Putative and known polysaccharide deacetylases (PDAs) from B. anthracis have key roles in resistance to host lysozyme, stabilization of the cell wall, biogenesis of peptidoglycan (PG) and for neutral polysaccharide modification and attachment to PG. Here we elucidated the physiological role of the putative PDA BA1836 from B. anthracis . The ba 1836 gene was expressed upon entrance into the stationary phase of growth and enhanced during the early stages of sporulation. The Δ ba 1836 knockout strain had normal growth rate, did not exhibit any significant alterations in PG pattern of stationary phase cells and was not sensitive to lysozyme, but showed a defect in cell separation. Strikingly, the Δ ba 1836 mutant strain exhibited a severe delay in spore development although mature spores were ultimately developed and had normal morphology. Additionally, digestion of Δ ba 1836 mutant spore PG with mutanolysin produced an almost identical muropeptide pattern compared to peptidoglycan from wild type spores, although the amount of all muropeptides was significantly reduced. Finally, knockout spores exhibited a lower germination rate. To our knowledge, BA1836 has a unique role, among the presently characterized PDAs from B. anthracis , in spore development and germination.
Keywords<i>Bacillus anthracis</i>Polysaccharide DeacetylasesBA1836SporesGerminationSpore Development
Mock, M. and Fouet, A. (2001) Anthrax. Annual Review of Microbiology, 55, 647-671. https://doi.org/10.1146/annurev.micro.55.1.647
Tan, I.S. and Ramamurthi, K.S. (2014) Spore Formation in Bacillus subtilis. Environmental Microbiology Reports, 6, 212-225. https://doi.org/10.1111/1758-2229.12130
Henriques, A.O. and Moran, C.P. (2007) Structure, Assembly, and Function of the Spore Surface Layers. Annual Review of Microbiology, 61, 555-588. https://doi.org/10.1146/annurev.micro.61.080706.093224
Driks, A. (2009) The Bacillus anthracis Spore. Molecular Aspects of Medicine, 30, 368-373. https://doi.org/10.1016/j.mam.2009.08.001
Errington, J. (2003) Regulation of Endospore Formation in Bacillus subtilis. Nature Reviews Microbiology, 1, 117-126. https://doi.org/10.1038/nrmicro750
Piggot, P.J. and Hilbert, D.W. (2004) Sporulation of Bacillus subtilis. Current Opinion in Microbiology, 7, 579-586. https://doi.org/10.1016/j.mib.2004.10.001
Boneca, I.G. (2005) The Role of Peptidoglycan in Pathogenesis. Current Opinion in Microbiology, 8, 46-53. https://doi.org/10.1016/j.mib.2004.12.008
Meador-Parton, J. and Popham, D.L. (2000) Structural Analysis of Bacillus subtilis Spore Peptidoglycan during Sporulation. Journal of Bacteriology, 182, 4491-4499. https://doi.org/10.1128/JB.182.16.4491-4499.2000
Gilmore, M.E., Bandyopadhyay, D., Dean, A.M., Linnstaedt, S.D. and Popham, D.L. (2004) Production of Muramic Delta-Lactam in Bacillus subtilis Spore Peptidoglycan. Journal of Bacteriology, 186, 80-89. https://doi.org/10.1128/JB.186.1.80-89.2004
