Journal of Microbiology and Biotechnology

The Korean Society for Microbiology and Biotechnology (한국미생물·생명공학회)
권호 표지
DOI QR코드

DOI QR Code

"A La Recherche" of Functions for the Spore Protein SASP-E from Bacillus subtilis

  • Ruzal, Sandra M. (Universidad de Buenos Aires, Facultad Ciencias Exactas y Naturales, Departamento de Quimica Biologica, Ciudad Universitaria) ;
  • Bustos, Patricia L. (Universidad de Buenos Aires, Facultad Ciencias Exactas y Naturales, Departamento de Quimica Biologica, Ciudad Universitaria) ;
  • Sanchez-Rivas, Carmen (Universidad de Buenos Aires, Facultad Ciencias Exactas y Naturales, Departamento de Quimica Biologica, Ciudad Universitaria)
  • Received : 2012.06.12
  • Accepted : 2012.08.28
  • Published : 2013.01.28
Download PDF
⟨ Previous Next ⟩

Abstract

We previously observed that Bacillus subtilis spores from sspE mutants presented a lower germination capacity in media containing high salt concentrations (0.9M NaCl). This deficiency was attributed to the absence of SASP-E (gamma-type small-acid-soluble protein), rich in osmocompatible amino acids released by degradation. Herein we observed that, in addition, this mutant spore presented a reduced capacity to use L-alanine as germinant (L-ala pathway), required longer times to germinate in calcium dipicolinate ($Ca^{2+}$-DPA), but germinated well in asparagine, glucose, fructose, and potassium chloride (AGFK pathway). Moreover, mild sonic treatment of mutant spores partially recovered their germination capacity in L-ala. Spore qualities were also altered, since sporulating colonies from the sspE mutant showed a pale brownish color, a higher adherence to agar plates, and lower autofluorescence, properties related to their spore coat content. Furthermore, biochemical analysis showed a reduced partition in hexadecane and a higher content of $Ca^{2+}$-DPA when compared with its isogenic wild-type control. Coat protein preparations showed a different electrophoretic pattern, in particular when detected with antibodies against CotG and CotE. The complementation with a wild-type sspE gene in a plasmid allowed for recovering the wild-type coat phenotype. This is the first report of a direct involvement of SASP-E in the spore coat assembly during the differentiation program of sporulation.

