1887

Abstract

The bacterial endospore is the most resilient biological structure known. Multiple protective integument layers shield the spore core and promote spore dehydration and dormancy. Dormancy is broken when a spore germinates and becomes a metabolically active vegetative cell. Germination requires the breakdown of a modified layer of peptidoglycan (PG) known as the spore cortex. This study reports and analyses of the SleL protein. SleL is a spore cortex lytic enzyme composed of three conserved domains: two N-terminal LysM domains and a C-terminal glycosyl hydrolase family 18 domain. Derivatives of SleL containing both, one or no LysM domains were purified and characterized. SleL is incapable of digesting intact cortical PG of either decoated spores or purified spore sacculi. However, SleL derivatives can hydrolyse fragmented PG substrates containing muramic-δ-lactam recognition determinants. The muropeptides that result from SleL hydrolysis are the products of -acetylglucosaminidase activity. These muropeptide products are small and readily released from the cortex matrix. Loss of the LysM domain(s) decreases both PG binding and hydrolysis activity but these domains do not appear to determine specificity for muramic-δ-lactam. When the SleL derivatives are expressed , those proteins lacking one or both LysM domains do not associate with the spore. Instead, these proteins remain in the mother cell and are apparently degraded. SleL with both LysM domains localizes to the coat or cortex of the endospore. The information revealed by elucidating the role of SleL and its domains in sporulation and germination is important in designing new spore decontamination methods. By exploiting germination-specific lytic enzymes, eradication techniques may be greatly simplified.

Loading

Article metrics loading...

/content/journal/micro/10.1099/mic.0.056630-0
2012-05-01
2020-08-05
Loading full text...

Full text loading...

/deliver/fulltext/micro/158/5/1359.html?itemId=/content/journal/micro/10.1099/mic.0.056630-0&mimeType=html&fmt=ahah

