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

4. Enrichment cultures

5. Strain (Plasmid)Pollutant P. putida mt-2 (pWW0)Toluene Pseudomonas sp, CF600 (pVI150)Phenol Burkholderia sp. RP007Phenantrene P. putida TMBTrimethyl benzene P. putida NAH (NAH7)Naphthalene Acinetobacter sp. ADP1Aryl esters R. eutropha (pJP4)2,4 D Pseudomonas sp. (pP51)Trichlorobenzene P. putida UCC22 (pTDN1)Aniline P. oleovorans (pOCT)n-alkanes Burkholderia cepacia LB400Biphenyl, PCBs Pseudomonas sp. VLB120Styrene

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

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

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

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

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

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

28. Reporter lacZ gene fusions

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

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 Differential gene Expression (DGE) reveals signals and promoters Condition A Condition B

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

34. Structural genes Enzymes Membrane proteins Transcriptional regulators

35. Regulation of translation

36. Example of feedback inhibition