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PatB1 is an O-acetyltransferase that decorates secondary cell wall polysaccharides

Nature Chemical Biology volume 14, pages 79–85 (2018)Cite this article

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Abstract

O-Acetylation of the secondary cell wall polysaccharides (SCWP) of the Bacillus cereus group of pathogens, which includes Bacillus anthracis, is essential for the proper attachment of surface-layer (S-layer) proteins to their cell walls. Using a variety of pseudosubstrates and a chemically synthesized analog of SCWP, we report here the identification of PatB1 as a SCWP O-acetyltransferase in Bacillus cereus. Additionally, we report the crystal structure of PatB1, which provides detailed insights into the mechanism of this enzyme and defines a novel subfamily of the SGNH family of esterases and lipases. We propose a model for the O-acetylation of SCWP requiring the translocation of acetyl groups from a cytoplasmic source across the plasma membrane by PatA1 and PatA2 for their transfer to SCWP by PatB1.

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Figure 1: Structure of SCWP from the Bacillus cereus group and organization of genes involved in its modification.
Figure 2: Enzymatic characterization of PatB1 as a SCWP O-acetyltransferase.
Figure 3: Structure of PatB188–396.
Figure 4: Identification of catalytic and oxyanion hole residues.
Figure 5: Proposed model for the O-acetylation of SCWP in the Bacillus cereus group of pathogens.

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  • 04 December 2017

    In the version of this article initially published online, the caption of Figure 5 incorrectly stated, "Although PatA2 resembles an acetyltransferase, its function and role remain to be determined." It should read "Although PatB2 resembles an acetyltransferase, its function and role remain to be determined." The error has been corrected in the PDF and HTML versions of this article.

References

  1. Liu, Y. et al. Genomic insights into the taxonomic status of the Bacillus cereus group. Sci. Rep. 5, 14082 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  2. Drobniewski, F.A. Bacillus cereus and related species. Clin. Microbiol. Rev. 6, 324–338 (1993).

    CAS  PubMed  PubMed Central  Google Scholar 

  3. Helgason, E. et al. Bacillus anthracis, Bacillus cereus, and Bacillus thuringiensis–one species on the basis of genetic evidence. Appl. Environ. Microbiol. 66, 2627–2630 (2000).

    CAS  PubMed  PubMed Central  Google Scholar 

  4. Fouet, A. & Moya, M. in Microbial Megaplasmids (ed. Schwartz, E.) 187–206 (Springer, 2008).

  5. Koch, R. Die Ätiologie der Milzbrand-Krankheit, begründet auf die Entwicklungsgeschichte des. Bacillus anthracis. Beitr. Biol. Pflanz. 2, 277–310 (1876).

    Google Scholar 

  6. Barras, V. & Greub, G. History of biological warfare and bioterrorism. Clin. Microbiol. Infect. 20, 497–502 (2014).

    CAS  PubMed  Google Scholar 

  7. Fouet, A. The surface of. Bacillus anthracis. Mol. Aspects Med. 30, 374–385 (2009).

    CAS  PubMed  Google Scholar 

  8. Fagan, R.P. & Fairweather, N.F. Biogenesis and functions of bacterial S-layers. Nat. Rev. Microbiol. 12, 211–222 (2014).

    CAS  PubMed  Google Scholar 

  9. Mesnage, S., Tosi-Couture, E., Mock, M., Gounon, P. & Fouet, A. Molecular characterization of the Bacillus anthracis main S-layer component: evidence that it is the major cell-associated antigen. Mol. Microbiol. 23, 1147–1155 (1997).

    CAS  PubMed  Google Scholar 

  10. Etienne-Toumelin, I., Sirard, J.C., Duflot, E., Mock, M. & Fouet, A. Characterization of the Bacillus anthracis S-layer: cloning and sequencing of the structural gene. J. Bacteriol. 177, 614–620 (1995).

    CAS  PubMed  PubMed Central  Google Scholar 

  11. Mesnage, S. et al. 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 (2000).

    CAS  PubMed  PubMed Central  Google Scholar 

  12. Kern, J., Ryan, C., Faull, K. & Schneewind, O. Bacillus anthracis surface-layer proteins assemble by binding to the secondary cell wall polysaccharide in a manner that requires csaB and tagO. J. Mol. Biol. 401, 757–775 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  13. Choudhury, B. et al. The structure of the major cell wall polysaccharide of Bacillus anthracis is species-specific. J. Biol. Chem. 281, 27932–27941 (2006).

    CAS  PubMed  Google Scholar 

  14. Leoff, C. et al. Structural elucidation of the nonclassical secondary cell wall polysaccharide from Bacillus cereus ATCC 10987. Comparison with the polysaccharides from Bacillus anthracis and B. cereus type strain ATCC 14579 reveals both unique and common structural features. J. Biol. Chem. 283, 29812–29821 (2008).

