Gene Regulation - Signalling

1. Gene Regulation - Signalling

2. Methylation Superhelicity Rearrangement Gene Regulation Replication DNA Coupling Initiation Elongation Termination Temporal Tandem Promoters Stability Amount Adenylation Transcription mRNA Translation Protein Efficiency Poor RBS Sequestering Riboswitches Stringent Response Processing Stability Status Signals: intra- and extracellular

3. Regulation via Signaling • Two component response- regulator system • Covalent modification of the effector by the modulator • Involves phospho-relay • Three component response-regulator system: Quorum sensing • Modification? • Involved diffusible molecules, the acyl-homoserine lactones

4. Two-Component Signal Transduction • Sense minute fluctuations in chemical and physical conditions • The process: • Stimulus detection • Signal processing including amplification • Integration of sensory inputs • Production of appropriate output responses • Two components: Sensor and Response Regulator • They have “transmitters” and “receivers” Sensor Response Regulator Input Signal Output Signal Receiver Output domain Input domain Transmitter

5. Sensor Response Regulator Input Signal Output Signal Receiver Output domain Input domain Transmitter Cytoplasm IM Two-Component Signal Transducer Components + P Periplasm • Transmitters and receivers communication involves phosphorylation activities • Transmitters: • Autokinase activity • Attach phosphate from ATP to histidine --> Histidine kinases • High-energy intermediate that transfers the Pi to and Aspartate residue on the “Receiver” • Localization • Transmitter: Cytoplasmic membrane • Receiver: Cytoplasm

6. Two-Component Signal Transducer Components • Three reactions: • (I) ATP + His <--> ADP + His ~ P • (II) His ~ P + Asp <--> His + Asp ~ P • (III) Asp ~ P + H2O <--> Asp + Pi • These reactions transduce information - also called the “phospho-relay’ • The g phosphoryl group in ATP is first transferred to a histidine side-chain in response to a stimulus (II) The phosphoryl group is then transferred from the phosphohistidine residue to an aspartate side-chain --> phosphorylation induced conformational change --> eliciting response stimulate or repress the transcription of specific genes (III) Finally the phosphoryl group from a phosphoaspartae residue is transferred to water • Always have to function in pairs • Widespread - prokaryotic and eukaryotic

7. Sensor Response Regulator Response Regulator Sensor Two-Component Signal Transduction System The genes for the two components are linked. Part of an operon

8. Output domain DNA-binding Domain Receiver Phosphorylation Domain Response Regulators Localized in the cytoplasm Stimulate or repress the transcription of specific genes - DNA-binding Domains Conserved regions N-terminal Phosphorylation Domain C-terminal DNA-binding Domain Example: OmpR circuitry Activates transcription of ompF and ompC Represses transcription of ompF Histidine kinases Reference Pages http://www.uni-kl.de/FB-Biologie/AG-Hakenbeck/TGrebe/HPK/Classification.htm

9. Osmoregulation • The proteins involved are • EnvZ Sensor Kinase • OmpR Response Regulator • Three reactions: • (I) Mg-ATP + EnvZ-His <--> ADP + EnvZ-His ~ P • (II) EnvZ- His ~ P + OmpR-Asp <--> EnvZ-His + OmpR-Asp ~ P • OmpR-Asp ~ P + H2O <--> OmpR-Asp + Pi The phosphorylated residues: His 243 Asp 55 http://info.bio.cmu.edu/Courses/03441/TermPapers/99TermPapers/TwoCom/osmoregmech.html

10. Sensor Kinases or Histidine Protein Kinases Cytoplasmic membrane protein with at least two membrane spanning domain Unconserved regions provide the specificity (80 %) Highly conserved regions EnvZ Sensory Domain ~115 residues Signal Transduction Domain IM H Box (His-243) Catalytic Domain ~270 residues N Box - Kinase domain G Box - ATP Binding Histidine phosphorylation is NOT intra-molecular, requires a second monomer Can also act as phosphatase - dephosphorylate the response regulators

