1. Lecture 1: Metabolism and Assembly Reactions Reading assignments in Text: Lengeler et al. 1999 Text:pages 110-113 Metabolic overview Text:pages 568-569, 573-574 Pili (Fimbriae) and flagella Text:pages 825-829 Surface virulence factors Candle Flame analogy Dynamic process that looks static Birth and death Self sustaining: Acquire food and remove wastes Wastes = CO H O 22 2 Food = -(CH ) - n O “respiration” 2 “Learning should not be the filling of a bucket, but the lighting of a flame.” - roughly W.B. Yeats

2. Metabolic overview 2 n Food = -(CH ) - Wastes = CO H O 22 E. coli ~4,000 genes / reactions ~ 50% are expressed ~2,000 proteins / reactions AssemblyFuellingBiosyn.Polymer. Lecture 1 Pili Flagella DNA RNA Proteins Lecture 2 Lectures 3,4 ATP NAD(P)H 12 Precursor Metabolites Assimilation reactions CNS P

3. Assembly reactions / general concepts Self assembly: protein folding ribosome assembly membrane bi-layers Directed assembly: Scaffolds(leader-peptides, pro-proteins) Protein chaperones (SecB, PapD, others) Ordered substrate production B C A Check-point control 1 2 3 Cell division DNA replication DNA segregation

5. Pili assembly Illustrates: Protein chaperones and “ordered substrate production” Many types: E. coli F-plasmid “sex-conjugation pili” Myxobacteria Social motility “gliding/type IV pili” E. coli Urinary track infection “pathogenic pili” “Pap” Structure IM Periplasmic space OM Genetic analysis Wt papA papG EM RB Cell clumping yes no ? ? PapG = lectin (Gal)  1-4(Gal) ? G papAA G Wt

6. Pap Genetic system DNA A Pilin “tube” G Tip “lectin” Other genes = 11 Genes ! RNA PrTer 3 Types of proteins: 6 structuralC = anchor A H = tube E F G = tip 2 regulatory On/Off “phase variation” On/Off 3 assembly J K ? PapD = chaperone

7. PapD periplasmic chaperone Ig-like fold selectively binds Pap proteins Binds secreted and unfolded Pap on IM Release at site on OM Folding at nucleation point Protein:Protein recognition

8. Assembled PapA programs released PapA folding Folding information released free

9. Pap assembly model IM OM General secretion, SecAY SecB PapD PapC G F G Ordered substrate production F G E E A A F G E E H A A A A F G E E PapH = cap Model explains: Sub-unit order Pili length (PapH) Other issues: Polar vs lateral placement Many pili types Pili retraction (locomotion) Pili sensory (touch) transduction

10. uropathogenic Escherichia coli E. coli (gold) tethered to the lumenal surface of bladder cells (blue).

11. Flagellum assembly Illustrate: Check-point control SalmonellaCaulobacter Flagella Flagellum Basal body assembled Cell wall in out Make filament ?

12. The flagellum organelle ~50 genes = ~20 structural ~30 regulatory + assembly ? Basal body Mot (M S P L-rings/rods) Turbine/rotary engine H+ Switch CheY~P Secretion system Hook Flagellar filament (universal joint) (propeller)

13. Leader peptide proteins use regular SecAY/SecB secretory system Lack leader sequences Secretion and placement ? “Protein pump” ATP Open? Capped Nucleation point Inside view Open site Accepts flagellin Cap = Hap (Hook associated protein) Hook Flagellum organelle assembly

14. “Type III secretion systems” Hook Hap “Protein pump” ATP Hap Flagellum growth/Hap movement

15. Secretion systems (Type III) Many: Type I Type II Type III Type IV Signal Recognition Particle/Receptor system Selective for Hemolysins/S-layer proteins General SecA,Y/SecB Selective for key pathogenic proteins “virulence factors” Selective, conjugation DNA/proteins or virulence proteins (like eukaryotes, e.g. yeast) in out Flagellum “Needle complex” of Type III sec Homologous, also similar to F 0 F 1 ATPases ATP = direct injection Toxins Yersinia pestis cytoplasm Macrophage cytoplasm “contact-dependent sec” Anti-phagocytic Anti-cytokine Pro-apoptosis

16. Shigella flexneri

17. Flagellum assembly check-point control Basal Body assembled Make filament Salmonella typhimurium Species specific controls Last of 4 check-points Trigger stage 1 gene expression ~50 genes in stages 1, 2, 3, 4 2 3 1 DNA 4 Hook and flagellin genes ? Passage from stage 3 to 4 ? Sensor ? Triggering RNA polymerase ? Sensing BB structure/function BB

18. Flagellum assembly check-point control MECHANISM Trigger stage 1 gene expression 2 3 1 DNA 4 Hook and flagellin genes 1, 2, 3 stages made structure proteins Plus Regulators Sigma factor-28 FlgM =Anti-Sigma FlgM looks/pumps like flagellin Sigma-28 stays inside the cell Together trigger transcription RNA Freed Sigma-28 binds RNA polymerase FlgM is pumped out Stage 4 protein synthesis