Prokaryotic promoters in the test tube and in the environment

1. Prokaryotic promoters in the test tube and in the environment Pseudomonas putida Víctor de Lorenzo Centro Nacional de Biotecnología CSIC

4. Enrichment cultures

5. Strain (Plasmid) Pollutant P. putida mt-2 (pWW0) Toluene Pseudomonas sp, CF600 (pVI150) Phenol Burkholderia sp. RP007 Phenantrene P. putida TMB Trimethyl benzene P. putida NAH (NAH7) Naphthalene Acinetobacter sp. ADP1 Aryl esters R. eutropha (pJP4) 2,4 D Pseudomonas sp. (pP51) Trichlorobenzene P. putida UCC22 (pTDN1) Aniline P. oleovorans (pOCT) n-alkanes Burkholderia cepacia LB400 Biphenyl,PCBs Pseudomonas sp. VLB120 Styrene

6. Evolution of biodegradative capabilities 1. Genes encoding catabolic enzymes 2. Regulatory systems responding to substrates 3. Coupling transcription to Physiological signals

7. Stages in the regulation of gene expression systems 1 inducer Expression of catabiolic genes addition + C 2 3 4 Growth

8. Evolutionary gravitation towards an optimal regulation Gain / capacity Window of activity Specificity Metabolic coupling

9. And now a reminder on promoters and gene expression in bacteria...

10. Gene expression is a multi-step process Transcription mRNA stability Translation

11. Overview of transcription and translation

12. Transcription is in itself a multi-step process

14. Stem-loop structure of some terminators

15. Bacterial genes are frequently clustered in operons

16. What is a promoter?

20. Structure of rifampin

21. Positive and negative regulation

22. Regulation of transcription by two-component regulatory systems

23. Regulation of transcription by quorum-sensing systems

24. Structures of some autoinducers in quorum-sensing systems

25. s factors determine promoter specificity Sigma 54 Sigma 70 family A class in its own! ECF Family Ec rpoE Ec rpoH Ec fecI Sigma 70-like Ec rpoD Ec rpoS Ec rpoF

26. Sigma 54 promoters • -12/-24 consensus sequence • Require an activator that binds far from +1 GG-10-GC

27. The -12/-24 site • Small compared to -10/-35 • GC rich (frequent in Pseudomonas)

28. Reporter lacZ gene fusions

29. Monitoring promoter activity… once at a time Accumulation of beta-galactosidase

30. The big leap forward: from genetics to genomics

31. Computer-assisted search for genes and promoters

32. Probing global gene expression with Gene arrays Condition A Condition B Differential gene Expression (DGE) reveals signals and promoters

33. Number of Regulators 450 400 350 300 250 200 150 100 50 0 cjej ctra tpal hinf xfas llact ecoli rpxx ccre mlep hpyl tmar mtub bhal bbur dra1 paer spyo vcho buch pmul bsub ureur nmen meslo cpneu aquae mycge mycpn staphyN synecho

34. 700 7000 600 6000 500 5000 2 400 4000 R = 0,9668 Num. ORFs Num. Regulators 300 3000 2 R = 0,8842 200 2000 100 1000 0 0 0 1 2 3 4 5 6 7 8 Genome Size (Mb) Structural genes Enzymes Membrane proteins Transcriptional regulators

35. Regulation of translation

36. Example of feedback inhibition

37. Example of feedback inhibition