The objective of this project is a detailed understanding of formation and maintenance of a dormant, resistant bacterial (Bacillus subtilis) spore; there are 3 major goals. I. The structure of the complex between alpha/beta type small, acid-soluble spore proteins (SASP) and DNA will be determined.
Specific aims are: a) probe DNA structure in a SASP/DNA complex; b) identify residues in a SASP/DNA complex involved in SASP-DNA or SASP-SASP interaction; c) study SASP's ability to associate two DNA helices side-by- side; d) use a SASP variant containing trp to study SASP- DNA interaction; e) construct a minimal functional SASP; f) continue collaborative efforts to obtain a high-resolution structure of a SASP/oligonucleotide complex; g) confirm this structure by analysis of appropriate mutant SASP; h) generate SASP altered in the site recognized and cleaved by the SASP specific protease (GPR), analyze their function in vivo, and their cleavage by GPR; and i) identify and characterize minor B. subtilis SASP and the genes coding for them. Il. The fall in intraforespore pH late in sporulation will be studied in detail.
Specific aims are: a) analyze the pH decrease in both wild type and spo mutants of B. subtilis; b) modulate depots of 3-phosphoglycerate (3PGA) in vegetative and sporulating cells and germinating spores by altering intracellular pH; c) analyze the effects of inactivation of the B. subtilis pgm gene (coding for phosphoglycerate mutase (PGM)) on growth, sporulation and spore 3PGA depots; d) overexpress B. subtilis PGM or a Mn independent PGM in B. subtilis and analyze the effect of overexpression on spore 3PGA depots; and e) determine the reason for the Mn dependence and pH sensitivity of B. subtilis PGM. III. Analysis of genes and proteins involved in peptidoglycan metabolism during sporulation and germination will be continued.
Specific aims are: a) analyze the sequence, regulation and function of additional genes encoding penicillin-binding proteins (PBP); b) with strains lacking one or more PBPs, analyze the cortex structure and heat resistance of their spores; and c) identify the enzyme(s) initiating cortex hydrolysis during spore germination. While this work will be carried out with B. subtilis, the knowledge gained will be applicable to other Gram-positive spore formers. A number of the latter organisms are significant agents of food spoilage as well as human disease, in particular food born disease, in large part because of the resistant nature of spores. Consequently, knowledge gained in the proposed work could have significant applications.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Method to Extend Research in Time (MERIT) Award (R37)
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Microbial Physiology and Genetics Subcommittee 2 (MBC)
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University of Connecticut
Schools of Dentistry
United States
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Setlow, Peter; Li, Lei (2015) Photochemistry and Photobiology of the Spore Photoproduct: A 50-Year Journey. Photochem Photobiol 91:1263-90
Plomp, Marco; Carroll, Alicia Monroe; Setlow, Peter et al. (2014) Architecture and assembly of the Bacillus subtilis spore coat. PLoS One 9:e108560
Setlow, Peter (2014) Spore Resistance Properties. Microbiol Spectr 2:
Wei, Jie; Shah, Ishita M; Ghosh, Sonali et al. (2010) Superdormant spores of bacillus species germinate normally with high pressure, peptidoglycan fragments, and bryostatin. J Bacteriol 192:1455-8
Ghosh, S; Setlow, P (2010) The preparation, germination properties and stability of superdormant spores of Bacillus cereus. J Appl Microbiol 108:582-90
Setlow, B; Peng, L; Loshon, C A et al. (2009) Characterization of the germination of Bacillus megaterium spores lacking enzymes that degrade the spore cortex. J Appl Microbiol 107:318-28
Paredes-Sabja, Daniel; Setlow, Peter; Sarker, Mahfuzur R (2009) SleC is essential for cortex peptidoglycan hydrolysis during germination of spores of the pathogenic bacterium Clostridium perfringens. J Bacteriol 191:2711-20
Ghosh, Sonali; Setlow, Peter (2009) Isolation and characterization of superdormant spores of Bacillus species. J Bacteriol 191:1787-97
Ghosh, Sonali; Zhang, Pengfei; Li, Yong-qing et al. (2009) Superdormant spores of Bacillus species have elevated wet-heat resistance and temperature requirements for heat activation. J Bacteriol 191:5584-91
Peng, Lixin; Chen, De; Setlow, Peter et al. (2009) Elastic and inelastic light scattering from single bacterial spores in an optical trap allows the monitoring of spore germination dynamics. Anal Chem 81:4035-42

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