Abstract
Elongation of rod-shaped bacteria is mediated by a dynamic peptidoglycan-synthetizing machinery called the Rod complex. Here we report that, in Bacillus subtilis, this complex is functional in the absence of all known peptidoglycan polymerases. Cells lacking these enzymes survive by inducing an envelope stress response that increases the expression of RodA, a widely conserved core component of the Rod complex. RodA is a member of the SEDS (shape, elongation, division and sporulation) family of proteins, which have essential but ill-defined roles in cell wall biogenesis during growth, division and sporulation. Our genetic and biochemical analyses indicate that SEDS proteins constitute a family of peptidoglycan polymerases. Thus, B. subtilis and probably most bacteria use two distinct classes of polymerase to synthesize their exoskeleton. Our findings indicate that SEDS family proteins are core cell wall synthases of the cell elongation and division machinery, and represent attractive targets for antibiotic development.
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References
Goffin, C. & Ghuysen, J. M. Multimodular penicillin-binding proteins: an enigmatic family of orthologs and paralogs. Microbiol. Mol. Biol. Rev. 62, 1079–1093 (1998)
Lombard, V., Golaconda Ramulu, H., Drula, E., Coutinho, P. M. & Henrissat, B. The carbohydrate-active enzymes database (CAZy) in 2013. Nucleic Acids Res. 42, D490–D495 (2014)
Matteï, P. J., Neves, D. & Dessen, A. Bridging cell wall biosynthesis and bacterial morphogenesis. Curr. Opin. Struct. Biol. 20, 749–755 (2010)
Packiam, M., Weinrick, B., Jacobs, W. R., Jr & Maurelli, A. T. Structural characterization of muropeptides from Chlamydia trachomatis peptidoglycan by mass spectrometry resolves “chlamydial anomaly”. Proc. Natl Acad. Sci. USA 112, 11660–11665 (2015)
McPherson, D. C. & Popham, D. L. Peptidoglycan synthesis in the absence of class A penicillin-binding proteins in Bacillus subtilis. J. Bacteriol. 185, 1423–1431 (2003)
Arbeloa, A. et al. Role of class A penicillin-binding proteins in PBP5-mediated β-lactam resistance in Enterococcus faecalis. J. Bacteriol. 186, 1221–1228 (2004)
Rice, L. B. et al. Role of class A penicillin-binding proteins in the expression of β-lactam resistance in Enterococcus faecium. J. Bacteriol. 191, 3649–3656 (2009)
Garner, E. C. et al. Coupled, circumferential motions of the cell wall synthesis machinery and MreB filaments in B. subtilis. Science 333, 222–225 (2011)
Domínguez-Escobar, J. et al. Processive movement of MreB-associated cell wall biosynthetic complexes in bacteria. Science 333, 225–228 (2011)
van Teeffelen, S. et al. The bacterial actin MreB rotates, and rotation depends on cell-wall assembly. Proc. Natl Acad. Sci. USA 108, 15822–15827 (2011)
Ruiz, N. Lipid flippases for bacterial peptidoglycan biosynthesis. Lipid Insights 8 (Suppl 1), 21–31 (2016)
Mercer, K. L. N. & Weiss, D. S. The Escherichia coli cell division protein FtsW is required to recruit its cognate transpeptidase, FtsI (PBP3), to the division site. J. Bacteriol. 184, 904–912 (2002)
Goehring, N. W., Gonzalez, M. D. & Beckwith, J. Premature targeting of cell division proteins to midcell reveals hierarchies of protein interactions involved in divisome assembly. Mol. Microbiol. 61, 33–45 (2006)
