How do bacteria respond to their environment?

1. How do bacteria respond to their environment? • MyxococcusStarvation Fruiting bodies • BacillusStarvation Spores • AnabaenaStarvation Heterocysts How is starvation sensed and interpreted?

2. Response of E. colito lactose deprivation Economize Don’t make enzymes for lactose utilization

3. How does E. coli sense starvation for lactose? + Lactose - Lactose lactose lac expression repressed (use other carbon source)

4. Response of E. colito nitrogen deprivation + Nitrogen - Nitrogen Protein synthesis Economy Fast growth Slow growth

5. Response of E. colito nitrogen deprivation • Stringent response • Make less ribosomal protein • Make less ribosomal RNA • Make less transfer RNA • Problem: Multiple outputs • Control scores of genes • Problem: Multiple inputs • Deficiency in ANY amino acid = starvation

6. How does E. coli sense starvation for nitrogen? Is there a protein that binds ALL amino acids? TheRibosome

7. How does E. coli sense starvation for nitrogen? Plenty of amino acids

8. How does E. coli sense starvation for nitrogen? Plenty of amino acids

9. How does E. coli sense starvation for nitrogen? Plenty of amino acids

10. How does E. coli sense starvation for nitrogen? Plenty of amino acids

11. How does E. coli sense starvation for nitrogen? Plenty of amino acids

12. How does E. coli sense starvation for nitrogen? Starvation for amino acid Stuck! Ribosomes can sense absence of any amino acid

13. How does E. coli sense starvation for nitrogen? ??? Stringent response Low alanine Low cysteine … Low valine One small molecular signal

14. How does E. coli sense starvation for nitrogen? What’s the signal? Translocation (requires energy) GTP

15. How does E. coli sense starvation for nitrogen? What’s the signal? Translocation Pi GDP

16. How does E. coli sense starvation for nitrogen? What’s the signal? pppGpp GTP RelA RelA- relaxed(no stringent response) ATP AMP

17. Is response to N-starvation mediated through pppGpp? Critical Questions 1. Does pppGpp affect genes of stringent response? 2.Does uncharged tRNA pppGpp ??? Ogawa & Sy (1977) J Biochem 82:947. Synthesis of guanosine polyphosphates (pppGpp and ppGpp) and its regulation by aminoacyl t-RNA

18. Does uncharged tRNA pppGpp? How to tell? • Experiment • Add uncharged tRNA to ribosomes • See if pppGpp increases Need: • In vitro system where charging of tRNA can be controlled • Assay for level of pppGpp

19. Does uncharged tRNA pppGpp? In vitro system (~Nirenberg & Matthaei) Ribosomes: From differential centrifugation tRNAs:store bought tRNA charging enzymes: From soluble fraction or or Amino acids: store bought RNAs: homopolymers, e.g. AAAAAAAAA... GTP and ATP: store bought

20. Does uncharged tRNA pppGpp? Assay for level of pppGpp GTP* ATP + pppGpp GTP Thin layer chromatography + (ATP) + GDP + ppGpp + (magic spot) + amino acids - amino acids

21. Does uncharged tRNA pppGpp? What’s required to get pppGpp? % GTP* (p)ppGpp Condition + Poly(A) - Poly(A) No aa’s 30.6 0.4 All aa’s 2.8 0.3 AAAAA… (AAA=lys) 8.9 0.4 Only lysine All but lysine 30.1 0.2 Maximal pppGpp depends only on absence of lysine

22. Does uncharged tRNA pppGpp? Yes Is [pppGpp] correlated with [lys-tRNA]? Dose-response curve

23. Does uncharged tRNA pppGpp? • In vitro, Lys induces pppGpp • In vitro, Lys induces Lys-tRNA • In vitro, Lys-tRNA correlated with pppGpp What happens in vivo? What does ppGpp do? Stuck!