to percent binding compared to that of the WT, for which protein binding, i.e., fluorescence units, was set as 100%. P values were calculated using an unpaired, twotailed Student’s t test. Microscopy. To visualize bacterial growth, spores were germinated and, at timed intervals, vegetative bacteria were fixed with buffered 4% formaldehyde. Fixed specimens were observed by light microscopy with a charge-coupled-device (CCD) camera on an Olympus IX81 microscope using a 100⫻ objective. To visualize surface proteins, newly germinated bacilli were incubated with rabbit antisera raised against purified Sap, EA1, and BslR (14, 37), and bound antibodies were detected with DyLight594-conjugated goat anti-rabbit secondary antibody (Fisher). Cells were counterstained with boron dipyrromethene (bodipy)-vancomycin (Invitrogen). A Leica SP5 STED-CW superresolution laser scanning confocal microscope with a 63⫻ objective was used to acquire digital micrographs of bright-field (BF) and fluorescence microscopy images, which then were merged using ImageJ. B. anthracis cells, stripped of their S-layers via boiling with 3 M urea and labeled with mCherry or mCherry hybrids, were visualized in the same manner. For visualization of FtsZ, spores were germinated at 40°C in BHI broth and incubated for either 4 or 6 h with shaking. Xylose was added to the culture medium at 0.02% (wt/vol) to induce ftsZ transcription during the final hour of incubation. Vegetative bacilli were sedimented by centrifugation, stained with 1:1,000 Hoechst 33342 (Invitrogen), mounted, and analyzed by microscopy. Im-

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ages were obtained with the Olympus IX81 microscope using a 100⫻ objective as described above.

RESULTS

Mutations affecting B. anthracis expression of tagO. Previous attempts to inactivate the tagO gene of B. anthracis were unsuccessful (4). We generated a merodiploid strain carrying endogenous tagO on the chromosome in addition to the plasmid-encoded tagO, placed under the constitutive hrpK promoter (tagO⫹/ ptagOC) (Fig. 1A and B). For allelic replacement, we used pJML82, a temperature-sensitive vector for replication containing the spectinomycin resistance marker (aad9) flanked by 1-kb DNA segments derived from sequences upstream or downstream of chromosomal tagO. pJML82 cointegrants into B. anthracis carrying an empty vector could not be resolved to yield colonies with viable bacilli. In contrast, pJML82 cointegrants into B. anthracis(ptagOC) were readily resolved. Oligonucleotides PI and PII with sequences flanking the tagO gene were designed to amplify tagO from the chromosome (Fig. 1A). PCR amplification of tagO from wild-type B. anthracis yielded a 1.1-kb DNA fragment (predicted size, 1,147 nucleotides) with a unique NheI restriction site (Fig. 1A and C, left image). The replacement of tagO with aad9 in B. anthracis JML360(tagO::aad9, ptagOC) generated a PCR fragment of 1,282 nucleotides; as expected, this PCR fragment was cleaved with KpnI but not with NheI (Fig. 1A and C). The chromosomal lesion of the tagO::aad9 mutation was verified by DNA sequencing. Bacteriophage CP-51 was used to generate lysates from the tagO::aad9(ptagOC) merodiploid strain for transduction experiments with the tagO::aad9 allele into B. anthracis carrying pJK4 vector, ptagOi, or pts-tagOi with a temperature-sensitive plasmid replicon. In the ptagOi and pts-tagOi plasmids, the expression of tagO is placed under the control of the IPTG-inducible spac promoter (4) (Fig. 1B). CP-51 transductants of tagO::aad9 were plated in the presence and absence of IPTG on BHI agar with spectinomycin. As a control, aliquots of the B. anthracis recipient strains were transduced with CP-51 phage lysate derived from the B. anthracis bslO::aad9 mutant, which carries a deletion of the nonessential BslO murein hydrolase encoding the blsO gene (32). Transduction of tagO::aad9 or bslO::aad9 CP-51 lysates generated spectinomycin-resistant colonies in B. anthracis ptagOi or B. anthracis pts-tagOi recipients (Fig. 1D). In contrast, CP-51 transduction of bslO::aad9 but not of tagO::aad9 generated spectinomycinresistant colonies in B. anthracis (WT) or B. anthracis(pJK4) (Fig. 1D). In 12 independent experiments, nine spectinomycin-resistant tagO::aad9 candidate colonies were isolated using the haploid (wild-type tagO, pJK4) recipient. When examined for genomic content, these isolates appeared to be the result of spontaneous mutations conferring spectinomycin resistance, as the aad9 resistance cassette could not be PCR amplified from their genomic content and they contained a wild-type tagO gene (Fig. 1A and C). Genomic DNA of 21 spectinomycin-resistant transductants into the merodiploid tagO⫹ (ptagOi) recipient was analyzed by PCR with the PI and PII primer pair. All PCR products were restricted by KpnI but not by NheI. A representative sample of such a restriction digest is shown in Fig. 1C. These data demonstrate that the tagO::aad9 mutation can be effectively crossed into tagO merodiploid recipients but not into haploid strains, suggesting that tagO is essential for vegetative growth under laboratory conditions.

