Lecture 8: Bacterial Differentiation: Sporulation Reading assignments in Text: Lengeler et al Lecture 8 Text: pages Sporulation Text: pages 627 Secondary metabolism Lecture 7 Text: pages Gut bugs Text: pages , 800 Bacterial Lux Text: pages Quorum sensing Text: pages 801 Bacteria and plants Text: pages Agrobacterium Text: pages 275 Toxic oxygen Text: pages 807 Nitrogen cycle Text: pages Rhizobia and Cyanobacteria

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

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

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)

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

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

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

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

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

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

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

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 ?

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

Streptomyces coelicolor “smell like soil”

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)

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