Role of the Putative Polysaccharide Deacetylase BA1836 from B. anthracis in Spore Development and Germination

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 ba1836 gene was expressed upon entrance into the stationary phase of growth and enhanced during the early stages of sporulation. The Δba1836 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 Δba1836 mutant strain exhibited a severe delay in spore development although mature spores were ultimately developed and had normal morphology. Additionally, digestion of Δba1836 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.

[1]  D. V. van Aalten,et al.  Structures of Bacillus subtilis PdaA, a family 4 carbohydrate esterase, and a complex with N‐acetyl‐glucosamine , 2004, FEBS letters.

[2]  K. E. Cole,et al.  Structure determination of BA0150, a putative polysaccharide deacetylase from Bacillus anthracis. , 2014, Acta crystallographica. Section F, Structural biology communications.

[3]  N. Bergman,et al.  Transcriptional Profiling of the Bacillus anthracis Life Cycle In Vitro and an Implied Model for Regulation of Spore Formation , 2006, Journal of bacteriology.

[4]  L. Marraffini,et al.  Targeting proteins to the cell wall of sporulating Bacillus anthracis , 2006, Molecular microbiology.

[5]  A. Moir,et al.  Spore germination , 2002, Cellular and Molecular Life Sciences CMLS.

[6]  V. Bouriotis,et al.  Carbohydrate esterase family 4 enzymes: substrate specificity. , 2003, Carbohydrate research.

[7]  P. Howell,et al.  Functional characterization of Staphylococcus epidermidis IcaB, a de-N-acetylase important for biofilm formation. , 2013, Biochemistry.

[8]  D Kafetzopoulos,et al.  Chitin deacetylases: new, versatile tools in biotechnology. , 2000, Trends in biotechnology.

[9]  J. Sekiguchi,et al.  Characterization of a polysaccharide deacetylase gene homologue (pdaB) on sporulation of Bacillus subtilis. , 2004, Journal of biochemistry.

[10]  R. Losick,et al.  Gene Conservation among Endospore-Forming Bacteria Reveals Additional Sporulation Genes in Bacillus subtilis , 2012, Journal of bacteriology.

[11]  G. Davies,et al.  Structure of a carbohydrate esterase from Bacillus anthracis , 2006, Proteins.

[12]  D Kafetzopoulos,et al.  Bioconversion of chitin to chitosan: purification and characterization of chitin deacetylase from Mucor rouxii. , 1993, Proceedings of the National Academy of Sciences of the United States of America.

[13]  M. Prevost,et al.  A critical role for peptidoglycan N-deacetylation in Listeria evasion from the host innate immune system , 2007, Proceedings of the National Academy of Sciences.

[14]  T. Sekizaki,et al.  Significant contribution of the pgdA gene to the virulence of Streptococcus suis , 2008, Molecular microbiology.

[15]  R. Losick,et al.  Peptide anchoring spore coat assembly to the outer forespore membrane in Bacillus subtilis , 2006, Molecular microbiology.

[16]  Xavier Robert,et al.  Deciphering key features in protein structures with the new ENDscript server , 2014, Nucleic Acids Res..

[17]  Jeffrey H. Miller.  Experiments in molecular genetics , 1972 .

[18]  W. Hillen,et al.  Inducible high-level expression of heterologous genes in Bacillus megaterium using the regulatory elements of the xylose-utilization operon , 1991, Applied Microbiology and Biotechnology.

[19]  P. Howell,et al.  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* , 2012, The Journal of Biological Chemistry.

[20]  P. Setlow,et al.  Analysis of the peptidoglycan structure of Bacillus subtilis endospores , 1996, Journal of bacteriology.

[21]  M. Mock,et al.  Developmental switch of S‐layer protein synthesis in Bacillus anthracis , 2002, Molecular microbiology.

[22]  P. Hanna,et al.  Understanding Bacillus anthracis pathogenesis. , 1999, Trends in microbiology.

[23]  P. Hanna,et al.  Amino Acid- and Purine Ribonucleoside-Induced Germination of Bacillus anthracis ΔSterne Endospores: gerS Mediates Responses to Aromatic Ring Structures , 2002, Journal of bacteriology.