    CAS  PubMed  PubMed Central  Google Scholar 

  15. Forsberg, L.S. et al. 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 a virulent B. anthracis CDC 684. Glycobiology 22, 1103–1117 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  16. Leoff, C. et al. Cell wall carbohydrate compositions of strains from the Bacillus cereus group of species correlate with phylogenetic relatedness. J. Bacteriol. 190, 112–121 (2008).

    CAS  PubMed  Google Scholar 

  17. Leoff, C. et al. Secondary cell wall polysaccharides of Bacillus anthracis are antigens that contain specific epitopes which cross-react with three pathogenic Bacillus cereus strains that caused severe disease, and other epitopes common to all the Bacillus cereus strains tested. Glycobiology 19, 665–673 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  18. Lunderberg, J.M. et al. 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 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  19. Kern, J. & Schneewind, O. BslA, the S-layer adhesin of B. anthracis, is a virulence factor for anthrax pathogenesis. Mol. Microbiol. 75, 324–332 (2010).

    CAS  PubMed  Google Scholar 

  20. Kern, V.J., Kern, J.W., Theriot, J.A., Schneewind, O. & Missiakas, D. Surface-layer (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 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  21. Nguyen-Mau, S.-M., Oh, S.Y., Kern, V.J., Missiakas, D.M. & Schneewind, O. Secretion genes as determinants of Bacillus anthracis chain length. J. Bacteriol. 194, 3841–3850 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  22. Hofmann, K.A superfamily of membrane-bound O-acyltransferases with implications for wnt signaling. Trends Biochem. Sci. 25, 111–112 (2000).

    CAS  PubMed  Google Scholar 

  23. Franklin, M.J. & Ohman, D.E. Mutant analysis and cellular localization of the AlgI, AlgJ, and AlgF proteins required for O acetylation of alginate in. Pseudomonas aeruginosa.J. Bacteriol. 184, 3000–3007 (2002).

    CAS  Google Scholar 

  24. Clarke, A.J., Strating, H. & Blackburn, N.T. in Glycomicrobiology (ed. Doyle, R.J.) 187–223 (Plenum Publishing Co. Ltd., 2000).

  25. Weadge, J.T., Pfeffer, J.M. & Clarke, A.J. Identification of a new family of enzymes with potential O-acetylpeptidoglycan esterase activity in both Gram-positive and Gram-negative bacteria. BMC Microbiol. 5, 49 (2005).

    PubMed  PubMed Central  Google Scholar 

  26. Laaberki, M.-H., Pfeffer, J., Clarke, A.J. & Dworkin, J. O-Acetylation of peptidoglycan is required for proper cell separation and S-layer anchoring in. Bacillus anthracis. J. Biol. Chem. 286, 5278–5288 (2011).

    CAS  PubMed  Google Scholar 

  27. Baker, P. et al. P. aeruginosa SGNH hydrolase-like proteins AlgJ and AlgX have similar topology but separate and distinct roles in alginate acetylation. PLoSPathog 10, e1004334 (2014).

    Google Scholar 

  28. Weadge, J.T. et al. Expression, purification, crystallization and preliminary X-ray analysis of Pseudomonas aeruginosa AlgX. Acta Crystallogr. Sect. F Struct. Biol. Cryst. Commun. 66, 588–591 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  29. Riley, L.M. et al. Structural and functional characterization of Pseudomonas aeruginosa AlgX: role of AlgX in alginate acetylation. J. Biol. Chem. 288, 22299–22314 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  30. Akoh, C.C., Lee, G.C., Liaw, Y.C., Huang, T.H. & Shaw, J.F. GDSL family of serine esterases/lipases. Prog. Lipid Res. 43, 534–552 (2004).

    CAS  PubMed  Google Scholar 

  31. Moynihan, P.J. & Clarke, A.J. Assay for peptidoglycan O-acetyltransferase: a potential new antibacterial target. Anal. Biochem. 439, 73–79 (2013).

    CAS  PubMed  Google Scholar 

  32. Moynihan, P.J. & Clarke, A.J. Substrate specificity and kinetic characterization of peptidoglycan O-acetyltransferase B from. Neisseria gonorrhoeae. J. Biol. Chem. 289, 16748–16760 (2014).

    CAS  PubMed  Google Scholar 

  33. Weadge, J.T. & Clarke, A.J. Identification and characterization of O-acetylpeptidoglycan esterase: a novel enzyme discovered in. Neisseria gonorrhoeae. Biochemistry 45, 839–851 (2006).