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FIG 1 tagO and BAS5051 genes of B. anthracis. (A) Organization of tagO and neighboring genes on the chromosome of B. anthracis Sterne 34F2. The arrowhead below gene BAS5051 denotes the bursa aurealis insertion site. Sites for restriction enzymes NheI and KpnI are shown as well as sites of hybridization of primers PI and PII. (B) Genetic layout of tagO complementing plasmids used in this study. P indicates promoters, and rep indicates genes involved in plasmid replication. The bottom two plasmids are identical, with the exception of pts-tagOi, containing thermosensitive (ts) replication protein F. (C) Disruption of the tagO gene following allelic recombination or transduction was confirmed by PCR amplification of the deleted locus with flanking primers PI and PII, followed by digestion with enzymes. Photographs of DNA gels are shown. (D) Transduction efficiency of the bslO or tagO allele into wild-type B. anthracis (WT) alone or carrying pJK4 (cloning plasmid), ptagOi, or thermosensitive pts-tagOi plasmids. Spectinomycin-resistant colonies were enumerated after incubation of plates at 30°C for 24 h with and without IPTG in the medium. The insertion of the correct alleles was verified as shown in panel C as well as by DNA sequencing. Phage lysate of bslO::aad9 was used as a control. Data from six independent experiments were averaged and plotted with their associated standard errors.

B. anthracis tagO::aad9(pts-tagOi) strain cannot form colonies under nonpermissive plasmid replication conditions. To analyze the role of tagO for B. anthracis vegetative growth, we took advantage of tagO::aad9 merodiploid strains carrying tagO on plasmids ptagOi and pts-tagOi (Fig. 1B). When the tagO gene is placed solely under the control of the IPTG-inducible spac promoter, leaky transcription occurs in B. anthracis growth medium, and strains do not require the addition of IPTG for vegetative growth (Fig. 1D). However, pts-tagOi carries the thermosensitive repF allele and cannot replicate at temperatures above 30°C, whereas ptagOi replicates under both low- and high-temperature conditions (4, 29). When propagated in liquid broth at 30°C, B. anthracis tagO::aad9(ptagOi) and tagO::aad9(pts-tagOi) strains grew at a similar rate and formed spores when inoculated into modified G medium (Fig. 2A and data not shown). Here too, the addition of IPTG was not required for B. anthracis tagO:: aad9(ptagOi) and tagO::aad9(pts-tagOi) mutant growth. Spore inoculation into rich broth and incubation at 40°C revealed that, in contrast to the B. anthracis tagO::aad9(ptagOi) variant, the tagO::aad9(pts-tagOi) variant was unable to grow beyond a small increase in the A600 (Fig. 2A); further, the tagO::aad9(ptstagOi) variant could not propagate colonies on BHI agar with 200 ␮g/ml spectinomycin incubated at 40°C. Bright-field (BF) microscopy of bacilli grown at 40°C revealed cell shape distortions in the B. anthracis tagO::aad9(pts-tagOi) strain but not in the B. anthracis tagO::aad9(ptagOi) strain. The tagO::aad9(pts-tagOi) mutant appeared for 3 to 4 h as short chains of vegetative forms. Subsequently, however, the tagO::aad9(pts-tagOi) variant increased in cell diameter and assumed a coccoid morphology (Fig. 2B).

