1. Gene Regulation - Signalling

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

3. 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 Regulation via Signaling

4. 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” Input domainTransmitter Sensor ReceiverOutput domain Response Regulator Input Signa l Output Signal Two-Component Signal Transduction

5. Cytoplasm IM Input domainTransmitter Sensor ReceiverOutput domain Response Regulator Input Signal Output Signal 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 + P Periplasm Two-Component Signal Transducer Components

6. Three reactions: (I)ATP + His ADP + His ~ P (II)His ~ P + Asp His + Asp ~ P (III)Asp ~ P + H 2 O Asp + P i These reactions transduce information - also called the “phospho-relay’ (I)The  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 Two-Component Signal Transducer Components

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

8. 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 Receiver Phosphorylation Domain Output domain DNA-binding Domain Response Regulators

9. http://info.bio.cmu.edu/Courses/03441/TermPapers /99TermPapers/TwoCom/osmoregmech.html The proteins involved are EnvZ Sensor Kinase OmpRResponse Regulator Three reactions: (I)Mg-ATP + EnvZ-His ADP + EnvZ-His ~ P (II) EnvZ- His ~ P + OmpR-Asp EnvZ-His + OmpR-Asp ~ P (III)OmpR-Asp ~ P + H 2 O OmpR-Asp + P i The phosphorylated residues: His 243 Asp 55 Osmoregulation

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

11. http://www.science.siu.edu/microbiology/micr425/425Notes/03- Osmoreg.html 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 OmpR Regulation of OmpF and OmpC

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

13. Bacterial Bioluminescence

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

15. 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) Thermodynamics

16. 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 Bioluminescent Enzymes Luciferases

17. FMNH 2 + O 2 + Fatty Aldehyde  FMN + Fatty Acid + H 2 O + hv (Light) Fatty-CHO  Fatty-COOH Reduced (energy rich)  oxidized (energy poor) FMNH 2  FMN both oxidized by O 2 Luciferase is a Mixed Function Oxidase Bacterial Luciferase Reaction

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

19. Proton Motive Force  1.ATP 2.Rotate Flagella 3.Active Transport 4.Reversed electron transport It costs energy to produce light ! Bacterial Bioluminescence A Shunt from Electron Transport

20. Luciferase Assay

21. Fast and Slow Luciferases The Enzyme Assay

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

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

24. 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. Phenomenon of Luciferase Induction

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

26. 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 The Inevitable Science Drill

27. Population density-dependent ORGrowth-phase-dependent Cell-Density Dependent Expression

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

29. 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 (10 7 /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 History

30. Euprymna scolopes

31. A network of cellular transduction mechanisms. Integrate signals from the bacterial environment to control gene expression, and thereby the bacterial phenotype. 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 Cell-to-cell Communication Quorum Sensing Molecules

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

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

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

35. 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) LuxI Family

36. 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 - - LuxR Family

37. LuxI-LuxR System

38. Quorum sensing systems - a sample

39. BioluminescenceConjugal transfer Vibrio anguillarum, V. fischeri, V. harveyiAgrobacterium tumefaciens Ti plasmid Plant-microbe interactionsSwarming motility Rhizobium leguminosarumSerratia liquefaciens R. meliloti - rhizosphere-specific genes Cell division Virulence determinants Escherichia 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 productionPolysaccharide production Erwinia carotovora - carbapenem,  -lactam antibioticKlebsiella 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 pathogenBacillus subtilis AHL molecules and their functions

40. 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 Quorum Sensing

41. Classical ParadigmCurrent Perspectives Bacteria exist as individuals.Bacteria can act in groups. Consistent phenotypes.Cell-density dependent phenotypes. One species, one disease. (Koch postulate) Multiple species, one disease. (Cross-talk) Non-interaction between host and pathogens Close intimacy between host and pathogens. Courtesy: KKF Changes in Perspectives

42. rhlIrhlR RhlIRhlR + Tester P. aeruginosa Passive Diffusion O O H N H O + Monitor E. coli P luxI -gfpluxR LuxR gfpluxR GFPLuxR GFP-based detection

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