    CAS  PubMed  Google Scholar 

  34. Moynihan, P.J. & Clarke, A.J. Mechanism of action of peptidoglycan O-acetyltransferase B involves a Ser-His-Asp catalytic triad. Biochemistry 53, 6243–6251 (2014).

    CAS  PubMed  Google Scholar 

  35. Goldschmidt, L., Cooper, D.R., Derewenda, Z.S. & Eisenberg, D. Toward rational protein crystallization: a web server for the design of crystallizable protein variants. Protein Sci. 16, 1569–1576 (2007).

    CAS  PubMed  PubMed Central  Google Scholar 

  36. Almog, O. et al. The 0.93A crystal structure of sphericase: a calcium-loaded serine protease from. Bacillus sphaericus. J. Mol. Biol. 332, 1071–1082 (2003).

    CAS  PubMed  Google Scholar 

  37. Heikinheimo, P., Goldman, A., Jeffries, C. & Ollis, D.L. Of barn owls and bankers: a lush variety of α/β hydrolases. Structure 7, R141–R146 (1999).

    CAS  PubMed  Google Scholar 

  38. Oh, S.Y., Lunderberg, J.M., Chateau, A., Schneewind, O. & Missiakas, D. Genes required for Bacillus anthracis secondary cell wall polysaccharide synthesis. J. Bacteriol. 199, e00613–e00616 (2016).

    PubMed  PubMed Central  Google Scholar 

  39. Moynihan, P.J. & Clarke, A.J. O-acetylation of peptidoglycan in gram-negative bacteria: identification and characterization of peptidoglycan O-acetyltransferase in. Neisseria gonorrhoeae. J. Biol. Chem. 285, 13264–13273 (2010).

    CAS  PubMed  Google Scholar 

  40. Larsen, N.A., Lin, H., Wei, R., Fischbach, M.A. & Walsh, C.T. Structural characterization of enterobactin hydrolase IroE. Biochemistry 45, 10184–10190 (2006).

    CAS  PubMed  Google Scholar 

  41. Franklin, M.J., Douthit, S.A. & McClure, M.A. Evidence that the algI/algJ gene cassette, required for O-acetylation of Pseudomonas aeruginosa alginate, evolved by lateral gene transfer. J. Bacteriol. 186, 4759–4773 (2004).

    CAS  PubMed  PubMed Central  Google Scholar 

  42. Little, D.J. et al. Combining in situ proteolysis and mass spectrometry to crystallize Escherichia coli PgaB. Acta Crystallogr. Sect. F Struct. Biol. Cryst. Commun. 68, 842–845 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  43. Mo, K.F. et al. 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 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  44. Otwinowski, Z. & Minor, W. Processing of X-ray diffraction data collected in oscillation mode. Methods Enzymol. 276, 307–326 (1997).

    CAS  Google Scholar 

  45. Pape, T. & Schneider, T.R. HKL2MAP: a graphical user interface for phasing with SHELX programs. J. Appl. Crystallogr. 37, 843–844 (2004).

    CAS  Google Scholar 

  46. Terwilliger, T.C. Automated main-chain model building by template matching and iterative fragment extension. ActaCrystallogr. D Biol. Crystallogr. 59, 38–44 (2003).

    Google Scholar 

  47. Terwilliger, T.C. et al. Iterative model building, structure refinement and density modification with the PHENIX AutoBuild wizard. ActaCrystallogr. D Biol. Crystallogr. 64, 61–69 (2008).

    CAS  Google Scholar 

  48. Emsley, P. & Cowtan, K. Coot: model-building tools for molecular graphics. ActaCrystallogr. D Biol. Crystallogr. 60, 2126–2132 (2004).

    Google Scholar 

  49. Afonine, P.V. et al. Towards automated crystallographic structure refinement with phenix.refine. ActaCrystallogr. D Biol. Crystallogr. 68, 352–367 (2012).

    CAS  Google Scholar 

  50. Painter, J. & Merritt, E.A. TLSMD web server for the generation of multi-group TLS models. J. Appl. Crystallogr. 39, 109–111 (2006).

    CAS  Google Scholar 

  51. Chen, V.B. et al. MolProbity: all-atom structure validation for macromolecular crystallography. ActaCrystallogr. D Biol. Crystallogr. 66, 12–21 (2010).

    CAS  Google Scholar 

  52. Gasteiger, E. et al. in The Proteomics Protocols Handbook (ed. Walker, J.M.) 571–607 (Humana Press, 2005).

  53. Chenna, R. et al. Multiple sequence alignment with the Clustal series of programs. Nucleic Acids Res. 31, 3497–3500 (2003).