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B. anthracis tagO::aad9(pts-tagOi) variant cannot assemble FtsZ rings under nonpermissive plasmid replication conditions. To visualize the process of cell division, B. anthracis wildtype and tagO::aad9(pts-tagOi) mutant strains were transformed with a plasmid expressing an ftsZ-gfp translational hybrid. Earlier work demonstrated that FtsZ and FtsZ-GFP assemble into a ring structure at the cell division septum of bacteria (40). As expected, FtsZ-GFP Z rings were detected via microscopy in wild-type B. anthracis 4 and 6 h following spore germination (Fig. 2C, white arrows). At 4 h postgermination, the B. anthracis tagO::aad9(ptstagOi) variant formed misshapen cells, which appeared shorter and wider than those of the wild type and eventually assumed a coccoid cell shape by 6 h of incubation (Fig. 2C). At 4 h postgermination, the B. anthracis tagO::aad9(pts-tagOi) vegetative cells frequently displayed FtsZ-GFP rings, although the Z rings (white arrows) were positioned at irregular intervals and some cells harbored diffuse FtsZ-GFP staining throughout the cytoplasm (Fig. 2C, green arrow). By 6 h, tagO-depleted bacilli did not oligomerize FtsZ-GFP, and some cells produced diffuse fluorescence staining (Fig. 2C, green arrow). Of note, FtsZ-GFP rings were no longer observed in the B. anthracis tagO::aad9(pts-tagOi) mutant at 6 h postgermination. These data suggest that the depletion of tagO does not immediately block cell division and initially affects expansions of the cell wall envelope, impacting its physiological shape and function. Eventually, tagO depletion altogether blocks cell division in B. anthracis. Depletion of tagO affects B. anthracis SCWP synthesis and S-layer assembly. The SCWP is tethered via acid-labile phosphodiester bonds to the cell wall peptidoglycan of B. anthracis (4).

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FIG 2 Limited expression of tagO causes growth and morphological defects. (A) Spores (1 ⫻ 106/ml) derived from tagO::aad9(ptagOi) and tagO::aad9(pts-

tagOi) strains were inoculated into BHI broth at 30 or 40°C, and vegetative growth was monitored as increased optical density at 600 nm (A600). Growth curves are representative graphs of three experimental replicates. (B) Bacilli were fixed at the specified time points following germination at 40°C and observed by bright-field (BF) microscopy. Scale bar, 10 ␮m. (C) Spores (1 ⫻ 106/ml) derived from wild-type and merodiploid tagO::aad9(pts-tagOi) strains carrying a plasmid expressing FtsZ-GFP were inoculated into BHI broth at 40°C. Bacilli were stained with Hoechst (DNA) at 4 and 6 h following germination at 40°C and observed by phase and fluorescence microscopy. White arrows identify Z rings, whereas green arrows identify cells with aberrant FtsZ-Gfp distribution. Scale bar, 5 ␮m.

