2. Stages of Sporulation in Bacillus subtilis Stage 0 Normal vegetative growth Stage II 1 Asymmetric septation Stage II 3 Engulfment Stage III Prespore protoplast Stage IV Cortex formation Stage V Coat formation Stage VI Maturation Stage VII Release Germination

3. Stages of Sporulation in Bacillus subtilis Stage 0Stage II 1 Stage II 3 Stage IIIStage IVStage VStage VIStage VII How is the process regulated? spo0spoIIIspoVIspoIVspoVspoII

4. Stages of Sporulation in Bacillus subtilis Stage 0Stage II 1 Stage II 3 Stage IIIStage IVStage VStage VIStage VII spo0spoIIIspoVIspoIVspoVspoII Golden age of Bacillus genetics Identify spo genes Clone and characterize spo genes Determine their interdependencies

5. Stages of Sporulation in Bacillus subtilis Stage 0Stage II 1 Stage II 3 Stage IIIStage IVStage VStage VIStage VII spo0spoIIIspoVIspoIVspoVspoII Errington and Mandelstam (1986) Care in using reporter gene (lacZ) How is regulation exerted? Dependence of spoIIA expression on other genes spoIIA

6. Goals of Errington and Mandelstam Measure spoIIA expression: wt vs spo mutants spoIIA spoIIAAspoIIABspoIIAC P sp lacZ P sp ß-galactosidase spoIIA::lacZ

7. Goals of Errington and Mandelstam P sp spoIIA::lacZ B. subtilis wild typespoIIA::lacZ B. subtilis spo0A43 B. subtilis spo0B136 B. subtilis spo0D8 B. subtilis spoIIAA562 B. subtilis spoIIB131 B. subtilis spoIID298 B. subtilis spoIIIA65 B. subtilis spoIIIB2 B. subtilis spoIIIC94 B. subtilis spoIVA67 B. subtilis spoIVB165 B. subtilis spoIVC23 B. subtilis spoVA89 B. subtilis spoVB91 B. subtilis spoVC134 B. subtilis spoVIA513 B. subtilis spoVIB520 B. subtilis spoVIC610 B. subtilis gerE36 Need a way to move around spoIIA::lacZ Measure spoIIA expression: wt vs spo mutants...

8. Goals of Errington and Mandelstam P sp spoIIA::lacZ Make spoIIA::lacZ transportable  105J19 cat  105J19: temperate phage Infection Lysis Lysogeny

9. Goals of Errington and Mandelstam Make spoIIA::lacZ transportable P sp spoIIA::lacZ  105J19 cat Wt (  105J19 spoIIAA::lac-cat)Wt (  105J19) lysogen recombination spo - lysogen Transducing phage... Select for Cm r

10. Goals of Errington and Mandelstam Measure expression from spoIIA::lacZ OH O O NO 2 HO OH O NO 2 Starve cells Break cells + ONPG ß-gal Conclusion?

11. Goals of Errington and Mandelstam Measure expression from spoIIA::lacZ Starve cells Break cells + MUG ß-gal OH O O O MUG Moral Beware of equating what is measured with what is sought

12. Induction of spoIIA::lacZ during sporulation ß gal activity (ONPG) Time after move to starvation medium 0 h 6 h (Figure 2a) wt spoIIA::lacZ wt

13. Induction of spoIIA::lacZ during sporulation ß gal activity (MUG) Time after move to starvation medium 0 h 6 h (Figure 2b) wt spoIIA::lacZ spoIIA + spoIIA - wt Moral Beware of side effects. Context is important.

14. Why does spoIIA::lacZ expression fall? P sp Transcriptional regulators transcriptiontranslation RNA degradation protein degradation RNA secondary structure Not well understood Presence of proteases Cm

15. Why does spoIIA::lacZ expression fall? ß gal activity (MUG) Time after move to starvation medium 0 h 3 h (Figure 3) Rate of protein degradation constant +Cm

16. Why does spoIIA::lacZ expression fall? P sp transcriptiontranslation RNA degradation protein degradation MUG RNA Time after starvation

17. Why controls spoIIA::lacZ expression? Table 2 Spo0 proteins SpoII proteins SpoIII proteins SpoIV proteins SpoV proteins

18. Why controls spoIIA::lacZ expression? Table 2 Spo0 proteins SpoII proteins SpoIII proteins SpoIV proteins SpoV proteins Nonregulatory protein 1. How does this work? Hint: Spo0AA is a sigma factor

19. Why controls spoIIA::lacZ expression? Table 2 Spo0 proteins SpoII proteins SpoIII proteins SpoIV proteins SpoV proteins 1. How does it get started? Environmental sensing + signal transduction