Keywords

References

  1. Camp, A. H. and R. Losick. 2008. A novel pathway of intercellular signaling in Bacillus subtilis involves a protein with similarity to a component of type III secretion channels. Mol. Microbiol. 6: 402-417.
  2. Clements, M. O. and A. Moir. 1998. Role of the gerI operon of Bacillus cereus 569 in the response of spores to germinants. J. Bacteriol. 180: 6729-6735.
  3. Cucchi, A. and C. Sanchez-Rivas. 1995. ssp Genes and spore osmotolerance in Bacillus thuringiensis israelensis and Bacillus sphaericus. Curr. Microbiol. 31: 228-233. https://doi.org/10.1007/BF00298379
  4. Cucchi, A. and C. Sanchez-Rivas. 1998. SASP (small, acidsoluble spore proteins) and spore properties in Bacillus thuringiensis israelensis and Bacillus sphaericus. Curr. Microbiol. 36: 220-225 https://doi.org/10.1007/s002849900298
  5. Driks, A. 2002. Proteins of the spore core and coat, in Bacillus subtilis and its closest relatives, pp. 527-536. Bacillus subtilis and its Closest Relatives. ASM Press, Washington, DC. A. L. Sonenshein, J. A. Hoch, and R. Losick (eds.).
  6. Ebmeier, S. E., I. S. Tan, and K. R. Clapham. 2012. Small proteins link coat and cortex assembly during sporulation in Bacillus subtilis. Mol. Microbiol. 84: 682-696. https://doi.org/10.1111/j.1365-2958.2012.08052.x
  7. Ferencko, L. and B. Rotman. 2010. Constructing fluorogenic Bacillus spores (F-spores) via hydrophobic decoration of coat proteins. PLoS One 19: e9283.
  8. Francesconi, S. C., T. J. MacAlister, B. Setlow, and P. Setlow. 1988. Immunoelectron microscopic localization of small, acidsoluble spore proteins in sporulating cells of Bacillus subtilis. J. Bacteriol. 170: 5963-5967.
  9. Hayes, C. S. and P. Setlow. 2001. An alpha/beta-type, small, acid-soluble spore protein which has very high affinity for DNA prevents outgrowth of Bacillus subtilis spores. J. Bacteriol. 183: 2662-2266. https://doi.org/10.1128/JB.183.8.2662-2666.2001
  10. Henriques, A. O., L. R. Melsen, and C. P. Moran Jr. 1998. Involvement of superoxide dismutase in spore coat assembly in Bacillus subtilis. J. Bacteriol. 180: 2285-2291.
  11. Henriques, A. O. and C. P. Moran Jr. 2007. Structure, assembly, and function of the spore surface layers. Annu. Rev. Microbiol. 61: 555-588. https://doi.org/10.1146/annurev.micro.61.080706.093224
  12. Hudson, K. D., B. M. Corfe, E. H. Kemp, I. M. Feavers, P. J. Coote, and A. Moir. 2001. Localization of GerAA and GerAC germination proteins in the Bacillus subtilis spore. J. Bacteriol. 183: 4317-4322. https://doi.org/10.1128/JB.183.14.4317-4322.2001
  13. Imamura, D., R. Kuwana, H. Takamatsu, and K. Watabe. 2010. Localization of proteins to different layers and regions of Bacillus subtilis spore coats. J. Bacteriol. 192: 518-524. https://doi.org/10.1128/JB.01103-09
  14. Johnson, M. J., D. A. Todd, P. S. Sheperd, and A. Moir. 2006. ExsY and CotY are required for the correct assembly of the exosporium and spore coat of Bacillus cereus. J. Bacteriol. 188: 7905-7913. https://doi.org/10.1128/JB.00997-06
  15. Kodama, T., T. Matsubayashi, T. Yanagihara, H. Komoto, K. Ara, K. Ozaki, et al. 2011. A novel small protein of Bacillus subtilis involved in spore germination and spore coat assembly. Biosci. Biotechnol. Biochem. 75: 1119-1128. https://doi.org/10.1271/bbb.110029
  16. Keijser, B. J. F., A. T. Beek, H. Rauwerda, F. Schuren, R. Montijn, H. van der Spek, and S. Brul. 2007. Analysis of temporal gene expression during Bacillus subtilis spore germination and outgrowth. J. Bacteriol. 189: 3624-3634. https://doi.org/10.1128/JB.01736-06
  17. Laemmli, U. K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227: 680-685 https://doi.org/10.1038/227680a0
  18. Lai, E. M., N. D. Phadke, M. T. Kachman, R. Giorno, S. Vazquez, J. A. Vazquez, J. R. Maddock, and A. Driks. 2003. Proteomic analysis of the spore coats of Bacillus subtilis and Bacillus anthracis. J. Bacteriol. 185: 1443-1454. https://doi.org/10.1128/JB.185.4.1443-1454.2003