Fay, A., Meyer, P. & Dworkin, J. Interactions between late-acting proteins required for peptidoglycan synthesis during sporulation. J. Mol. Biol. 399, 547–561 (2010)
Fraipont, C. et al. The integral membrane FtsW protein and peptidoglycan synthase PBP3 form a subcomplex in Escherichia coli. Microbiology 157, 251–259 (2011)
Wei, Y., Havasy, T., McPherson, D. C. & Popham, D. L. Rod shape determination by the Bacillus subtilis class B penicillin-binding proteins encoded by pbpA and pbpH. J. Bacteriol. 185, 4717–4726 (2003)
Henriques, A. O., Glaser, P., Piggot, P. J. & Moran, C. P., Jr. Control of cell shape and elongation by the rodA gene in Bacillus subtilis. Mol. Microbiol. 28, 235–247 (1998)
Daniel, R. A., Williams, A. M. & Errington, J. A complex four-gene operon containing essential cell division gene pbpB in Bacillus subtilis. J. Bacteriol. 178, 2343–2350 (1996)
Gamba, P., Veening, J. W., Saunders, N. J., Hamoen, L. W. & Daniel, R. A. Two-step assembly dynamics of the Bacillus subtilis divisome. J. Bacteriol. 191, 4186–4194 (2009)
Ishino, F. et al. Peptidoglycan synthetic activities in membranes of Escherichia coli caused by overproduction of penicillin-binding protein 2 and rodA protein. J. Biol. Chem. 261, 7024–7031 (1986)
Söding, J. Protein homology detection by HMM-HMM comparison. Bioinformatics 21, 951–960 (2005)
Schmidt, G., Mannel, D., Mayer, H., Whang, H. Y. & Neter, E. Role of a lipopolysaccharide gene for immunogenicity of the enterobacterial common antigen. J. Bacteriol. 126, 579–586 (1976)
Wilkinson, R. G., Gemski, P., Jr & Stocker, B. A. D. Non-smooth mutants of Salmonella typhimurium: differentiation by phage sensitivity and genetic mapping. J. Gen. Microbiol. 70, 527–554 (1972)
Telenti, A. et al. The emb operon, a gene cluster of Mycobacterium tuberculosis involved in resistance to ethambutol. Nat. Med. 3, 567–570 (1997)
Lizak, C., Gerber, S., Numao, S., Aebi, M. & Locher, K. P. X-ray structure of a bacterial oligosaccharyltransferase. Nature 474, 350–355 (2011)
Kus, J. V. et al. Modification of Pseudomonas aeruginosa Pa5196 type IV Pilins at multiple sites with D-Araf by a novel GT-C family Arabinosyltransferase, TfpW. J. Bacteriol. 190, 7464–7478 (2008)
Ye, X. Y. et al. Better substrates for bacterial transglycosylases. J. Am. Chem. Soc. 123, 3155–3156 (2001)
Gampe, C. M., Tsukamoto, H., Wang, T.-S. A., Walker, S. & Kahne, D. Modular synthesis of diphospholipid oligosaccharide fragments of the bacterial cell wall and their use to study the mechanism of moenomycin and other antibiotics. Tetrahedron 67, 9771–9778 (2011)
Robins, W. P., Faruque, S. M. & Mekalanos, J. J. Coupling mutagenesis and parallel deep sequencing to probe essential residues in a genome or gene. Proc. Natl Acad. Sci. USA 110, E848–E857 (2013)
Mascher, T., Hachmann, A. B. & Helmann, J. D. Regulatory overlap and functional redundancy among Bacillus subtilis extracytoplasmic function σ factors. J. Bacteriol. 189, 6919–6927 (2007)
Salzberg, L. I., Luo, Y., Hachmann, A. B., Mascher, T. & Helmann, J. D. The Bacillus subtilis GntR family repressor YtrA responds to cell wall antibiotics. J. Bacteriol. 193, 5793–5801 (2011)
Liechti, G. W. et al. A new metabolic cell-wall labelling method reveals peptidoglycan in Chlamydia trachomatis. Nature 506, 507–510 (2014)
Pilhofer, M. et al. Discovery of chlamydial peptidoglycan reveals bacteria with murein sacculi but without FtsZ. Nat. Commun. 4, 2856 (2013)