We hypothesized that some, or perhaps all, SCWP molecules are anchored to the cell wall by TagO-initiated murein linkage units. Earlier work established a protocol for SEC-HPLC purification of SCWP that had been extracted with HF treatment from purified murein sacculi of B. anthracis (9). Spores of the B. anthracis wild type and tagO::aad9(pts-tagOi) merodiploid variant were incubated at 40°C (the nonpermissive temperature for pts-tagOi) in BHI broth for germination and vegetative expansion over 5 h. Murein sacculi of vegetative bacilli were extracted with HF, and the SCWP was analyzed by SEC-HPLC. The SCWP of wild-type B. anthracis eluted as a large absorbance peak at 206 nm, whereas tagO::aad9 mutant bacilli produced only small amounts of SCWP (Fig. 3A). To determine whether tagO expression is essential for S-layer assembly, spores of the B. anthracis wild type and tagO::aad9(ptstagOi) merodiploid variant were incubated at 40°C in BHI broth for germination and vegetative expansion over 2 and 5 h. Cultures were centrifuged to separate the extracellular medium (M fraction) from vegetative bacilli in the sediment. S-layer proteins associated with bacilli were solubilized with 3 M urea extraction (S fraction). Sedimented bacteria subsequently were broken with glass beads to generate the cellular lysate (C fraction). Proteins in all three fractions (M, S, and C) were analyzed by Coomassiestained SDS-PAGE, which identified Sap S-layer protein assembly after 2 h of incubation in wild-type but not in tagO::aad9(ptstagOi) mutant bacilli (Fig. 3B). After 5 h of vegetative expansion, Sap was detected in the S-layer fraction of tagO::aad9(pts-tagOi) mutant bacilli, although the S-layer abundance was diminished compared to that of wild-type B. anthracis (Fig. 3B). Taken together, these data suggest that the tagO::aad9 mutation diminishes SCWP synthesis and S-layer assembly in B. anthracis and that

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FIG 3 Depletion of tagO causes SCWP synthesis and S-layer assembly defects in B. anthracis. (A) Spores derived from B. anthracis wild-type (WT) and tagO::aad9(pts-tagOi) strains were germinated at 40°C, and vegetative forms were expanded for 5 h. Murein sacculi were isolated and treated with hydrofluoric acid (HF). Released SCWP was separated by SEC-HPLC, and absorbance was recorded at 206 nm (mAu). The SEC-HPLC data are representative of three experimental replicates. (B) Spores derived from B. anthracis wildtype (WT) and tagO::aad9(pts-tagOi) strains were germinated at 40°C, and vegetative forms expanded for 2 and 5 h. B. anthracis cultures were centrifuged to separate the extracellular medium (M) from the bacterial sediment. The S-layer (S) of bacilli was extracted by boiling in 3 M urea. Extracted cells (C) were broken in a bead beater. Proteins in all fractions were precipitated with TCA, washed in acetone, separated on 10% SDS-PAGE, and stained with Coomassie.

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FIG 4 Limited expression of tagO causes decreased binding of the Sap S-layer. Spores from the B. anthracis wild type (WT), sap, eag, and bslR mutants and tagO::aad9/ptagOI and tagO::aad9/pts-tagOI variants were germinated in BHI broth at 40°C and incubated for 2, 5, or 8 h, fixed in formalin, and stained with anti-Sap antibodies (A and B), anti-EA1 antibodies (C), or anti-BslR antibodies (D). Antibody binding and bacterial architecture were revealed with DyLight594conjugated anti-rabbit secondary antibody and bodipy-vancomycin (bodipy-van), respectively. Bright-field and fluorescence microscopy images were acquired. Data sets were merged to reveal the location of the proteins in relation to the cell septa (bodipy-van). The sap, eag, and bslR mutants were stained as controls for the specificity of antibody binding. Scale bar, 10 ␮m.