[24]  S. Stibitz,et al.  Improvements to a Markerless Allelic Exchange System for Bacillus anthracis , 2015, PloS one.

[25]  J. Errington.  Regulation of endospore formation in Bacillus subtilis , 2003, Nature Reviews Microbiology.

[26]  D. Hilbert,et al.  Sporulation of Bacillus subtilis. , 2004, Current opinion in microbiology.

[27]  A. Driks.  Maximum shields: the assembly and function of the bacterial spore coat. , 2002, Trends in microbiology.

[28]  R. Losick,et al.  A feeding tube model for activation of a cell-specific transcription factor during sporulation in Bacillus subtilis. , 2009, Genes & development.

[29]  S. Foster,et al.  A Polysaccharide Deacetylase Gene (pdaA) Is Required for Germination and for Production of Muramic δ-Lactam Residues in the Spore Cortex of Bacillus subtilis , 2002, Journal of bacteriology.

[30]  A. Tomatsidou,et al.  Polysaccharide deacetylases serve as new targets for the design of inhibitors against Bacillus anthracis and Bacillus cereus. , 2018, Bioorganic & medicinal chemistry.

[31]  A. Tomasz,et al.  Isolation and analysis of cell wall components from Streptococcus pneumoniae. , 2012, Analytical biochemistry.

[32]  S. Stibitz,et al.  Routine Markerless Gene Replacement in Bacillus anthracis , 2006, Infection and Immunity.

[33]  M. Mock,et al.  Contribution of individual toxin components to virulence of Bacillus anthracis , 1991, Infection and immunity.

[34]  M. Kokkinidis,et al.  Structure determination through homology modelling and torsion-angle simulated annealing: application to a polysaccharide deacetylase from Bacillus cereus. , 2013, Acta crystallographica. Section D, Biological crystallography.

[35]  A. Tomasz,et al.  The pgdA Gene Encodes for a PeptidoglycanN-Acetylglucosamine Deacetylase in Streptococcus pneumoniae * , 2000, The Journal of Biological Chemistry.

[36]  J. Dworkin.  Protein Targeting during Bacillus subtilis Sporulation. , 2014, Microbiology spectrum.

[37]  P. Berche,et al.  Optimization of green fluorescent protein expression vectors for in vitro and in vivo detection of Listeria monocytogenes. , 2000, Research in microbiology.

[38]  A. W. Schüttelkopf,et al.  Structure and metal-dependent mechanism of peptidoglycan deacetylase, a streptococcal virulence factor , 2005, Proceedings of the National Academy of Sciences of the United States of America.

[39]  A. Fouet,et al.  Distinct functions of polysaccharide deacetylases in cell shape, neutral polysaccharide synthesis and virulence of Bacillus anthracis , 2013, Molecular microbiology.

[40]  Michael Y. Galperin,et al.  Genomic determinants of sporulation in Bacilli and Clostridia: towards the minimal set of sporulation-specific genes , 2012, Environmental microbiology.

[41]  V. Fischetti,et al.  A Novel Spore Protein, ExsM, Regulates Formation of the Exosporium in Bacillus cereus and Bacillus anthracis and Affects Spore Size and Shape , 2010, Journal of bacteriology.

[42]  S. Ho,et al.  Gene splicing by overlap extension: tailor-made genes using the polymerase chain reaction. , 2013, BioTechniques.

[43]  S. Foster,et al.  Analysis of the role of bacterial endospore cortex structure in resistance properties and demonstration of its conservation amongst species , 2001, Journal of applied microbiology.

[44]  M. Freeman,et al.  New lives for old: evolution of pseudoenzyme function illustrated by iRhoms , 2012, Nature Reviews Molecular Cell Biology.

[45]  J. Goepfert,et al.  A sporulation medium for Bacillus anthracis. , 1974, The Journal of applied bacteriology.

[46]  D. Popham.  Specialized peptidoglycan of the bacterial endospore: the inner wall of the lockbox , 2002, Cellular and Molecular Life Sciences CMLS.