van Teeseling, M. C. F. et al. Anammox Planctomycetes have a peptidoglycan cell wall. Nat. Commun. 6, 6878 (2015)
Jeske, O. et al. Planctomycetes do possess a peptidoglycan cell wall. Nat. Commun. 6, 7116 (2015)
Vollmer, J. et al. Requirement of lipid II biosynthesis for cell division in cell wall-less Wolbachia, endobacteria of arthropods and filarial nematodes. Int. J. Med. Microbiol. 303, 140–149 (2013)
Sham, L. T. et al. Bacterial cell wall. MurJ is the flippase of lipid-linked precursors for peptidoglycan biogenesis. Science 345, 220–222 (2014)
Meeske, A. J. et al. MurJ and a novel lipid II flippase are required for cell wall biogenesis in Bacillus subtilis. Proc. Natl Acad. Sci. USA 112, 6437–6442 (2015)
Vasudevan, P., McElligott, J., Attkisson, C., Betteken, M. & Popham, D. L. Homologues of the Bacillus subtilis SpoVB protein are involved in cell wall metabolism. J. Bacteriol. 191, 6012–6019 (2009)
Vasudevan, P., Weaver, A., Reichert, E. D., Linnstaedt, S. D. & Popham, D. L. Spore cortex formation in Bacillus subtilis is regulated by accumulation of peptidoglycan precursors under the control of sigma K. Mol. Microbiol. 65, 1582–1594 (2007)
Gibson, D. G. et al. Enzymatic assembly of DNA molecules up to several hundred kilobases. Nat. Methods 6, 343–345 (2009)
Adachi, M. et al. Degradation and reconstruction of moenomycin A and derivatives: dissecting the function of the isoprenoid chain. J. Am. Chem. Soc. 128, 14012–14013 (2006)
Fujita, M. Temporal and selective association of multiple sigma factors with RNA polymerase during sporulation in Bacillus subtilis. Genes Cells 5, 79–88 (2000)
Lin, D. C., Levin, P. A. & Grossman, A. D. Bipolar localization of a chromosome partition protein in Bacillus subtilis. Proc. Natl Acad. Sci. USA 94, 4721–4726 (1997)
Kruse, A. C. et al. Structure and dynamics of the M3 muscarinic acetylcholine receptor. Nature 482, 552–556 (2012)
Eiamphungporn, W. & Helmann, J. D. The Bacillus subtilis σM regulon and its contribution to cell envelope stress responses. Mol. Microbiol. 67, 830–848 (2008)
Baba, T. et al. Construction of Escherichia coli K-12 in-frame, single-gene knockout mutants: the Keio collection. Mol. Syst. Biol. 2, 0008 (2006)
Acknowledgements
We thank past and present members of the Rudner and Bernhardt super group for advice and encouragement, members of the Walker and Kahne labs for help with PGT assays, D. Perlstein for initial characterization of a PGT activity in the aPBP quadruple mutant, and X. Wang for plasmids. Support for this work comes from the National Institute of Health Grants GM073831 (D.Z.R.), RC2 GM092616 (D.Z.R.), AI083365 (T.G.B.), AI099144 (T.G.B. and S.W.), and Center for Excellence in Translational Research (U19 AI109764) (D.Z.R., T.G.B., A.C.K., S.W., D.K., J.J.M.).
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The project was conceived by A.J.M., T.U., T.G.B., and D.Z.R. Experiments were carried out by A.J.M. and E.P.R. MutSeq library preparation and analyses were performed by E.P.R. and W.P.R. with assistance from J.J.M. A.C.K. assisted in protein purification. D.K. and S.W. provided synthetic lipid II and moenomycin. The manuscript was written by A.J.M., T.G.B., and D.Z.R.
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Reviewer Information Nature thanks S. Withers and the other anonymous reviewer(s) for their contribution to the peer review of this work.