most, but not all, SCWP synthesis occurs in a TagO-dependent manner. Depletion of tagO affects Sap S-layers in B. anthracis. Spores of B. anthracis wild-type strain and tagO::aad9(ptagOi) and tagO:: aad9(pts-tagOi) merodiploid variants were incubated at 40°C. At timed intervals of 2 or 5 h, bacilli were stained with bodipy-vancomycin and with rabbit antibodies raised against purified Sap and secondary antibody conjugates (Fig. 4). Fluorescence microscopy revealed bodipy-vancomycin deposition at cell division septa of wild-type B. anthracis and Sap staining throughout the bacterial envelope at 2 and 5 h postgermination (Fig. 4A and B). The S-layer protein EA1 and BslR, an S-layer-associated protein, are assembled into the B. anthracis envelope during late-stage logarithmic growth (33) and deposited near cell division septa of bacilli incubated for 8 h in germination medium (Fig. 4C and D). After 2 h of temperature-induced depletion of tagO, B. anthracis tagO:: aad9(pts-tagOi) did not yield immunofluorescence staining with Sap antibodies. By 5 h, tagO::aad9(pts-tagOi) bacilli formed coccoid cells with weak but significant Sap-specific immunofluorescence and increased bodipy-vancomycin staining compared to the fluorescence signals generated by the ⌬sap mutant (Fig. 4C and D). We presume that the B. anthracis tagO::aad9(pts-tagOi) mutant, similar to the B. subtilis ⌬tagO and S. aureus ⌬tagO mutants,

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cannot properly localize factors involved in peptidoglycan synthesis and cytokinesis (PBPs and MreB), causing the accumulation of lipid II precursors and uncrosslinked peptidoglycan with cell wall pentapeptides (D-Ala-D-Ala), which each bind bodipy-vancomycin (20, 41). After 8 h of vegetative expansion, tagO::aad9(ptstagOi) bacilli displayed mislocalized EA1- and BslR-specific immunofluorescence (Fig. 4C and D). Taken together, these data suggest that the depletion of tagO diminishes SCWP synthesis and Sap S-layer assembly, while the deposition of EA1 and BslR occurs in a tagO-independent and SCWP-dependent manner. These experimental results imply that the SCWP can be assembled in a tagO-dependent and tagO-independent manner; the purpose of the latter may be the deposition of S-layer and S-layer-associated proteins at discrete sites in the bacterial envelope. Binding of SLH domains to the envelope of tagO mutant B. anthracis. To quantify the binding capacity of B. anthracis SCWP produced with or without tagO for S-layer proteins, spores of the B. anthracis wild type and tagO::aad9(ptagOi) and tagO::aad9(ptstagOi) merodiploid variants were germinated and incubated at 40°C for 5 h to induce tagO depletion. Vegetative cells were washed and stripped of S-layer proteins by extraction with urea (39). Bacilli then were incubated with purified SapSLH-mCherry or EA1SLH-mCherry, hybrid polypeptides with the SLH domains of

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TABLE 1 Relative binding of protein hybrids to stripped mutant bacilli compared to that of wild-type bacilli Protein binding (%) to stripped B. anthracis varianta Protein hybrid

csaB mutant

SapSLH-mCherry EA1SLH-mCherry PlyGCBD-mCherry

6 ⫾ 16 5 ⫾ 10 94 ⫾ 3

P value

tagO::aad9(pts-tagOi) mutant

1 ⫻ 10⫺6 6.5 ⫻ 10⫺11 0.07

58 ⫾ 9 36 ⫾ 4 39 ⫾ 2

P value

tagO::aad9(ptagOi) mutant

P value

8.3 ⫻ 10⫺4 3.5 ⫻ 10⫺9 2.8 ⫻ 10⫺12

119 ⫾ 9 103 ⫾ 4 93 ⫾ 4

0.07 0.57 0.06

a Binding of fluorescent proteins was assessed by fluorescence measurement; means and associated standard errors were derived from six independent experimental determinations. Arbitrary units of fluorescence were converted to percent binding. Binding to wild-type bacilli was set as 100% for a given hybrid. The number of bacilli was normalized between experiments. In all cases, binding of mCherry alone was ⬍5%.