11. OmpR Regulation of OmpF and OmpC • OmpR bindig Sites: High and Low affinity • Low OP • OmpR-P form binds to the high affinity site --> activate ompF transcription • High OP • Increase in OmpR-P, • Bind to both high & LOW affinity sites --> inhibit ompF transcription • Bind to ompC promoter --> activate transcription • Activate micF expression --> inhibit ompF translation http://www.science.siu.edu/microbiology/micr425/425Notes/03-Osmoreg.html

12. Gene Regulation: Regulation of Luminesence in Marine Bacteria Light Production Costs Energy and Is Regulated…..

13. Bacterial Bioluminescence

14. Bioluminescence Chemiluminescence from an Enzyme Lets Light Up Some Terms: Fluorescence Phosphorescence Luminescence Energy Source? Excited States? Excited State Life Times?

15. Thermodynamics Energy Change During a Chemical Reaction – (regardless of the energy source: chemical or light) Substrate(s)  Product(s) + Energy or Substrate(s) + Energy  Product(s)

16. Bioluminescent Enzymes Luciferases In honor of Lucifer ! In Greek Mythology Lucifer is the Bearer of Light In Christian Mythology…. Luciferase = Enzyme Catalyzing Bioluminescent Reaction Luciferin = small molecule whose excited state produces light

17. Bacterial Luciferase Reaction FMNH2 + O2 + Fatty Aldehyde  FMN + Fatty Acid + H2O + hv (Light) Fatty-CHO  Fatty-COOH Reduced (energy rich)  oxidized (energy poor) FMNH2 FMN both oxidized by O2 Luciferase is a Mixed Function Oxidase

18. Planck-Einstein Equation E = hν Energy = Planck’s Constant x vibrational frequency of light The smaller the Wavelength (λ), the larger the v.

19. Bacterial Bioluminescence A Shunt from Electron Transport • Proton Motive Force  • ATP • Rotate Flagella • Active Transport • Reversed electron transport It costs energy to produce light !

20. Luciferase Assay

21. Fast and Slow Luciferases The Enzyme Assay

22. Is Luciferases Constitutive or Inducible? Nealson, Platt and Hastings, 1970. Their Photobacterium fischeri MAV was reclassified Beneckea harveyi and now Vibrio harveyi MAV.

23. Is Luciferases Constitutive or Inducible? Shewanella woodyi Photobacterium phosphoreum WSU

24. Phenomenon of Luciferase Induction is called AUTOINDUCTION bacterial growth results in production of “autoinducer”…. a small diffusible molecule that when it accumulates in the medium to sufficient concentration  induces the synthesis of luciferase. Nealson, Platt and Hastings, 1970 Also called Quorum Sensing (since 90’s) … the molecules are called AutoInducers. AI-1, AI-2, etc.

25. Quorum The number of members of a group or an organization required to be present to transact business legally, usually a majority. - Webster Quorum Sensing - 1995

26. The Inevitable Science Drill Widespread phenomena but have to go through the drill: “Every novel idea in science passes through three stages: 1. First people say it isn’t true, 2.Then they say it’s true but not important, and 3. Finally they say it’s true and important, but not new” Peter Greenberg What was unique? Covalent Modification is not NEW – but new for bacteria Diffusible chemicals to mediate regulation is NEW for bacteria

27. 1000 10 ) 7 100 1 A 660 nm LUCIFERASE ACTIVITY (qps X 10 10 0.1 1 0.01 0 2 4 6 8 10 TIME (hours) Cell-Density Dependent Expression Population density-dependent OR Growth-phase-dependent

28. Quorum sensing Luminescent bacteria in marine environment Why do they produce light?? The genes that regulate luminescence are lux genes Intracellular communication