[47]  D. Popham,et al.  Production of muramic delta-lactam in Bacillus subtilis spore peptidoglycan. , 2004, Journal of bacteriology.

[48]  E. Eliopoulos,et al.  Two Putative Polysaccharide Deacetylases Are Required for Osmotic Stability and Cell Shape Maintenance in Bacillus anthracis , 2015, The Journal of Biological Chemistry.

[49]  P. Setlow.  Summer meeting 2013 – when the sleepers wake: the germination of spores of Bacillus species , 2013, Journal of applied microbiology.

[50]  S. Foster,et al.  Peptidoglycan N-Acetylglucosamine Deacetylases from Bacillus cereus, Highly Conserved Proteins in Bacillus anthracis* , 2005, Journal of Biological Chemistry.

[51]  S. Withers,et al.  Structure and mechanism of chitin deacetylase from the fungal pathogen Colletotrichum lindemuthianum. , 2006, Biochemistry.

[52]  J. Mandelstam,et al.  Commitment to sporulation in Bacillus subtilis and its relationship to development of actinomycin resistance. , 1969, The Biochemical journal.

[53]  Søren Brunak,et al.  Prediction of twin-arginine signal peptides , 2005, BMC Bioinformatics.

[54]  A. Fouet,et al.  Production and cell surface anchoring of functional fusions between the SLH motifs of the Bacillus anthracis S‐layer proteins and the Bacillus subtilis levansucrase , 1999, Molecular microbiology.

[55]  S. Pergantis,et al.  Unusual α-Carbon Hydroxylation of Proline Promotes Active-Site Maturation. , 2017, Journal of the American Chemical Society.

[56]  P. Trieu-Cuot,et al.  Plasmid transfer by conjugation from Escherichia coli to Gram‐positive bacteria , 1987 .

[57]  K. Ramamurthi,et al.  Spore formation in Bacillus subtilis. , 2014, Environmental microbiology reports.

[58]  Henrik Nielsen,et al.  Predicting Secretory Proteins with SignalP. , 2017, Methods in molecular biology.

[59]  A. Driks.  The Bacillus anthracis spore. , 2009, Molecular aspects of medicine.

[60]  T. Koehler,et al.  Regulation of the Bacillus anthracis protective antigen gene: CO2 and a trans-acting element activate transcription from one of two promoters , 1994, Journal of bacteriology.

[61]  H. Agaisse,et al.  Structural and functional analysis of the promoter region involved in full expression of the cryIIIA toxin gene of Bacillus thuringiensis , 1994, Molecular microbiology.

[62]  D. Popham,et al.  Structural Analysis of Bacillus subtilisSpore Peptidoglycan during Sporulation , 2000, Journal of bacteriology.

[63]  A. Brzozowski,et al.  Structure and Activity of Two Metal Ion-dependent Acetylxylan Esterases Involved in Plant Cell Wall Degradation Reveals a Close Similarity to Peptidoglycan Deacetylases* , 2006, Journal of Biological Chemistry.

[64]  D. Popham,et al.  Spore cortex formation in Bacillus subtilis is regulated by accumulation of peptidoglycan precursors under the control of sigma K , 2007, Molecular microbiology.

[65]  S. Cutting,et al.  Structural and functional studies on an FtsH inhibitor from Bacillus subtilis. , 2000, Biochimica et biophysica acta.

[66]  I. Boneca,et al.  The role of peptidoglycan in pathogenesis. , 2005, Current opinion in microbiology.

[67]  A. Krogh,et al.  Predicting transmembrane protein topology with a hidden Markov model: application to complete genomes. , 2001, Journal of molecular biology.

[68]  A. Henriques,et al.  Structure, assembly, and function of the spore surface layers. , 2007, Annual review of microbiology.

[69]  B. Janes,et al.  The role of Bacillus anthracis germinant receptors in germination and virulence , 2010, Molecular microbiology.

[70]  T. Tatsuta,et al.  SpoVM, a small protein essential to development in Bacillus subtilis, interacts with the ATP-dependent protease FtsH , 1997, Journal of bacteriology.