Sap or EA1 fused to the fluorescent reporter mCherry (4, 9). Following incubation, bacilli were washed and protein binding was quantified by fluorometry or visualized by fluorescence microscopy (Table 1 and Fig. 5). As controls, mCherry alone did not bind to urea-extracted wild-type bacilli, whereas SapSLH-mCherry and EA1SLH-mCherry bound SCWP in the envelope of wild-type B. anthracis but not in the ⌬csaB mutant; the csaB mutation blocks ketal-pyruvylation of the SCWP, a modification necessary for SCWP-SLH domain association (4). In tagO-depleted bacilli, the binding of SapSLH-mCherry and EA1SLH-mCherry to tagO:: aad9(pts-tagOi) vegetative bacilli was diminished (Fig. 5). In contrast, tagO::aad9/ptagOi bacilli bound to the SLH domains of SapSLH-mCherry and EA1SLH-mCherry in a manner indistinguishable from that of wild-type bacilli (Table 1). Previous work demonstrated that bacteriophage lysins of B. anthracis, PlyG and PlyL, associate with the SCWP through their C-terminal cell wall binding domain (CBD) in a manner independent of csaB-mediated ketal-pyruvylation (38, 42–44). We generated purified PlyGCBD-mCherry, where the C-terminal CBD of PlyG (38) is fused to the mCherry reporter, to assess the overall abundance and subcellular distribution of the SCWP (with or without ketal-pyruvate) in wild-type and tagO::aad9(pts-tagOi) vegetative forms. PlyGCBD-mCherry associated with the SCWP of wild-type bacilli and appeared uniformly distributed throughout

FIG 5 Capacity of S-layer-stripped bacilli to retain SCWP-binding proteins with or without tagO expression. Spores from B. anthracis Sterne (WT), csaB mutant, or tagO::aad9(ptagOi) and tagO::aad9(pts-tagOi) variants were germinated in BHI broth at 40°C for 5 h. Bacilli then were treated with 3 M urea to remove S-layer proteins attached to the bacterial envelope. Stripped bacilli were incubated with purified reporter hybrids SapSLH-mCherry, EA1SLHmCherry, or PlyGCBD-mCherry and washed, and bright-field and fluorescence microscopy images were acquired. The csaB mutant served as a control for the loss of S-layer binding. Scale bar, 10 ␮m.

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the bacterial envelope (Fig. 5). The abundance and uniform distribution pattern of PlyGCBD-mCherry deposition in the bacterial envelope was not affected in csaB mutant B. anthracis (Table 1 and Fig. 5). As expected, the overall chain length of the B. anthracis csaB mutant is increased compared to that of the wild type (2, 32). Depletion of tagO in tagO::aad9(pts-tagOi) mutant bacilli decreased the binding of PlyGCBD-mCherry to the bacterial envelope by 61%, in agreement with the hypothesis that TagO is required for the synthesis and cell wall attachment of some, but not all, SCWP (Table 1). Fluorescence microscopy experiments suggest that residual SCWP attachment in tagO-depleted bacilli occurs near the cell septa but not throughout the envelope of the coccoid bacilli that are formed in the absence of TagO (Fig. 5). Taken together, these data indicate that TagO activity is required for the synthesis and cell wall deposition of the SCWP in the cylindrical envelope of B. anthracis, whereas TagO-independent SCWP synthesis and deposition appears to occur near cell septa. In agreement with this model, tagO-depleted bacilli display a large defect in the assembly of the Sap S-layer protein along the cylindrical axis of the B. anthracis envelope, whereas the deposition of EA1 and BslR near cell septa is only moderately affected (Fig. 4). Contribution of BA5436 (BAS5051) deacetylase to SCWP synthesis. Balomenou et al. reported that B. anthracis lacking the polysaccharide deacetylase BA5436 (⌬BA5436 mutant) produces long chains of vegetative bacilli and that HF extraction of peptidoglycan did not release SCWP in this mutant (22). Further, the ⌬BA5436 mutant assembled reduced amounts of S-layer proteins (Sap and EA1) in the bacterial envelope, as determined by urea extraction and SDS-PAGE analysis of these polypeptides (22). Nevertheless, ⌬BA5436 mutant bacilli did not display defects in growth, which was surprising, as studies of gneZ, gneY (12, 14), and tagO (this report) suggest that SCWP synthesis is essential for B. anthracis growth. Of note, the structural gene for BA5436 (BAS5051 in B. anthracis Sterne) is located immediately adjacent to tagO (BAS5050). We isolated a bursa aurealis insertion at position 412 of the 737-bp BAS5051 open reading frame (ORF) (32, 34) and transduced the mutation into wild-type B. anthracis (Fig. 1A). Further, a temperature-sensitive plasmid construct was generated to delete the entire ORF in B. anthracis, yielding the ⌬BAS5051 variant (Fig. 1A). The ⌬BAS5051 and BAS5051::bursa aurealis mutants did not display growth or sporulation defects (data not shown). HF extracts of murein sacculi were examined by SEC-HPLC for SCWP production (Fig. 6A); wild-type, BAS5051:: bursa aurealis, and ⌬BAS5051 mutant strains of B. anthracis released SCWP with similar abundance. Cultures of wild-type B. anthracis or its BAS5051::bursa aurealis and ⌬BAS5051 variants were grown to an A600 of 2 and subjected to S-layer analysis on

