1. Lecture 8: Bacterial Differentiation: Sporulation Reading assignments in Text: Lengeler et al. 1999 Lecture 8 Text: pages 586-601 Sporulation Text: pages 627 Secondary metabolism Lecture 7 Text: pages 792-799 Gut bugs Text: pages 276-277, 800 Bacterial Lux Text: pages 498-499 Quorum sensing Text: pages 801 Bacteria and plants Text: pages 865-868 Agrobacterium Text: pages 275 Toxic oxygen Text: pages 807 Nitrogen cycle Text: pages 851-862 Rhizobia and Cyanobacteria

2. Lecture Overview Bacterial populations (lab conditions) Metabolism GROWTHGROWTH Bacteria as single cells (“cell cycles”) Differentiation Symbiosis Sporulation

3. Bacillus subtilis growth and sporulation time Log(N) inoculate 1 log growth, symmetric cell division 1 2 Gain motility 2 3 Gain genetic competence 3 4 Sporulate, asymmetric cell div 4 Relatives: B. cereus, B. anthracis ~7 hours VII stages Genetics: Spo >200 genes/proteins III Stage (0, I, II, III, IV, V, VI) B Gene (A, B, C …) Quorum sensing

5. 0 B. subtilis sporulation stages M D 2 chromosomes, axial fibers I2 cells, cell wall IIEngulfment IIIDouble membrane IV Cortex V-VI Coat VII Spore maturation Mother cell lysis (DNA)

6. Bacillus subtilis engulfment SpoIIIE-GFP fusion protein (pink) Membranes (red) DNA (blue)

7. Sigma factor saga I Log/ veg. Growth  A 0  H II  K IV VII V-VI  E  F  E  F Inactive Active  G Inactive III Active ? decision ? activation ? Signals Check-points

9. Deciding to sporulate  A  H ? sporulate 1 1 Starvation: What is monitored? [GTP] = (C, N, P) 2 Cell density: 2 ? “quorum sensing” peptides Food low, [cell] low Food low, [cell] high Ok ? Trouble ahead ! 3 3 Chromosomes: 2 undamaged, complete, and polar placement. Not ppGpp

10. Decision committee Histidine Kinases KinA, KinB Spo0FSpo0B Spo0A ~P relay Inputs 1, 2, 3, ? ~~~~~~~ Spo0A~P ~P TGNCGAA DNA New genes mRNA New genes:  H ~P relay genes (+ feedback) (+ feed back ) “developmental switch”  E  F (inactive, “in waiting”) Asymmetric division

12. Activating the sigma factors  E SpoIIAASpoIIAB  F DNA New genes mRNA Spo0A~P Anti-sigma (ATP) SpoIIAASpoIIAB  F Anti-Anti-sigma (ADP) Released in “daughter” forespore compartment Pro-Sigma Protein SpoIIE Pro- cut off Active Sigma MotherDaughter Many genes SpoIIGA, SpoIIR proteases

13. Sigma-K activation in mother cell Split gene 42 kb Recombinase gene Joined gene Pro-sigma-K protein Mother Daughter sigma-K Pro-

15. Sigma factor summary I Log/ veg. Growth  A 0  H II  K IV VII V-VI  E  F  E  F Inactive Active  G Inactive III Active decision activation Signals Check-points

16. B. subtilis versus E. coli  s  5 Spores Smaller cells Fewer ribosomes Compact DNA Hard cortex Cyclopropane fatty acids Programmed mother cell death Cell death Genetic competence (sex) Hypervariation ?

18. Other sporulating bacteria Bacillus thuringiensis Crystal toxin Clostrium botulinum Neurotoxins1 gram kills a billion hamsters “Jurassic Park” experiment Endospores2 cells make 1 spore Exospores1 cell makes 1 spore (not as durable) Streptomyces coelicolorGram(+), fungus-like myceleum, antibiotics Myxococcus xanthusGram(-), wolf-pack gliding, social interactions

20. Streptomyces coelicolor “smell like soil”

22. Myxococcus xanthus 1 “wolf pack” “big stomach” feeding behavior, ~10 5 cells S-motility Type IV pili A-motilitySlime jets starvation A-signal (Quorum sensing) 2 Aggregate Ripple /waves Travel Germinate C-signal (contact, packing, alignment) Motto Eat together, spore together, move together 3 Fruiting Body (exospores)

23. Stigmatella aurantiaca fruiting body on a piece of decaying forest wood (scanning electron micrograph)