Extended data figures and tables
Extended Data Figure 1 Rod complex dynamics in the presence of different antibiotics.
a, b, Representative kymographs showing cessation of GFP–Mbl particle movement in wild type (a) and the quadruple (Δ4) aPBP mutant (b) upon treatment with 10 μg ml−1 ampicillin. c, Rod complex motion is unaffected by moenomycin treatment. Cells expressing GFP–Mbl were pre-treated with 1 μg ml−1 moenomycin for 15 min then imaged in the presence of moenomycin. Representative kymographs of individual GFP–Mbl particles are shown. d, Maximum intensity projection of the time-lapse in Supplementary Video 5. Scale bars, 1 μm. Data are representative of 3 biological replicates.
Extended Data Figure 2 Conserved neighbourhood architecture for loci encoding SEDS proteins and bPBPs.
a, Diagrams depicting the genomic context of genes encoding SEDS proteins (red) in a diverse set of bacterial taxa. Genes encoding bPBPs are depicted in blue and are frequently located adjacent to SEDS loci. These SEDS–bPBP pairs are often found in the context of the mreBCD operon (faded pink), suggesting that these orthologues function in cell elongation. SEDS and bPBP loci are also frequently present in the cluster of cell wall synthesis and cell division genes exemplified by the E. coli dcw cluster (faded green) and these orthologues probably function in cell division. Unrelated genes are shown as white triangles. Phylogenetic tree was constructed in PhyLoT (http://phylot.biobyte.de) and visualized in iToL (http://itol.embl.de/). b, Histogram showing the genetic distance (on log10 scale) between 2,958 SEDS loci (red) and the nearest bPBP locus (blue). Two commonly observed SEDS–bPBP neighbourhood architectures are depicted. Distances between SEDS and the nearest recA gene are shown in yellow as a negative control. SEDS and bPBP loci were identified using tblastn with five diverse members of each family used as the query.
Extended Data Figure 3 RodA overexpression partially suppresses the phenotypes of the quadruple aPBP mutant.
a, Growth curves of wild type (WT), the quadruple (∆4) aPBP mutant, and the ∆4 mutant overexpressing rodA-his10, representative of three biological replicates. b, Quantification of indicated cytological phenotypes, n = 500. Error bars denote s.e.m. c, Live–dead (propidium iodide) staining of strains analysed in a. Dead cells or cells with membrane integrity defects were visualized by fluorescence microscopy. Images representative of 3 biological replicates. Scale bars, 5 μm. d, Immunoblot analysis of RodA–His10 levels for the three strains in a as well as the ∆4 strain overexpressing nonfunctional mutants W105A and D280A. A fusion of his10 to rodA at its native locus was used to assess wild-type RodA levels (lane 2). Sigma A (σA) levels are shown to control for loading. e, Detergent solubilization of RodA–His10 from B. subtilis membranes using CHAPS. Anti-His immunoblot showing the relative amounts of solubilized RodA–His10 after overnight incubation with 2% CHAPS and ultracentrifugation at 100,000g.
Extended Data Figure 4 Polymers synthesized by RodA in vitro are susceptible to muramidase digestion.
a, To determine whether the products of RodA activity are glycan strands, their susceptibility to cleavage by the muramidase mutanolysin was investigated. Mutanolysin specifically cleaves the β(1,4) linkage between N-acetylmuramic acid and N-acetylglucosamine in peptidoglycan chains. 0.2 μM Flag–RodA was incubated with 4 μM synthetic lipid II for 1 h, then quenched by boiling for 2 min. The products were subjected to overnight digestion with mutanolysin (0.1 mg ml−1) at 37 °C, and analysed by SDS–PAGE. Lipid II, and undigested RodA products are shown for comparison. Data representative of 2 technical replicates. b, The reaction catalysed by RodA can be inhibited by vancomycin (50 μg ml−1), which binds and sequesters the lipid II substrate. Graph denotes the mean from 3 technical replicates, error bars show s.e.m.