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FIG 6 Requirement of BAS5051 deacetylase for SCWP synthesis and S-layer assembly. (A) Equal amounts of purified murein sacculi from B. anthracis wild-type (WT) or isogenic ⌬BAS5051 and BAS5051::bursa variants were treated with hydrofluoric acid. Released SCWP was separated by SEC-HPLC, and absorbance was recorded at 206 nm (mAu). (B) Cultures of B. anthracis Sterne (WT) or ⌬BAS5051 and BAS5051::bursa isogenic variants were grown to the mid-log phase and centrifuged to separate the extracellular medium (M) from the bacterial sediment. The S-layer (S) of bacilli was extracted by boiling in 3 M urea. Extracted cells (C) were broken in a bead beater. Proteins in all fractions were precipitated with TCA, washed in acetone, and separated over 10% SDS-PAGE gels for Coomassie staining or immunoblotting with rabbit antisera raised against purified Sap (␣Sap) or EA1 (␣EA1).

Coomassie-stained SDS-PAGE and immunoblotting for S-layer proteins Sap and EA1. Similar total amounts and a similar distribution of Sap and EA1 in the medium and S-layer were detected by Coomassie-stained SDS-PAGE and immunoblotting (Fig. 6B). Taken together, these data suggest that mutations in BAS5051 do not affect B. anthracis growth, SCWP synthesis, or S-layer assembly. DISCUSSION

Unlike S. aureus or B. subtilis, B. anthracis does not synthesize glycerol (GroP) or ribitol (RibP) phosphate WTA (5). Accordingly, homologs of tarI, tarJ, tarL, tagB, tagD, or tagF, genes whose products catalyze precursor activation and polymerization of wall teichoic acid, are absent from the genome of B. anthracis (45). Nevertheless, the genome of B. anthracis encodes homologs of tagO and tagA (tagA1 and tagA2), which are involved in the synthesis of cell wall linkage units (-P-GlcNAc-ManNAc-R) that tether teichoic acid repeat units to peptidoglycan (46, 47). A genetic linkage between tagA and csaB genes and the presence of acid-labile SCWP led us to hypothesize that the tagO gene of B. anthracis is involved in the synthesis of the SCWP (4). Here, this hypothesis was tested and validated. We used, for the first time, an experimental system of temperature-induced gene depletion in B. anthracis and demonstrated that the tagO::aad9 mutant cannot grow without tagO expression. Shortly after spore germination and depletion of tagO, tagO mutants exhibit defective Z ring for-