Popham, D.L. (2002) Specialized Peptidoglycan of the Bacterial Endospore: The Inner Wall of the Lockbox. Cellular and Molecular Life Sciences, 59, 426-433. https://doi.org/10.1007/s00018-002-8435-5
Dowd, M.M., Orsburn, B. and Popham, D.L. (2008) Cortex Peptidoglycan Lytic Activity in Germinating Bacillus anthracis Spores. Journal of Bacteriology, 190, 4541-4548. https://doi.org/10.1128/JB.00249-08
Dworkin, J. (2014) Protein Targeting during Bacillus subtilis Sporulation. Microbiology Spectrum, 2, TBS-0006-2012. https://doi.org/10.1128/microbiolspec.TBS-0006-2012
Vollmer, W. and Tomasz, A. (2000) The pgdA Gene Encodes for a Peptidoglycan N-Acetylglucosamine Deacetylase in Streptococcus pneumoniae. The Journal of Biological Chemistry, 275, 20496-20501. https://doi.org/10.1074/jbc.M910189199
Boneca, I.G., Dussurget, O., Cabanes, D., Nahori, M.A., Sousa, S., Lecuit, M., et al. (2007) A Critical Role for Peptidoglycan N-Deacetylation in Listeria Evasion from the Host Innate Immune System. Proceedings of the National Academy of Sciences of the United States of America, 104, 997-1002. https://doi.org/10.1073/pnas.0609672104
Fittipaldi, N., Sekizaki, T., Takamatsu, D., de la Cruz Dominguez-Punaro, M., Harel, J., Bui, N.K., et al. (2008) Significant Contribution of the pgdA Gene to the Virulence of Streptococcus suis. Molecular Microbiology, 70, 1120-1135. https://doi.org/10.1111/j.1365-2958.2008.06463.x
Atrih, A. and Foster, S.J. (2001) Analysis of the Role of Bacterial Endospore Cortex Structure in Resistance Properties and Demonstration of Its Conservation amongst Species. Journal of Applied Microbiology, 91, 364-372. https://doi.org/10.1046/j.1365-2672.2001.01394.x
Driks, A. (2002) Maximum Shields: The Assembly and Function of the Bacterial Spore Coat. Trends in Microbiology, 10, 251-254. https://doi.org/10.1016/S0966-842X(02)02373-9
Hanna, P.C. and Ireland, J.A. (1999) Understanding Bacillus anthracis Pathogenesis. Trends in Microbiology, 7, 180-182. https://doi.org/10.1016/S0966-842X(99)01507-3
Moir, A., Corfe, B.M. and Behravan, J. (2002) Spore Germination. Cellular and Molecular Life Sciences, 59, 403-409. https://doi.org/10.1007/s00018-002-8432-8
Ireland, J.A. and Hanna, P.C. (2002) Amino Acid- and Purine Ribonucleoside-Induced Germination of Bacillus anthracis DeltaSterne Endospores: gerS Mediates Responses to Aromatic Ring Structures. Journal of Bacteriology, 184, 1296-1303. https://doi.org/10.1128/JB.184.5.1296-1303.2002
Setlow, P. (2013) When the Sleepers Wake: The Germination of Spores of Bacillus Species. Journal of Applied Microbiology, 115, 1251-1268. https://doi.org/10.1111/jam.12343
Moir, A. and Cooper, G. (2015) Spore Germination. Microbiology Spectrum, 3. https://doi.org/10.1128/microbiolspec.TBS-0014-2012
Psylinakis, E., Boneca, I.G., Mavromatis, K., Deli, A., Hayhurst, E., Foster, S.J., et al. (2005) Peptidoglycan N-acetylglucosamine Deacetylases from Bacillus cereus, Highly Conserved Proteins in Bacillus anthracis. The Journal of Biological Chemistry, 280, 30856-30863. https://doi.org/10.1074/jbc.M407426200
