1. Sporulation Vegetative cell divides asymmetrically: septum (new cell wall) produced near one end

2. Sporulation Larger cell (“mother cell”) engulfs smaller (“forespore”) mother cell forespore

3. Sporulation The forespore now has two membrane layers

4. Sporulation Mother cell DNA is breaking down--this cell is “programmed” to die

5. Sporulation Mother cell and forespore both produce spore coat layers of protein and other materials core cortex coat

6. Sporulation Mother cell lyses and releases spore

7. Spore resistance  Heat (many species’ spores can be boiled)  Drying  Organic solvents  Salts  Lysozyme and other degradative enzymes  UV

8. Sporulation

9. Spore structure Core DNA SASPs protect against heat, UV, oxidation Dehydration protects against heat, oxidation, chemical reactions Cortex Peptidoglycan; protects against organic solvents Coat Protein layers; protects against lysozyme and many small molecules Exosporium Protein and polysaccharide Functions in sieving, attachment, enzymes, ??

10. Assembly of the spore coat B. subtilis spore 500 nm

11. How do we know what proteins are in the spore coat? Lai et al., Proteomic analysis of the spore coats of B. subtilis and B. anthracis, J. Bacteriol. 185:1443 (2003)  Spore protein extract  SDS-PAGE  Coomassie-stained proteins

12. Density-gradient centrifugation Bacillus sporulated in DSM 50% Renografin (1.2 g/ml) 20% Renografin (1.08 g/ml) 12,000 × g diatrizoate cell debris cells and phase-dark spores phase-bright spores

13. MALDI-MS identification of proteins protein fragment (e.g., with protease) ionize determine mass with mass spectrometer

14. 2D Gel electrophoresis

15. high m.w. low m.w. + – most negative most positive

16. How did we learn about the roles of morphogenetic proteins? Little and Driks, Functional analysis of the B. subtilis morphogenetic spore coat protein CotE, Mol. Microbiol. 42:1107 (2001)  Spore protein extract  SDS-PAGE  Western Blot mutations in cotE

17. How did we learn about the roles of morphogenetic proteins? Ozin et al., Morphogenetic proteins SpoVID and SafA form a complex during assembly of the Bacillus subtilis spore coat, J. Bacteriol. 182:1828 (2000)  Spore protein extract  immunoprecipitation  SDS-PAGE  Western Blot (anti-SpoIVD) wt spoVID mutant wt spoVID mutant safA mutant

18. How did we learn about the roles of morphogenetic proteins? Bauer et al., Functional regions of the Bacillus subtilis spore coat morphogenetic protein CotE, J. Bacteriol. 181:7043 (1999)  in situ immunofluorescence  anti-CotE antibody wtcotE mutant spoIVA mutant

20. What about the exosporium?  Little known about function  20+ known proteins and glycoproteins in B. anthracis BclA is major surface protein ExsFA and ExsFB exosporium proteins not found in B. subtilis  Contains enzymes that may modify germinants Alanine racemase Inosine hydrolase