References

  1. Albrink W. S.. ( 1961;). Pathogenesis of inhalation anthrax. Bacteriol Rev25:268–273[PubMed]
    [Google Scholar]
  2. Atrih A., Zöllner P., Allmaier G., Foster S. J.. ( 1996;). Structural analysis of Bacillus subtilis 168 endospore peptidoglycan and its role during differentiation. J Bacteriol178:6173–6183[PubMed]
    [Google Scholar]
  3. Atrih A., Zöllner P., Allmaier G., Williamson M. P., Foster S. J.. ( 1998;). Peptidoglycan structural dynamics during germination of Bacillus subtilis 168 endospores. J Bacteriol180:4603–4612[PubMed]
    [Google Scholar]
  4. Austin B. P., Nallamsetty S., Waugh D. S.. ( 2009;). Hexahistidine-tagged maltose-binding protein as a fusion partner for the production of soluble recombinant proteins in Escherichia coli. Methods Mol Biol498:157–172 [CrossRef][PubMed]
    [Google Scholar]
  5. Buist G., Steen A., Kok J., Kuipers O. P.. ( 2008;). LysM, a widely distributed protein motif for binding to (peptido)glycans. Mol Microbiol68:838–847 [CrossRef][PubMed]
    [Google Scholar]
  6. Chen Y., Miyata S., Makino S., Moriyama R.. ( 1997;). Molecular characterization of a germination-specific muramidase from Clostridium perfringens S40 spores and nucleotide sequence of the corresponding gene. J Bacteriol179:3181–3187[PubMed]
    [Google Scholar]
  7. Chen Y., Fukuoka S., Makino S.. ( 2000;). A novel spore peptidoglycan hydrolase of Bacillus cereus: biochemical characterization and nucleotide sequence of the corresponding gene, sleL . J Bacteriol182:1499–1506 [CrossRef][PubMed]
    [Google Scholar]
  8. Costa T., Isidro A. L., Moran C. P. Jr, Henriques A. O.. ( 2006;). Interaction between coat morphogenetic proteins SafA and SpoVID. J Bacteriol188:7731–7741 [CrossRef][PubMed]
    [Google Scholar]
  9. Dowd M. M., Orsburn B., Popham D. L.. ( 2008;). Cortex peptidoglycan lytic activity in germinating Bacillus anthracis spores. J Bacteriol190:4541–4548 [CrossRef][PubMed]
    [Google Scholar]
  10. Foster S. J., Johnstone K.. ( 1987;). Purification and properties of a germination-specific cortex-lytic enzyme from spores of Bacillus megaterium KM. Biochem J242:573–579[PubMed]
    [Google Scholar]
  11. Gerhardt P., Marquis R. E.. ( 1989;). Spore thermoresistance mechanisms. Regulation of Prokaryotic Development43–63 Smith I., Slepecky R. A., Setlow P.. Washington, DC: American Society for Microbiology;
    [Google Scholar]
  12. Heffron J. D., Orsburn B., Popham D. L.. ( 2009;). Roles of germination-specific lytic enzymes CwlJ and SleB in Bacillus anthracis . J Bacteriol191:2237–2247 [CrossRef][PubMed]
    [Google Scholar]
  13. Heffron J. D., Lambert E. A., Sherry N., Popham D. L.. ( 2010;). Contributions of four cortex lytic enzymes to germination of Bacillus anthracis spores. J Bacteriol192:763–770 [CrossRef][PubMed]
    [Google Scholar]
  14. Heffron J. D., Sherry N., Popham D. L.. ( 2011;). In vitro studies of peptidoglycan binding and hydrolysis by the Bacillus anthracis germination-specific lytic enzyme SleB. J Bacteriol193:125–131 [CrossRef][PubMed]
    [Google Scholar]
  15. Hu K., Yang H., Liu G., Tan H.. ( 2007;). Cloning and identification of a gene encoding spore cortex-lytic enzyme in Bacillus thuringiensis . Curr Microbiol54:292–295 [CrossRef][PubMed]
    [Google Scholar]
  16. Imamura D., Kuwana R., Takamatsu H., Watabe K.. ( 2010;). Localization of proteins to different layers and regions of Bacillus subtilis spore coats. J Bacteriol192:518–524 [CrossRef][PubMed]
    [Google Scholar]
  17. Ishikawa S., Yamane K., Sekiguchi J.. ( 1998;). Regulation and characterization of a newly deduced cell wall hydrolase gene (cwlJ) which affects germination of Bacillus subtilis spores. J Bacteriol180:1375–1380[PubMed]
    [Google Scholar]
  18. Janes B. K., Stibitz S.. ( 2006;). Routine markerless gene replacement in Bacillus anthracis . Infect Immun74:1949–1953 [CrossRef][PubMed]
    [Google Scholar]
  19. Kapust R. B., Tözsér J., Fox J. D., Anderson D. E., Cherry S., Copeland T. D., Waugh D. S.. ( 2001;). Tobacco etch virus protease: mechanism of autolysis and rational design of stable mutants with wild-type catalytic proficiency. Protein Eng14:993–1000 [CrossRef][PubMed]
    [Google Scholar]
  20. Kim H. U., Goepfert J. M.. ( 1974;). A sporulation medium for Bacillus anthracis . J Appl Bacteriol37:265–267 [CrossRef][PubMed]