Extended Data Figure 5 Critical amino acid residues in RodA identified by MutSeq.
a, Topological map of the RodA protein. The extent to which each amino acid residue tolerated mutations based on the MutSeq screen are indicated. Residues that tolerated a spectrum of amino acid changes are shown in grey. Residues that did not tolerate any mutations are shown in red. Residues that only tolerated conservative changes (conservation of charge, hydrophobicity, or functional groups) are in purple. Residues that had limited mutability but tolerated a non-conservative substitution are shown in pink. The complete data set can be found in Supplementary Table 1. b, Multiple sequence alignment (created using ESPRIPT: http://espript.ibcp.fr/) of 14 diverse SEDS proteins with W105 and D280 residues highlighted.
Extended Data Figure 6 Validation of critical amino acids identified in the MutSeq screen.
a, Schematic of the strain used to test a subset of critical amino acid residues in rodA identified by MutSeq. A wild-type copy of rodA was placed under IPTG-inducible control at an ectopic chromosomal locus (ycgO) and the native copy of rodA was deleted. The mutant alleles to be tested were placed under xylose-inducible control at a second ectopic locus (amyE). As a positive control, a wild-type allele under xylose control was integrated at the second locus. The empty vector was used as a negative control. b, Immunoblot analysis of the RodA mutants expressed as His10-tagged fusions under xylose-inducible control, representative of two biological replicates. ParB levels are shown to control for loading. c, Growth curves of strains expressing mutant rodA alleles. Each strain was grown at 37 °C in CH medium in the presence of 500 μM IPTG to maintain expression of wild-type rodA. Cultures were then washed 3 times in medium lacking inducer, diluted to OD600 of 0.02 in CH medium with 10 mM xylose, and growth was monitored. Growth curves are representative of 2 biological replicates. d, Morphological phenotypes of the strains analysed in c were examined by fluorescence microscopy at the indicated time points (below the images) after resuspension in xylose-containing medium. Fluorescent images of cell membranes stained with TMA-DPH and phase contrast images are shown. A mutation in the highly conserved residue E288 that was found to be mutable by Mutseq was included as a negative control. Consistent with the MutSeq analysis, substitution to alanine (E288A) supported wild-type growth rates and had no effect on cell morphology.
Extended Data Figure 7 RodA overexpression suppresses the synthetic lethality of ∆sigM and Δ4 aPBP mutant.
a, LB agar plates onto which a ∆sigM ∆4 aPBP strain with an IPTG-inducible allele of rodA was streaked in the presence and absence of 15 μM IPTG and incubated at 37 °C overnight. b, The rodA allele containing mutations in its SigM-dependent promoter (rodA PΔsigM) grows in a manner indistinguishable from wild type in the absence of moenomycin. Wild type and the rodA PΔsigM mutant were grown in LB and OD600 was monitored continuously. c, The rodA PΔsigM mutant has a normal rod-shaped morphology. Phase contrast image of cells with the rodA PΔsigM promoter mutant grown to mid-exponential phase in LB medium. All data representative of 2 biological replicates. Scale bar, 1 μm.
Extended Data Figure 8 SEDS proteins and bPBPs are more widely conserved than aPBPs.
Phylogenetic tree showing distribution of SEDS proteins, bPBPs, and aPBPs in a diverse set of 1,773 bacterial taxa. The amino acid sequences of five members of each family were used as queries in a BLASTp search against the NCBI ‘nr’ database with an e-value cutoff of 10−4. The phylogenetic tree was constructed using PhyloT (http://phylot.biobyte.de/) and BLASTp results were plotted against the tree. The occurrence of a SEDS protein is indicated in red, a bPBP in blue, and an aPBP in green. The tree was visualized and annotated using iToL (http://itol.embl.de/). Clades whose genomes contain a SEDS protein and bPBP, but lack aPBPs, are indicated. Mycoplasma, which has no peptidoglycan, lacks all three.