    CAS  PubMed  PubMed Central  Google Scholar 

  54. Yu, N.Y. et al. PSORTb 3.0: improved protein subcellular localization prediction with refined localization subcategories and predictive capabilities for all prokaryotes. Bioinformatics 26, 1608–1615 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  55. Käll, L., Krogh, A. & Sonnhammer, E.L.L.A combined transmembrane topology and signal peptide prediction method. J. Mol. Biol. 338, 1027–1036 (2004).

    PubMed  Google Scholar 

  56. Petersen, T.N., Brunak, S., von Heijne, G. & Nielsen, H. SignalP 4.0: discriminating signal peptides from transmembrane regions. Nat. Methods 8, 785–786 (2011).

    CAS  PubMed  Google Scholar 

  57. Krogh, A., Larsson, B., von Heijne, G. & Sonnhammer, E.L.L. Predicting transmembrane protein topology with a hidden Markov model: application to complete genomes. J. Mol. Biol. 305, 567–580 (2001).

    CAS  PubMed  Google Scholar 

  58. Claros, M.G. & von Heijne, G. TopPred II: an improved software for membrane protein structure predictions. Comput. Appl. Biosci. 10, 685–686 (1994).

    CAS  PubMed  Google Scholar 

  59. Remmert, M., Biegert, A., Hauser, A. & Söding, J. HHblits: lightning-fast iterative protein sequence searching by HMM-HMM alignment. Nat. Methods 9, 173–175 (2011).

    PubMed  Google Scholar 

  60. Laemmli, U.K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680–685 (1970).

    CAS  Google Scholar 

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Acknowledgements

We thank D. Brewer and A. Charchoglyan of the Mass Spectrometry Facility (Advanced Analysis Centre, University of Guelph) for expert technical assistance and advice, both R. Pfoh and N.C. Bamford of the Howell laboratory for their assistance in X-ray data collection, and C. Jones for technical assistance with some of the enzyme assays. P.J. Moynihan, J.M. Pfeffer, J.C. Whitney and J.T. Weadge are thanked for helpful discussions in the early stages of this research. These studies were supported in part by operating grants from the Canadian Institutes of Health Research (CIHR) to A.J.C. (TGC 114045) and P.L.H. (MOP 43998) and the Canadian Glycomics Network (http://dx.doi.org/10.13039/501100009056). J.L. was supported in part by graduate scholarships from the University of Toronto, the Ontario Graduate Scholarship Program, and CIHR. P.L.H. is the recipient of a Canada Research Chair. The National Synchrotron Light Source beam line X29A is supported by the United States Department of Energy Office of Biological and Environmental Research and the National Institutes of Health National Centre for Research Resources.

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Author notes

  1. Dustin J Little

    Present address: Department of Biochemistry and Biomedical Sciences, McMaster University, Hamilton, Ontario, Canada

Authors and Affiliations

  1. Department of Molecular and Cellular Biology, University of Guelph, Guelph, Ontario, Canada

    David Sychantha & Anthony J Clarke

  2. Program in Molecular Medicine, Research Institute, The Hospital for Sick Children, Toronto, Ontario, Canada

    Dustin J Little & P Lynne Howell

  3. Department of Biochemistry, University of Toronto, Ontario, Canada

    Dustin J Little & P Lynne Howell

  4. Complex Carbohydrate Research Center, University of Georgia, Athens, Georgia, USA

    Robert N Chapman & Geert-Jan Boons

  5. Photon Science Division, Brookhaven National Laboratory, Upton, New York, USA

    Howard Robinson

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Contributions

D.S. conceived all experiments and performed all gene cloning, site-directed mutagenesis and expression experiments; purification of recombinant proteins; enzyme kinetic and MS analyses; crystallization of proteins; collection and analysis of crystallographic data; and solving of crystal structures; and made structural figures and tables; D.J.L. and P.L.H. oversaw the collection and analysis of crystallographic data; R.N.C. and G.-J.B. synthesized the SCWP model compound, MGG, and performed all NMR experiments; H.R. collected crystallographic data; A.J.C. helped conceive the cloning, protein purification, kinetic and MS experiments, analyzed data and oversaw the work of D.S. D.S. and A.J.C. wrote the paper with contributions from all authors.

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Correspondence to Anthony J Clarke.

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Supplementary Results, Supplementary Tables 1–4 and Supplementary Figures 1–18 (PDF 9039 kb)

Life Sciences Reporting Summary (PDF 129 kb)

Supplementary Note 1

Synthesis of MGG (PDF 602 kb)

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Sychantha, D., Little, D., Chapman, R. et al. PatB1 is an O-acetyltransferase that decorates secondary cell wall polysaccharides. Nat Chem Biol 14, 79–85 (2018). https://doi.org/10.1038/nchembio.2509

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