    [Google Scholar]
  21. Kodama T., Takamatsu H., Asai K., Kobayashi K., Ogasawara N., Watabe K.. ( 1999;). The Bacillus subtilis yaaH gene is transcribed by SigE RNA polymerase during sporulation, and its product is involved in germination of spores. J Bacteriol181:4584–4591[PubMed]
    [Google Scholar]
  22. Kodama T., Takamatsu H., Asai K., Ogasawara N., Sadaie Y., Watabe K.. ( 2000;). Synthesis and characterization of the spore proteins of Bacillus subtilis YdhD, YkuD, and YkvP, which carry a motif conserved among cell wall binding proteins. J Biochem128:655–663[PubMed][CrossRef]
    [Google Scholar]
  23. Lambert E. A., Popham D. L.. ( 2008;). The Bacillus anthracis SleL (YaaH) protein is an N-acetylglucosaminidase involved in spore cortex depolymerization. J Bacteriol190:7601–7607 [CrossRef][PubMed]
    [Google Scholar]
  24. Leighton T. J., Doi R. H.. ( 1971;). The stability of messenger ribonucleic acid during sporulation in Bacillus subtilis . J Biol Chem246:3189–3195[PubMed]
    [Google Scholar]
  25. Liu H., Bergman N. H., Thomason B., Shallom S., Hazen A., Crossno J., Rasko D. A., Ravel J., Read T. D.. & other authors ( 2004;). Formation and composition of the Bacillus anthracis endospore. J Bacteriol186:164–178 [CrossRef][PubMed]
    [Google Scholar]
  26. Makino S., Moriyama R.. ( 2002;). Hydrolysis of cortex peptidoglycan during bacterial spore germination. Med Sci Monit8:RA119–RA127[PubMed]
    [Google Scholar]
  27. Makino S., Ito N., Inoue T., Miyata S., Moriyama R.. ( 1994;). A spore-lytic enzyme released from Bacillus cereus spores during germination. Microbiology140:1403–1410 [CrossRef][PubMed]
    [Google Scholar]
  28. Marchler-Bauer A., Anderson J. B., Cherukuri P. F., DeWeese-Scott C., Geer L. Y., Gwadz M., He S., Hurwitz D. I., Jackson J. D.. & other authors ( 2005;). CDD: a Conserved Domain Database for protein classification. Nucleic Acids Res33:Database issue)D192–D196 [CrossRef][PubMed]
    [Google Scholar]
  29. McKenney P. T., Driks A., Eskandarian H. A., Grabowski P., Guberman J., Wang K. H., Gitai Z., Eichenberger P.. ( 2010;). A distance-weighted interaction map reveals a previously uncharacterized layer of the Bacillus subtilis spore coat. Curr Biol20:934–938 [CrossRef][PubMed]
    [Google Scholar]
  30. Meador-Parton J., Popham D. L.. ( 2000;). Structural analysis of Bacillus subtilis spore peptidoglycan during sporulation. J Bacteriol182:4491–4499 [CrossRef][PubMed]
    [Google Scholar]
  31. Miyata S., Moriyama R., Miyahara N., Makino S.. ( 1995;). A gene (sleC) encoding a spore-cortex-lytic enzyme from Clostridium perfringens S40 spores; cloning, sequence analysis and molecular characterization. Microbiology141:2643–2650 [CrossRef][PubMed]
    [Google Scholar]
  32. Mock M., Fouet A.. ( 2001;). Anthrax. Annu Rev Microbiol55:647–671 [CrossRef][PubMed]
    [Google Scholar]
  33. Moir A., Smith D. A.. ( 1990;). The genetics of bacterial spore germination. Annu Rev Microbiol44:531–553 [CrossRef][PubMed]
    [Google Scholar]
  34. Moriyama R., Hattori A., Miyata S., Kudoh S., Makino S.. ( 1996a;). A gene (sleB) encoding a spore cortex-lytic enzyme from Bacillus subtilis and response of the enzyme to l-alanine-mediated germination. J Bacteriol178:6059–6063[PubMed]
    [Google Scholar]
  35. Moriyama R., Kudoh S., Miyata S., Nonobe S., Hattori A., Makino S.. ( 1996b;). A germination-specific spore cortex-lytic enzyme from Bacillus cereus spores: cloning and sequencing of the gene and molecular characterization of the enzyme. J Bacteriol178:5330–5332[PubMed]
    [Google Scholar]
  36. Nallamsetty S., Waugh D. S.. ( 2007;). A generic protocol for the expression and purification of recombinant proteins in Escherichia coli using a combinatorial His6-maltose binding protein fusion tag. Nat Protoc2:383–391 [CrossRef][PubMed]
    [Google Scholar]
  37. Nicholson W. L., Setlow P.. ( 1990;). Sporulation, germination, and outgrowth. Molecular Biological Methods for Bacillus391–450 Harwood C. R., Cutting S. M.. Chichester, UK: Wiley;
    [Google Scholar]
  38. Nicholson W. L., Munakata N., Horneck G., Melosh H. J., Setlow P.. ( 2000;). Resistance of Bacillus endospores to extreme terrestrial and extraterrestrial environments. Microbiol Mol Biol Rev64:548–572 [CrossRef][PubMed]
    [Google Scholar]