Supplementary information
Supplementary Data
This file contains Supplementary Table 1, mutational frequencies for the RodA coding sequence detected by MutSeq. The table shows the type and count of each possible point mutation in the RodA nucleotide sequence detected in the MutSeq analysis. Observed counts for each mutation are presented in bold and the standard error of the mean for the three replicates is shown. For each category of nucleotide change, we calculated the average number of detected synonymous mutations (silent mutation rate) and, where possible, the average number of detected nonsense mutations (nonsense mutation rate). These two values represent upper and lower bounds for the functionality of each mutant. Based on these data, each amino acid was categorized as "mutable" (grey), "limited mutability" (pink), "conservative changes only" (purple), or “immutable” (red). Sheet 1 presents the average values for the three replicates. C to A and G to T transversions were heavily overrepresented due to artifacts in library preparation and are excluded from the analysis. The raw and complete datasets from each replicate are presented in Sheets 2-4 of this file. (XLSX 865 kb)
Supplementary Data
This file contains Supplementary Table 2, mass spectrometry analysis of purified RodA samples. Purified preparations of B. subtilis RodA(WT), RodA(W105A), and RodA(D280A) used in Figure 4 were analyzed by microcapillary liquid chromatography - mass spectrometry (LC/MS/MS). For RodA and contaminating E. coli proteins, the number of unique peptides, total number of peptides, and sum intensity values are reported. RodA was estimated to constitute ~40% of the total protein in each purification. Since the RodA protein contains 10 TM segments and therefore generates few tryptic peptides that can be detected by LC/MS/MS, this is likely to be an underestimate. E. coli PBP1B, PBP1C, and MtgA were absent from the expression strain and were not detected in any of the samples. The levels of E. coli PBP1A were very low (estimated to be 0.03-0.04% of the total protein in each sample) and were similar in the three purifications. No individual E. coli contaminant represented more than 3.45% of the total protein per sample. Furthermore, in all cases, the levels of each contaminating protein were similar in the three purifications. The top 25 contaminating proteins in the RodA(WT) purification were among the top 40 contaminants in the two RodA mutant purifications. (XLSX 132 kb)
Supplementary Data
This file contains Supplementary Table 3, a list of B. subtilis and E. coli strains used in the study. (XLSX 38 kb)
Supplementary Data
This file contains Supplementary Table 4, a list of plasmids used in the study (XLSX 38 kb)
Supplementary Data
This file contains Supplementary Table 5, a list of oligonucleotide primers used in the study. (XLSX 28 kb)
Supplementary Data
This file contains Supplementary Figure 1, original gel source images. (PDF 1192 kb)
GFP-Mbl dynamics in wild-type B. subtilis
Cells (strain BDR2434) were imaged on a CH agarose pad. 2 minute timelapse video displayed at 20 frames per second. Data representative of four biological replicates. (AVI 3239 kb)
GFP-Mbl dynamics in the quadruple aPBP mutant
Cells (strain BAM321) were imaged on a CH agarose pad. 4 minute timelapse video displayed at 20 frames per second. Data representative of four biological replicates. (AVI 11843 kb)
GFP-Mbl dynamics in wild-type B. subtilis treated with vancomycin
Cells (strain BDR2434) were imaged on a CH agarose pad 4 minutes after the addition of vancomycin (final concentration ~50μg/ml). 4 minute timelapse video displayed at 20 frames per second. Data representative of four biological replicates. (AVI 11165 kb)
GFP-Mbl dynamics in the quadruple aPBP mutant treated with vancomycin
Cells (strain BAM321) were imaged on a CH agarose pad 4 minutes after the addition of vancomycin (final concentration ~50μg/ml). 4 minute timelapse video displayed at 20 frames per second. Data representative of four biological replicates. (AVI 13359 kb)
GFP-Mbl dynamics in wild-type B. subtilis treated with moenomycin
Cells (strain BDR2436) were pre-treated with 1μg/ml moenomycin and imaged on a CH agarose pad containing moenomycin. 4 minute timelapse video displayed at 20 frames per second. Data representative of two biological replicates. (AVI 11332 kb)
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Meeske, A., Riley, E., Robins, W. et al. SEDS proteins are a widespread family of bacterial cell wall polymerases. Nature 537, 634–638 (2016). https://doi.org/10.1038/nature19331
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DOI: https://doi.org/10.1038/nature19331
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