  19. Magge, A., B. Setlow, A. E. Cowan, and P. Setlow. 2009. Analysis of dye binding by and membrane potential in spores of Bacillus species. J. Appl. Microbiol. 106: 814-824. https://doi.org/10.1111/j.1365-2672.2008.04048.x
  20. Mason, J. M., R. H. Hackett, and P. Setlow. 1988. Studies on the regulation of expression of genes coding for small acidsoluble proteins of Bacillus subtilis spores using lacZ gene fusions. J. Bacteriol. 170: 230-244.
  21. Meisner, J., X. Wang, M. Serrano, A. O. Henriques, and C. P. Moran Jr. 2008. A channel connecting the mother cell and forespore during bacterial endospore formation. Proc. Natl. Acad. Sci. USA 105: 15100-15105. https://doi.org/10.1073/pnas.0806301105
  22. McKenney, P. T., A. Driks, H. A. Eskandarian, P. Grabowski, J. Guberman, K. H. Wang, et al. 2010. A distance-weighted interaction map reveals a previously uncharacterized layer of the Bacillus subtilis spore coat. Curr. Biol. 20: 934-938. https://doi.org/10.1016/j.cub.2010.03.060
  23. Moir, A. 2006. How do spores germinate? J. Appl. Microbiol. 101: 526-530. https://doi.org/10.1111/j.1365-2672.2006.02885.x
  24. Moir, A., B. M. Corfe, and J. Behravan. 2002. Spore germination. Cell Mol. Life Sci. 59: 403-409. https://doi.org/10.1007/s00018-002-8432-8
  25. Paidhungat, M. and P. Setlow. 2001. Localization of a germinant receptor protein (GerBA) to the inner membrane of Bacillus subtilis spores. J. Bacteriol. 183: 3982-3990. https://doi.org/10.1128/JB.183.13.3982-3990.2001
  26. Paidhungat, M. and P. Setlow. 2001. Spore germination and outgrowth in Bacillus subtilis and its closest relatives, pp. 537-548. In A. L. Sonenshein, J. A. Hoch, and R. Losick (eds.). Bacillus subtilis and its Closest Relatives. ASM Press, Washington, DC.
  27. Paidhungat, M., K. Ragkousi, and P. Setlow. 2001. Genetic requirements for induction of germination of spores of Bacillus subtilis by $Ca^{2+}$-dipicolinate. J. Bacteriol. 183: 4886-4893. https://doi.org/10.1128/JB.183.16.4886-4893.2001
  28. Rotman, Y. and M. L. Fields. 1968. A modified reagent for dipicolinic acid analysis. Anal. Biochem. 22: 168. https://doi.org/10.1016/0003-2697(68)90272-8
  29. Ruzal, S. M., A. F. Alice, and C. Sanchez-Rivas. 1994. Osmoresistance of spores from Bacillus subtilis and the effect of ssp mutations. Microbiology. 140: 2173-2177. https://doi.org/10.1099/13500872-140-8-2173
  30. Schaeffer, P., J. Miller, and J. Aubert. 1965. Catabolic repression of bacterial sporulation. Proc. Natl. Acad. Sci. USA 554: 701-711
  31. Setlow, B., P. G. Wahome, and P. Setlow. 2008. Release of small molecules during germination of spores of Bacillus species. J. Bacteriol. 190: 4759-4763. https://doi.org/10.1128/JB.00399-08
  32. Tovar-Rojo, F., R. M. Cabrera-Martinez, B. Setlow, and P. Setlow. 2003. Studies on the mechanism of the osmoresistance of spores of Bacillus subtilis. J. Appl. Microbiol. 95: 167-179. https://doi.org/10.1046/j.1365-2672.2003.01958.x
  33. Vary, J. C. and H. O. Halvorson. 1965. Kinetics of germination of Bacillus spores. J. Bacteriol. 89: 1340-1347.
  34. Vyas, J., J. Cox, B. Setlow, W. H. Coleman, and P. Setlow. 2011. Extremely variable conservation of $\gamma$-type small, acidsoluble proteins from spores of some species in the bacterial order Bacillales. J. Bacteriol. 193: 1884-1892. https://doi.org/10.1128/JB.00018-11
  35. Vepachedu, V. R. and P. Setlow. 2007. Role of SpoVA proteins in release of dipicolinic acid during germination of Bacillus subtilis spores triggered by dodecylamine or lysozyme. J. Bacteriol. 189: 1565-1572. https://doi.org/10.1128/JB.01613-06
  36. Wootton, J. C. and M. Drummond. 1989. The Q linker: A class of interdomain sequences found in bacterial multidomain regulatory proteins. Protein Eng. 2: 535-543. https://doi.org/10.1093/protein/2.7.535