Caufrier, F., Martinou, A., Dupont, C. and Bouriotis, V. (2003) Carbohydrate Esterase Family 4 Enzymes: Substrate Specificity. Carbohydrate Research, 338, 687-692. https://doi.org/10.1016/S0008-6215(03)00002-8
Kafetzopoulos, D., Martinou, A. and Bouriotis, V. (1993) Bioconversion of Chitin to Chitosan: Purification and Characterization of Chitin Deacetylase from Mucor Rouxii. Proceedings of the National Academy of Sciences of the United States of America, 90, 2564-2568. https://doi.org/10.1073/pnas.90.7.2564
Tsigos, I., Martinou, A., Kafetzopoulos, D. and Bouriotis, V. (2000) Chitin Deacetylases: New, Versatile Tools in Biotechnology. Trends in Biotechnology, 18, 305-312. https://doi.org/10.1016/S0167-7799(00)01462-1
Balomenou, S., Fouet, A., Tzanodaskalaki, M., Couture-Tosi, E., Bouriotis, V. and Boneca, I.G. (2013) Distinct Functions of Polysaccharide Deacetylases in Cell Shape, Neutral Polysaccharide Synthesis and Virulence of Bacillus anthracis. Molecular Microbiology, 87, 867-883. https://doi.org/10.1111/mmi.12137
Arnaouteli, S., Giastas, P. andreou, A., Tzanodaskalaki, M., Aldridge, C., Tzartos, S.J., et al. (2015) Two Putative Polysaccharide Deacetylases Are Required for Osmotic Stability and Cell Shape Maintenance in Bacillus anthracis. The Journal of Biological Chemistry, 290, 13465-13478. https://doi.org/10.1074/jbc.M115.640029
Balomenou, S., Koutsioulis, D., Tomatsidou, A., Tzanodaskalaki, M., Petratos, K. and Bouriotis, V. (2018) Polysaccharide Deacetylases Serve as New Targets for the Design of Inhibitors against Bacillus anthracis and Bacillus cereus. Bioorganic & Medicinal Chemistry, 26, 3845-3851. https://doi.org/10.1016/j.bmc.2018.06.045
Fukushima, T., Yamamoto, H., Atrih, A., Foster, S.J. and Sekiguchi, J. (2002) A Polysaccharide Deacetylase Gene (pdaA) Is Required for Germination and for Production of Muramic Delta-Lactam Residues in the Spore Cortex of Bacillus subtilis. Journal of Bacteriology, 184, 6007-6015. https://doi.org/10.1128/JB.184.21.6007-6015.2002
Fukushima, T., Tanabe, T., Yamamoto, H., Hosoya, S., Sato, T., Yoshikawa, H., et al. (2004) Characterization of a Polysaccharide Deacetylase Gene Homologue (pdaB) on Sporulation of Bacillus subtilis. The Journal of Biochemistry, 136, 283-291. https://doi.org/10.1093/jb/mvh151
Kim, H.U. and Goepfert, J.M. (1974) A Sporulation Medium for Bacillus anthracis. Journal of Applied Bacteriology, 37, 265-267. https://doi.org/10.1111/j.1365-2672.1974.tb00438.x
Sterlini, J.M. and Mandelstam, J. (1969) Commitment to Sporulation in Bacillus subtilis and Its Relationship to Development of Actinomycin Resistance. Biochemical Journal, 113, 29-37. https://doi.org/10.1042/bj1130029
Janes, B.K. and Stibitz, S. (2006) Routine Markerless Gene Replacement in Bacillus anthracis. Infection and Immunity, 74, 1949-1953. https://doi.org/10.1128/IAI.74.3.1949-1953.2006
Pezard, C., Berche, P. and Mock, M. (1991) Contribution of Individual Toxin Components to Virulence of Bacillus anthracis. Infection and Immunity, 59, 3472-3477.