  39. Ozin A. J., Henriques A. O., Yi H., Moran C. P. Jr. ( 2000;). Morphogenetic proteins SpoVID and SafA form a complex during assembly of the Bacillus subtilis spore coat. J Bacteriol182:1828–1833 [CrossRef][PubMed]
    [Google Scholar]
  40. Pace C. N., Vajdos F., Fee L., Grimsley G., Gray T.. ( 1995;). How to measure and predict the molar absorption coefficient of a protein. Protein Sci4:2411–2423 [CrossRef][PubMed]
    [Google Scholar]
  41. Paidhungat M., Setlow B., Daniels W. B., Hoover D., Papafragkou E., Setlow P.. ( 2002;). Mechanisms of induction of germination of Bacillus subtilis spores by high pressure. Appl Environ Microbiol68:3172–3175 [CrossRef][PubMed]
    [Google Scholar]
  42. Popham D. L., Setlow P.. ( 1993;). The cortical peptidoglycan from spores of Bacillus megaterium and Bacillus subtilis is not highly cross-linked. J Bacteriol175:2767–2769[PubMed]
    [Google Scholar]
  43. Popham D. L., Helin J., Costello C. E., Setlow P.. ( 1996a;). Muramic lactam in peptidoglycan of Bacillus subtilis spores is required for spore outgrowth but not for spore dehydration or heat resistance. Proc Natl Acad Sci U S A93:15405–15410 [CrossRef][PubMed]
    [Google Scholar]
  44. Popham D. L., Helin J., Costello C. E., Setlow P.. ( 1996b;). Analysis of the peptidoglycan structure of Bacillus subtilis endospores. J Bacteriol178:6451–6458[PubMed]
    [Google Scholar]
  45. Sekiguchi J., Akeo K., Yamamoto H., Khasanov F. K., Alonso J. C., Kuroda A.. ( 1995;). Nucleotide sequence and regulation of a new putative cell wall hydrolase gene, cwlD, which affects germination in Bacillus subtilis . J Bacteriol177:5582–5589[PubMed]
    [Google Scholar]
  46. Setlow P.. ( 2000;). Resistance of bacterial spores. Bacterial Stress Responses217–230 Storz G., Hengge-Aronis R.. Washington, DC: American Society for Microbiology;
    [Google Scholar]
  47. Setlow P.. ( 2003;). Spore germination. Curr Opin Microbiol6:550–556 [CrossRef][PubMed]
    [Google Scholar]
  48. Setlow B., Melly E., Setlow P.. ( 2001;). Properties of spores of Bacillus subtilis blocked at an intermediate stage in spore germination. J Bacteriol183:4894–4899 [CrossRef][PubMed]
    [Google Scholar]
  49. Setlow B., Peng L., Loshon C. A., Li Y. Q., Christie G., Setlow P.. ( 2009;). Characterization of the germination of Bacillus megaterium spores lacking enzymes that degrade the spore cortex. J Appl Microbiol107:318–328 [CrossRef][PubMed]
    [Google Scholar]
  50. Shao X., Jiang M., Yu Z., Cai H., Li L.. ( 2009;). Surface display of heterologous proteins in Bacillus thuringiensis using a peptidoglycan hydrolase anchor. Microb Cell Fact8:48 [CrossRef][PubMed]
    [Google Scholar]
  51. Steen A., Buist G., Horsburgh G. J., Venema G., Kuipers O. P., Foster S. J., Kok J.. ( 2005;). AcmA of Lactococcus lactis is an N-acetylglucosaminidase with an optimal number of LysM domains for proper functioning. FEBS J272:2854–2868 [CrossRef][PubMed]
    [Google Scholar]
  52. Terwisscha van Scheltinga A. C., Armand S., Kalk K. H., Isogai A., Henrissat B., Dijkstra B. W.. ( 1995;). Stereochemistry of chitin hydrolysis by a plant chitinase/lysozyme and X-ray structure of a complex with allosamidin: evidence for substrate assisted catalysis. Biochemistry34:15619–15623 [CrossRef][PubMed]
    [Google Scholar]
  53. van Aalten D. M., Komander D., Synstad B., Gåseidnes S., Peter M. G., Eijsink V. G.. ( 2001;). Structural insights into the catalytic mechanism of a family 18 exo-chitinase. Proc Natl Acad Sci U S A98:8979–8984 [CrossRef][PubMed]
    [Google Scholar]
  54. van den Ent F., Löwe J.. ( 2006;). RF cloning: a restriction-free method for inserting target genes into plasmids. J Biochem Biophys Methods67:67–74 [CrossRef][PubMed]
    [Google Scholar]
  55. Wang K. H., Isidro A. L., Domingues L., Eskandarian H. A., McKenney P. T., Drew K., Grabowski P., Chua M. H., Barry S. N.. & other authors ( 2009;). The coat morphogenetic protein SpoVID is necessary for spore encasement in Bacillus subtilis . Mol Microbiol74:634–649 [CrossRef][PubMed]
    [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/micro/10.1099/mic.0.056630-0
Loading
/content/journal/micro/10.1099/mic.0.056630-0
Loading

Data & Media loading...

Most cited this month Most Cited RSS feed

This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error