29. History • 60s:The first report on cell-density dependent phenomenon • in the marine bacterium Vibrio fisheri and V. harveyi • by Nealson, Hastings, Eberhard, Makemson. • 70s: V. fisheri lives in specific light organs (Euprymma ecolopsis) as pure culture (107/ml). • Bioluminescence phenomenon - at stationary phase called autoinduction • 70’s-80s: The details of the regulation and mechanisms of luminescence were worked out. • The genes involved in autoinduction, luxR and luxI, were cloned. • Other effectors: Arginine, glucose (c-AMP), iron, oxygen, salt. • 90s: The term quorum sensing was coined. • Discovery of LuxR-LuxI systems in many gram negative bacteria. • Homology of LasR in Pseudomonas aeruginosa to LuxR. • Discovery of anti-quorum sensing molecules - Furanone

30. Euprymna scolopes

31. Cell-to-cell Communication A network of cellular transduction mechanisms. Integrate signals from the bacterial environment to control gene expression, and thereby the bacterial phenotype. Quorum Sensing Molecules Relies on the accumulation of small diffusible, extracellular signaling molecules to modulate the transcription of target operons. Autoinducers/Communication Molecules/Bacterial Pheromones. Interaction between autoinducers and a positive transcriptional activator. Three-component system

32. Three Component System Signaling molecules (Acyl homoserine lactone/ AHL) and many others Synthetase (LuxI homologue) Quorum-sensing Signal Generators - “I” Regulator (LuxR homologue) Signal Receptors - “R”

33. Lux system - Vibrio fischeri Genes involved in the luminescence

34. LuxI and LuxR Family Found in diverse microorganisms Affects various physiologic functions Genes are linked but NOT co-transcribed Divergently or independently transcribed

35. LuxI Family Synthetase for autoinducers forms amide bond between acyl-acyl carrier protein and S-adenosyl methionine N-acyl-homoserine lactones (AHLs) Differ in the structure of their N-acyl side chains (sat/unsat) Vary between 4 to 14 carbons Examples: N-butanoyl-L homoserine lactone (BHL) N-hexanoyl-L-homoserine lactone (HHL) N-octanoyl-L-homoserine lactone (OHL)

36. LuxR Family Transcriptional regulators lux Box Centered around -40 R N S T G Y A X G A T N X T R C A S R T G G G T G T A G G A T G G T G C A G G T A A C C A A A A C A T T T T C C C C - -

37. LuxI-LuxR System

38. Quorum sensing systems - a sample

39. AHL molecules and their functions BioluminescenceConjugal transfer Vibrio anguillarum, V. fischeri, V. harveyi Agrobacterium tumefaciens Ti plasmid Plant-microbe interactionsSwarming motility Rhizobium leguminosarum Serratia liquefaciens R. meliloti - rhizosphere-specific genes Cell division Virulence determinantsEscherichia coli Pseudomonas aeruginosa - elastase, pyocyanin etc Ralstonia solanacearum - a phytopathogen Extracellular lipase synthesis Aeromonas hydrophila - serine proteases Streptomyces lividans 66 Vibrio cholerae - HA/protease Bordetella pertussis - pertussis toxin Pigment production Erwinia carotovora - a phytopathogen Chromobacterium violaceum - violacein Staphylococcus aureus Antibiotic production Polysaccharide production Erwinia carotovora - carbapenem, b-lactam antibiotic Klebsiella pneumoniae Rhodobacter sphaeroides Escherichia coli - colanic acid Pseudomonas aureofaciens- 3 phenazine antibiotics Erwinia stewartii - Stewart's wilt of sweet corn Genetic competence Gram +ve lactic acid bacteria - antimicrobial peptide Streptococcus pneumoniae Pseudomonas solanacearum - plant pathogen Bacillus subtilis

40. Quorum Sensing • A cell-density dependent phenomenon. • A global mode of gene regulation, including virulence gene expression. • Alters bacterial behavior/phenotypes. • Involves intra-, inter-species and inter-kingdom communication. • Bacteria act as a community

41. Changes in Perspectives Courtesy: KKF

42. rhlR rhlI + O Monitor E. coli O RhlR RhlI N H H O Passive Diffusion luxR PluxI-gfp luxR gfp + LuxR LuxR GFP GFP-based detection Tester P. aeruginosa

43. (i) (a) (b) (d) (c) (ii) Cell-density dependent expression with GFP monitor