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

Phospo - transfer Reactions T 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

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

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

Histidine Kinase Kinase Catalytic Core: ~ 350 amino acids in length dimerization domain ATP/ADP-binding and phosphotransfer domain phosphatase activity found in some His P N-G 1 -F-G 2

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

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

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. Asp P

Modular Organization of tcs E. coli osmoregulation E. coli Anoxic Redox Regulation E. coli chemotaxis B. subtilis sporulation His P Asp P N-G 1 -F-G 2 His P N-G 1 -F-G 2 His P N-G 1 -F-G 2 Asp P P His P P Asp P N-G 1 -F-G 2 Asp P EnvZ OmpR His P Asp P His P N-G 1 -F-G 2 Asp P ArcB ArcA SpoOA SpoOB KinA KinB SpoOF CheA CheY CheB

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

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.

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

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)

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.

1)High osmotic pressure changes the conformation of the outer segment of EnvZ sensor protein. 2)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.

 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.)

 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 …….

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 Cyclic-di-gmp-mediated regulation in bacteria

2 GTP c-di-GMP 2 GMP 2 PP i diguanylate cyclase diguanylate phosphodiesterase GGDEF EAL

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

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

Vrg6 Vrg73 Vrg18 BvgAS Bvg+Bvg- 25 o C Nicotinic Acid MgSO 4  bvgAS

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

D HH CM OM ATP ADP ATP ADP D K I N H H K I N D H H ~ P BvgS ~ P 37 o C D BvgA HTH D

D HH CM OM ATP ADP ATP ADP D K I N H H K I N D H H BvgS ~ P ~ P 37 o C D BvgA HTH D

D HH CM OM ATP ADP ATP ADP D K I N H H K I N D H H D BvgA HTH D BvgS ~ P ~ P 37 o C

D HH CM OM ATP ADP ATP ADP D K I N H H K I N D H H BvgA BvgS D HTH D ~ P ~ P Virulence genes 37 o C

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

BvgR P vrg6 vrg18 vrg24 vrg53 vrg73 P P P P bvgAbvgS bvgR AUG GUA BvgA BvgS PP

BvgA RisS RisA BvgR MgSO 4 Nicotinic Acid Temperature - ? + +

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

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

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

Growth of gevA Mutant

GGDEFEAL PAS TM 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.

GGDEFEAL PAS TM GevA AAAAA AAL