Trieu-Cuot, P., Carlier, C., Martin, P. and Courvalin, P. (1987) Plasmid Transfer by Conjugation from Escherichia coli to Gram-Positive Bacteria. FEMS Microbiology Letters, 48, 289-294. https://doi.org/10.1111/j.1574-6968.1987.tb02558.x
Plaut, R.D. and Stibitz, S. (2015) Improvements to a Markerless Allelic Exchange System for Bacillus anthracis. PLoS ONE, 10, e0142758. https://doi.org/10.1371/journal.pone.0142758
Fortinea, N., Trieu-Cuot, P., Gaillot, O., Pellegrini, E., Berche, P. and Gaillard, J.L. (2000) Optimization of Green Fluorescent Protein Expression Vectors for in Vitro and in Vivo Detection of Listeria monocytogenes. Research in Microbiology, 151, 353-360. https://doi.org/10.1016/S0923-2508(00)00158-3
Rygus, T. and Hillen, W. (1991) Inducible High-Level Expression of Heterologous Genes in Bacillus megaterium Using the Regulatory Elements of the Xylose-Utilization Operon. Applied Microbiology and Biotechnology, 35, 594-599. https://doi.org/10.1007/BF00169622
Koehler, T.M., Dai, Z. and Kaufman-Yarbray, M. (1994) Regulation of the Bacillus anthracis Protective Antigen Gene: CO2 and a Trans-Acting Element Activate Transcription from One of Two Promoters. Journal of Bacteriology, 176, 586. https://doi.org/10.1128/jb.176.3.586-595.1994
Agaisse, H. and Lereclus, D. (1994) Structural and Functional Analysis of the Promoter Region Involved in Full Expression of the cryIIIA Toxin Gene of Bacillus thuringiensis. Molecular Microbiology, 13, 97-107. https://doi.org/10.1111/j.1365-2958.1994.tb00405.x
Horton, R.M., Cai, Z.L., Ho, S.N. and Pease, L.R. (1990) Gene Splicing by Overlap Extension: Tailor-Made Genes Using the Polymerase Chain Reaction. Biotechniques, 8, 528-535.
Camp, A.H. and Losick, R. (2009) A Feeding Tube Model for Activation of a Cell-Specific Transcription Factor during Sporulation in Bacillus subtilis. Genes & Development, 23, 1014-1024. https://doi.org/10.1101/gad.1781709
Miller, J. (1972) Experiments in Molecular Genetics. Cold Spring Harbor Laboratory Press, Cold Spring Harbor.
Traag, B.A., Pugliese, A., Eisen, J.A. and Losick, R. (2013) Gene Conservation among Endospore-Forming Bacteria Reveals Additional Sporulation Genes in Bacillus subtilis. Journal of Bacteriology, 195, 253-260. https://doi.org/10.1128/JB.01778-12
Mesnage, S., Tosi-Couture, E. and Fouet, A. (1999) Production and Cell Surface Anchoring of Functional Fusions between the SLH Motifs of the Bacillus anthracis S-Layer Proteins and the Bacillus subtilis Levansucrase. Molecular Microbiology, 31, 927-936. https://doi.org/10.1046/j.1365-2958.1999.01232.x
Popham, D.L., Helin, J., Costello, C.E. and Setlow, P. (1996) Analysis of the Peptidoglycan Structure of Bacillus subtilis Endospores. Journal of Bacteriology, 178, 6451-6458. https://doi.org/10.1128/jb.178.22.6451-6458.1996
Bui, N.K., Eberhardt, A., Vollmer, D., Kern, T., Bougault, C., Tomasz, A., et al. (2012) Isolation and Analysis of Cell Wall Components from Streptococcus pneumoniae. Analytical Biochemistry, 421, 657-666. https://doi.org/10.1016/j.ab.2011.11.026
Mignot, T., Mesnage, S., Couture-Tosi, E., Mock, M. and Fouet, A. (2002) Developmental Switch of S-Layer Protein Synthesis in Bacillus anthracis. Molecular Microbiology, 43, 1615-1627. https://doi.org/10.1046/j.1365-2958.2002.02852.x
