Two - Component Signal Transduction modular stimulas-response systems

1. Two - Component Signal Transduction modular stimulas-response systems • Response Regulator: conserved receiver domain + specific effecter domain • Histidine Kinase Sensor: conserved kinase core (transmitter domain) + specific sensory domain

2. T Phospo - transfer Reactions The -phosphoryl group is transferred to the conserved histidine side chain of the HK. The RR catalyzes the transfer of the phosphoryl group from the phospho-His residue to the conserved aspartic acid side chain of the RR. Finally the phosphoryl group is transferred from the phospho-Asp residue to water in a hydrolysis reaction

3. genomic distribution • E. coli: 30 HKs (5 hybrids) and 32 RRs • Synechocystis sp: 80 • Mycoplasma sp: 0 • Bacillus subtilis: 70 • Haemophilus influenza: 9 • Helicobacter pylori: 11

4. Histidine Kinase • Most are periplasmic membrane receptors. • Function as homodimers: autophosphorylation is a bimolecular event. • Periplasmic, N-terminal binding domain. • Transmembrane domain. • Linker domain. • Histidine-containing phosphotransfer domain • C-terminal kinase core. • CheA & NtrB are soluble, cytoplasmic HKs

5. P His N-G1-F-G2 Histidine Kinase Kinase Catalytic Core: ~ 350 amino acids in length dimerization domain ATP/ADP-binding and phosphotransfer domain phosphatase activity found in some

6. P His Histidine Kinase Histidine-containing phosphotransfer domain ~ 120 amino acids in length Histidine residue No kinase or phosphatase activity

7. Histidine Kinase • Sensing domain: N-terminal domain that senses external stimuli Usually periplasmic receptor - not always In many cases the ligand or stimulas is unknown Little or no sequence similarity. • Transmembrane and Linker Domains: Poorly understood Critical for propagation of signal from periplasmic binding domain to kinase core

8. P Asp Response Regulator • N-terminal Receiver or Regulatory domain • C-terminal Effector domain: DNA-binding transcriptional regulator enzymatic activity (CheB or RegA) protein-protein interactions • Catalyze the transfer of phosphryl group from phospho-HK to conserved aspartic acid: phosphorylation results in conformational change of response regulator. • Many also catalyze auto-dephosphorylation.

9. Modular Organization of tcs OmpR EnvZ P P P P P P P P P P P P P P ArcB ArcA Asp Asp Asp Asp Asp Asp Asp His His His His His His His N-G1-F-G2 N-G1-F-G2 N-G1-F-G2 N-G1-F-G2 N-G1-F-G2 KinA SpoOF SpoOB SpoOA KinB CheY CheA CheB E. coli osmoregulation E. coli Anoxic Redox Regulation E. coli chemotaxis B. subtilis sporulation

10. Modular Organization of tcs • Phosphotransfer Systems: His --> Asp • Phosphorelay Systems: His --> Asp --> His --> Asp Added complexity provides for multiple regulatory checkpoints and points of integration between signaling pathways

11. Regulatory Mechanisms The whole point of signal transduction is regulation. The signaling pathway provides steps at which the flow of information can be modulated. • Regulation of the Histidine Kinase: Autokinase activity either stimulated or repressed by specific stimulas. RR phosphatase activity of the histidine kinase can be modulated. • Regulation of the Response Regulator: Phosphorylation by cognate HK Dephosphorylation by specific phosphatases Stimulation of intrinsic autophosphatase activity. • Inhibition of phosphotransfer • Regulation of the expression of the two-component proteins.

12. Integration of Signals i • Five related HKs are capable of phosphorylating the RR SpoOF (KinA, KinB, KinC, KinD and KinE). • KinA, KinB, KinC, KinD and KinE share sequence similarities surrounding the phosphorylatable histidine residue but differ in their sensing domains. • RapE is expressed during vegetative growth. • RapA and RapB are induced by the ComA/ComP TCS • Therefore sporulation is prevented during vegetative growth and competence development

13. Integration of Signals II • ResD/ResE regulates expression of genes required for anaerobic respiration. • PhoP/PhoR regulates expression of genes required for phosphate uptake. • When phosphate is low, phosphorylated PhoP induces expression of res operon while repressing the PhoP-independent promoter. • Phosphorylated ResD activates phoP-phoR expression (positive feedback loop)

14. Integration of Signals III • The product of the udg gene is required for both the Pmr-regulated modification of LPS and the Rcs-dependent production of capsule. • Both PmrA and RcsB can bind and activate transcription from the ugd promoter. • PmrD activates PmrA post-transcriptionally independently of PmrB in response to Mg++. • The ugd gene is expressed in response to Mg++, Fe+++ OR cell envelope stress.