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mation and cannot complete the cell division cycle. Instead, these cells form misshapen coccoid cells with reduced amounts of SCWP and are defective in their Sap S-layer protein assembly. After prolonged incubation, the tagO mutant eventually generates limited amounts of SCWP and supports the assembly of S-layer protein in the vicinity of cell septa. These experiments also revealed that not all SCWP synthesis and envelope attachment is dependent on tagO expression. Even though tagO mutant coccoid cells cannot grow, the mutant attaches SCWP at cell contact sites, which also serve as assembly sites for the EA1 S-layer protein and the BslR S-layer-associated protein. It is well established that the distribution of S-layer proteins and S-layer-associated proteins in the envelope of B. anthracis is not homogeneous and occurs in an organized manner (2, 32, 33, 48). Two models have been entertained to explain this phenomenon. SLH domains of S-layer and S-layer-associated proteins are similar, but not identical, in sequence, suggesting they have discrete differences in affinity for SCWP molecules with or without modifications (49), for example, PatA1-PatB1 and PatA2-PatB2 acetylation (9). Do discrete differences in the affinity of SLH domains for specific SCWP molecules function as determinants for their subcellular distribution? If this model (here designated orchestrated assembly) were correct, one also would predict that SCWP molecules with discrete modifications were deposited at unique locations in the bacterial envelope. To the best of our knowledge, experimental support for this conjecture was missing and now is provided via the fluorescence microscopy experiments involving PlyGCBD-mCherry and wild-type as well as tagO mutant bacilli (Fig. 4). On the other hand, Sap and EA1 are transported across the bacterial envelope by a unique secretory pathway, defined by-products of the secA2, slaP, and slaQ genes (39, 50). Also, mutations that affect Sap S-layer protein expression impact the distribution of the other S-layer (EA1) and S-layer-associated proteins (BSLs) in the envelope; these observations imply that abundant secretion and assembly of Sap blocks sites on the SCWP that then cannot be occupied by other SLH domain proteins (the preferred seating model) (33). Our finding that tagO mutant bacilli synthesize small amounts of SCWP near cell septa, thereby maintaining assembly sites for EA1 and BslR but not Sap, provides new support for the orchestrated assembly model. We predict that specific LCP enzymes, catalysts of the phosphodiester linkage tethering teichoic acids and polysaccharides to peptidoglycan (20, 51), also play a key role in the assembly of the SCWP at discrete sites. For example, tagOmediated SCWP attachment and subsequent assembly of Sap Slayers may require specific LCP enzymes from a cast of six different catalysts in B. anthracis (LcpB1-4, LcpC, and LcpD) (37), whereas tagO-independent attachment of the SCWP would involve others. If so, some LCP enzymes may not utilize (-P-GlcNAc-ManNAc-SCWP) as a substrate and instead recognize SCWP assembled on undecaprenol-phosphate with or without amino sugars. In this regard, the conservation of two different tagA alleles in the genome of B. anthracis provides circumstantial evidence for the existence of another murein linkage unit. TagO is inhibited by tunicamycin, which at higher concentrations also inhibits MraY, an enzyme that links Park’s nucleotide (UDP-MurNAc-Ala-iGlu-Dpm-Ala-Ala) and undecaprenol-phosphate to initiate peptidoglycan synthesis (52). Even at very low concentrations, tunicamycin is an effective antibiotic for B. anthracis (4). Tunicamycin toxicity in verte-

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brates, caused by the inhibition of protein oligosaccharide-transferase (OST) (53), prohibits its clinical use. As supported by evidence in this report, SCWP synthesis and modification of peptidoglycan in Gram-positive bacteria with secondary polymers is essential for the replication of these microbes. Therefore, small molecules that specifically block TagO, TagA, or LCP enzymes may be clinically useful for the therapy of infections with antibiotic-resistant Gram-positive bacteria such as Clostridium difficile, Enterococcus faecium, and Staphylococcus aureus.

12.

13.

14.

ACKNOWLEDGMENTS We thank Saleem Khan for the GFP-producing plasmids, Derek Elli for assistance, and members of our laboratory for discussion and comments on the manuscript. J.M.L. and M.L.Z. are trainees of the Medical Scientist Training Program at the University of Chicago and are supported by a National Institutes of Health (NIH) training grant (GM07281). J.M.L. is the recipient of an NIH Ruth L. Kirschstein National Research Service Award (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.).

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Journal of Bacteriology

November 2015 Volume 197 Number 22

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

Bacillus anthracis elaborates a linear secondary cell wall polysaccharide (SCWP) that retains surface (S)-layer and associated proteins via their S-la...
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