Carr, K.A., Lybarger, S.R. anderson, E.C., Janes, B.K. and Hanna, P.C. (2010) The Role of Bacillus anthracis Germinant Receptors in Germination and Virulence. Molecular Microbiology, 75, 365-375. https://doi.org/10.1111/j.1365-2958.2009.06972.x
Fazzini, M.M., Schuch, R. and Fischetti, V.A. (2010) A Novel Spore Protein, ExsM, Regulates Formation of the Exosporium in Bacillus cereus and Bacillus anthracis and Affects Spore Size and Shape. Journal of Bacteriology, 192, 4012-4021. https://doi.org/10.1128/JB.00197-10
Krogh, A., Larsson, B., von Heijne, G. and Sonnhammer, E.L. (2001) Predicting Transmembrane Protein Topology with a Hidden Markov Model: Application to Complete Genomes. Journal of Molecular Biology, 305, 567-580. https://doi.org/10.1006/jmbi.2000.4315
Bendtsen, J.D., Nielsen, H., Widdick, D., Palmer, T. and Brunak, S. (2005) Prediction of Twin-Arginine Signal Peptides. BMC Bioinformatics, 6, 167. https://doi.org/10.1186/1471-2105-6-167
Nielsen, H. (2017) Predicting Secretory Proteins with SignalP. Methods in Molecular Biology, 1611, 59-73. https://doi.org/10.1007/978-1-4939-7015-5_6
Blair, D.E., Schuttelkopf, A.W., MacRae, J.I. and van Aalten, D.M. (2005) Structure and Metal-Dependent Mechanism of Peptidoglycan Deacetylase, a Streptococcal Virulence Factor. Proceedings of the National Academy of Sciences of the United States of America, 102, 15429-15434. https://doi.org/10.1073/pnas.0504339102
Fadouloglou, V.E., Balomenou, S., Aivaliotis, M., Kotsifaki, D., Arnaouteli, S., Tomatsidou, A., et al. (2017) Unusual α-Carbon Hydroxylation of Proline Promotes Active-Site Maturation. Journal of the American Chemical Society, 139, 5330-5337. https://doi.org/10.1021/jacs.6b12209
Fadouloglou, V.E., Kapanidou, M., Agiomirgianaki, A., Arnaouteli, S., Bouriotis, V., Glykos, N.M., et al. (2013) Structure Determination through Homology Modelling and Torsion-Angle Simulated Annealing: Application to a Polysaccharide Deacetylase from Bacillus cereus. Acta Crystallographica. Section D, Biological Crystallography, 69, 276-283. https://doi.org/10.1107/S0907444912045829
Giastas, P., Andreou, A., Papakyriakou, A., Koutsioulis, D., Balomenou, S., Tzartos, S.J., et al. (2018) Structures of the Peptidoglycan N-Acetylglucosamine Deacetylase Bc1974 and Its Complexes with Zinc Metalloenzyme Inhibitors. Biochemistry, 57, 753-763. https://doi.org/10.1021/acs.biochem.7b00919
Oberbarnscheidt, L., Taylor, E.J., Davies, G.J. and Gloster, T.M. (2007) Structure of a Carbohydrate Esterase from Bacillus anthracis. Proteins, 66, 250-252. https://doi.org/10.1002/prot.21217
Blair, D.E. and van Aalten, D.M. (2004) Structures of Bacillus subtilis PdaA, a Family 4 Carbohydrate Esterase, and a Complex with N-acetylglucosamine. FEBS Letters, 570, 13-19. https://doi.org/10.1016/j.febslet.2004.06.013
Strunk, R.J., Piemonte, K.M., Petersen, N.M., Koutsioulis, D., Bouriotis, V., Perry, K., et al. (2014) Structure Determination of BA0150, a Putative Polysaccharide Deacetylase from Bacillus anthracis. Acta Crystallographica Section F: Structural Biology Communications, 70, 156-159. https://doi.org/10.1107/S2053230X13034262
Taylor, E.J., Gloster, T.M., Turkenburg, J.P., Vincent, F., Brzozowski, A.M., Dupont, C., et al. (2006) Structure and Activity of Two Metal Ion-Dependent Acetylxylan Esterases Involved in Plant Cell Wall Degradation Reveals a Close Similarity to Peptidoglycan Deacetylases. The Journal of Biological Chemistry, 281, 10968-10975. https://doi.org/10.1074/jbc.M513066200