15. High osmotic pressure changes the conformation of the outer segment of EnvZ sensor protein. The change is transmitted inwards and EnvZ phosphorylates itself using ATP. It then transfers the phosphate group to OmpR. The OmpR-P form binds DNA.

16. When OP is low, there is only a trace of OmpR-P, but this is sufficient to bind to the high affinity site in front of the ompF gene and activate transcription. • At high OP, the concentration of OmpR-P rises and it can now occupy the low affinity sites. This stops transcription of the ompF gene and activates transcription of the ompC gene. • In addition the micF gene is transcribed to give MicF RNA. This binds to the front of the ompF message and prevents translation. Thus whenever expression of ompC is increased, expression of ompF is decreased. (Actually micF is more probably important for temperature control than for osmoregulation.)

17. CheA is HK that phosphorylates RRs CheY and CheB. • Phosphorylation of CheA stimulated by unoccupied receptors (requires CheW). • Phosphorylated CheY binds the flagellar motor and stimulates CW rotation of the motor which results in enhanced tumbling. • CheZ is a phosphatase that dephosphorylates CheY • Upon phosphorylation by CheA, CheB removes methyl groups from MCP resulting sensory adaptation. • Ligand bound MCP undergoes conformational change that inhibits autophosphorylation of CheA…….

18. Cyclic-di-gmp-mediated regulation in bacteria The discovery of c-diGMP dates back to work published by Moshe Benziman on the regulation of cellulose biosynthesis in Gluconacetobacter xylinum (formerly called Acetobacter xylinum) and Agrobacterium tumefaciens. In two landmark papers, published in 1987 and 1998, Benziman and colleagues first described the identification of c-diGMP as an allosteric regulator of cellulose synthase (CS) CS activity is almost completely dependent on the presence of c-diGMP

21. diguanylate cyclase diguanylate phosphodiesterase 2 GMP c-di-GMP 2 GTP 2 PPi EAL GGDEF

22. Cyclic di-GMP as a Bacterial 2nd Messenger diguanylate cyclase diguanylate phosphodiesterase 2 GMP c-di-GMP 2 GTP 2 PPi EAL GGDEF EAL Activity of Effector Protein

25. BrkA Fimbrea FHA Tracheal Colonization Factor Pertactin Ptl Adenylate Cyclase Toxin Pertussis Toxin Tracheal Cytotoxic Toxin

26. Vrg18 Vrg73 Vrg6 BvgAS Bvg+ Bvg- 25oC Nicotinic Acid MgSO4 DbvgAS

27. OM D P B B P D CM K I N H BvgS D H D BvgA HTH

28. OM CM K K I I H H P N N ~ P D HTH 37oC ATP ATP ~ H H ADP ADP BvgS D D D H H D BvgA HTH

29. OM CM K K I I H H P N N D HTH 37oC ATP ATP H H ADP ADP BvgS ~ ~ D D P D H H D BvgA HTH

30. OM CM K K I I H H P N N ~ D HTH 37oC ATP ATP H H ADP ADP BvgS D D D ~ P H H D BvgA HTH

31. OM CM K K I I H H N N P P ~ ~ D HTH 37oC ATP ATP H H ADP ADP BvgS D D D H H Virulence genes D BvgA HTH

32. OM CM K K I I H H N N D HTH 25°C or 37°C + SO or Niacin H H BvgS D D D H H D HTH Virulence genes BvgA

33. bvgR bvgA bvgS AUG AUG GUA P P BvgA BvgS P BvgR vrg6 P vrg18 P vrg24 P vrg53 P vrg73

34. vrg MgSO 4 ? - Nicotinic Acid Temperature RisS BvgS RisA BvgA + BvgR +

35. Cyclic di-GMP as a Bacterial 2nd Messenger diguanylate cyclase diguanylate phosphodiesterase 2 GMP c-di-GMP 2 GTP 2 PPi EAL GGDEF EAL Activity of Effector Protein BvgR EAL

37. GGDEF/EAL proteins in Bacillus anthracis BA 0548 EAL BA 0628 EAL BA 2533 EAL BA 3879 EAL BA 4203 EAL BA 4263 BA 5543 EAL BA 5593 EAL BA 5664

38. Virulence of GGDEF/EAL Mutants gevA = GGDEF/EAL virulence regulator A

39. Growth of gevA Mutant

40. TM PAS GGDEF EAL GevA PAS domains act as sensory modules for oxygen tension, redox potential or light intensities. The domain functions through protein-protein interactions or through binding cofactors within their hydrophobic cores to regulate protein-protein interactions in response to stimuli.

41. TM PAS GGDEF EAL GevA AAL AAAAA