Blair, D.E., Hekmat, O., Schuttelkopf, A.W., Shrestha, B., Tokuyasu, K., Withers, S.G., et al. (2006) Structure and Mechanism of Chitin Deacetylase from the Fungal Pathogen Colletotrichum lindemuthianum. Biochemistry, 45, 9416-9426. https://doi.org/10.1021/bi0606694
Robert, X. and Gouet, P. (2014) Deciphering Key Features in Protein Structures with the New ENDscript Server. Nucleic Acids Research, 42, W320-W324. https://doi.org/10.1093/nar/gku316
Bergman, N.H. anderson, E.C., Swenson, E.E., Niemeyer, M.M., Miyoshi, A.D. and Hanna, P.C. (2006) Transcriptional Profiling of the Bacillus anthracis Life Cycle in Vitro and an Implied Model for Regulation of Spore Formation. Journal of Bacteriology, 188, 6092-6100. https://doi.org/10.1128/JB.00723-06
Brunsing, R.L., La Clair, C., Tang, S., Chiang, C., Hancock, L.E., Perego, M., et al. (2005) Characterization of Sporulation Histidine Kinases of Bacillus anthracis. Journal of Bacteriology, 187, 6972-6981. https://doi.org/10.1128/JB.187.20.6972-6981.2005
Marraffini, L.A. and Schneewind, O. (2006) Targeting Proteins to the Cell Wall of Sporulating Bacillus anthracis. Molecular Microbiology, 62, 1402-1417. https://doi.org/10.1111/j.1365-2958.2006.05469.x
Vasudevan, P., Weaver, A., Reichert, E.D., Linnstaedt, S.D. and Popham, D.L. (2007) Spore Cortex Formation in Bacillus subtilis Is Regulated by Accumulation of Peptidoglycan Precursors under the Control of Sigma K. Molecular Microbiology, 65, 1582-1594. https://doi.org/10.1111/j.1365-2958.2007.05896.x
Ramamurthi, K.S., Clapham, K.R. and Losick, R. (2006) Peptide Anchoring Spore Coat Assembly to the Outer Forespore Membrane in Bacillus subtilis. Molecular Microbiology, 62, 1547-1557. https://doi.org/10.1111/j.1365-2958.2006.05468.x
Cutting, S. anderson, M., Lysenko, E., Page, A., Tomoyasu, T., Tatematsu, K., et al. (1997) SpoVM, a Small Protein Essential to Development in Bacillus subtilis, Interacts with the ATP-Dependent Protease FtsH. Journal of Bacteriology, 179, 5534-5542. https://doi.org/10.1128/jb.179.17.5534-5542.1997
Prajapati, R.S., Ogura, T. and Cutting, S.M. (2000) Structural and Functional Studies on an FtsH Inhibitor from Bacillus subtilis. Biochimica et Biophysica Acta, 1475, 353-359. https://doi.org/10.1016/S0304-4165(00)00089-1
Galperin, M.Y., Mekhedov, S.L., Puigbo, P., Smirnov, S., Wolf, Y.I. and Rigden, D.J. (2012) Genomic Determinants of Sporulation in Bacilli and Clostridia: Towards the Minimal Set of Sporulation-Specific Genes. Environmental Microbiology, 14, 2870-2890. https://doi.org/10.1111/j.1462-2920.2012.02841.x
Adrain, C. and Freeman, M. (2012) New Lives for Old: Evolution of Pseudoenzyme Function Illustrated by iRhoms. Nature Reviews Molecular Cell Biology, 13, 489-498. https://doi.org/10.1038/nrm3392
Pokrovskaya, V., Poloczek, J., Little, D.J., Griffiths, H., Howell, P.L. and Nitz, M. (2013) Functional Characterization of Staphylococcus epidermidis IcaB, a de-N-Acetylase Important for Biofilm Formation. Biochemistry, 52, 5463-5471. https://doi.org/10.1021/bi400836g
Little, D.J., Poloczek, J., Whitney, J.C., Robinson, H., Nitz, M. and Howell, P.L. (2012) The Structure- and Metal-Dependent Activity of Escherichia coli PgaB Provides Insight into the Partial de-N-acetylation of Poly-β-1,6-N-acetyl-D-glucosamine. The Journal of Biological Chemistry, 287, 31126-31137. https://doi.org/10.1074/jbc.M112.390005