Week 2

• a two-component signal transduction system (TCS) converts external signals into a phosphorelay. The two components that make up the system are the initial receptor and response regulator

  
• the more TCSs an organism has the better it will be able to survive and grow in different environments. TCSs allow bacteria to sense and adapt to changing environment
• in a TCS the signal binds to the the sensor histidine kinase. the kinase then auto-phosphorylates on its histidine residue. The Pi of the phospho-histidine is then transferred to the next protein in the phosphorelay, specifically on its aspartate residue.

  
• you can increase the regulation of a TCS system by adding connectors or phosphodonors

  
• We can have TCSs that are not part of the membrane but inside the cell (an example would be the KinA TCS)

  
• Regulation of the Histidine Kinase: Autokinase activity is either stimulated or repressed by a specific stimulus. RR phosphatase activity of the histidine kinase can be modulated.

  
• there are 5 related histidine kinases that are capable of phosphorylating the RR SpoOF KinA, KinB, KinC, KinD and KinE

  
• The Spo sequence started by Kin receptors begins by activating the Kin receptor. when activated Kin histidine kinase will auto-phosphorylate. The phosphate is then passed on to SpoOF then spoOB and finally SpoOA.

  
• Sporulation is prevented during vegetative growth and competence development.

  
• Anaerobic respiration using TCS: this scenario uses PhoR as the initial histidine kinase which responds to the change in phosphate concentration. Upon activation, it auto-phosphorylates and hands the phosphate group to PhoP which turns ON and becomes the transcription factor that makes ResD. ResE activated by low oxygen levels turns auto-phosphorylates and gives the phosphate to ResD which becomes a transcription factor for phoR and phoP. these 2 are required for phosphate uptake.

  
• Two-component systems allow bacteria to sense extracellular cues
• Extracellular cues activate histidine kinase that auto-phosphorylates and transfers phosphate to a response regulator - either directly or through a relay
• Phosphorylated response regulators have an affect on a cellular process - could be transcriptional or translational
• Two-component systems often display positive or negative feedback loop
• Many bacteria carry appendages such as pilus and flagellum
• Bacteria will move closer or further from the stimulus depending on what it corresponds to. It does this by using sensors that elicit a response inside the cell which in turn causes motility

  
• the flagellum is composed of: The basal body, hook, and the filament

  
• The basal body anchors the flagellum to the cell envelope

  
• The hook of the flagellum connects the basal body to the filament
• The filament is a long polymer comprised of the flagellin subunits.

  
• the basal body (flagellum anchor) has a rotor and stator component which allows for rotation. the energy for rotation is derived from the proton motive force

  
• The flagella allows for 2 modes of swimming: running and tumbling. the bacteria is running when the flagellum is rotating ccw. it will tumble when rotating cw.
• In the absence of nutrients, bacteria use their flagella to tumble frequently in order to sample their surroundings.

  
• when a nutrient gradient is encountered, bacteria use their flagella to run towards higher concentrations of nutrients.

  
• MCPs are transmembrane chemoreceptors associated with the flagella.

  
• Upon activation of MCP, CheA is inhibited and dephosphorylated. This allows the FliM to be free of Chey-P which allows the flagella to turn ccw (run)

  
• When nutrients are low, the Che system signals for tumbling. CheA autophosphorylates and gives the phosphate to CheY and CheB. CheY-P then attaches itself to the FliM of the flagellum basal body which causes cw rotation (tumbling).

  
• Studying Chemotaxis [Swimming assay] --> quantifies the ability of flagellated bacteria to move through semi-solid media containing a chemical attractant. These are phenotypic read-outs but don't inform on molecular details.
• Western blot analysis on chemotaxis: can be used to monitor covalent modifications of proteins. MCP methylation status of swimming bacteria can be assayed using western blotting. If running then the MCP would be heavily methylated.

  
• Bacteria with multiple flagella have the ability to swarm. Swarming motility describes flagellar movement across a solid surface. Swarming requires increased cell-cell contact and the production of surfactants.

  
• A motility pilus is a bacterial appendage involved in surface adherence and movement. A conjugative pilus facilitates DNA transfer between bacteria.

  
• Bacteria that use a pilus for motility typically produce many pili on their cell surface. In contrast to flagella, pili act as a molecular grappling hook by extending and retracting from the bacterial cell surface.
• Pili are used for a form of motility known as twitching

  
• Bacteria can sense the presence of solid surfaces. To attach to solid surfaces, many bacteria use type IV pili (T4P). The initial attachment event is accomplished by an extended type IV pilus.\

  
• Type IV pili are very important in the context of infection because some bacteria need to adhere to the surfaces of eukaryotic cells to cause

  
• T4P motility: After facilitating surface adherence, T4P is also used for movement along a surface (twitching motility). The force required for T4P-mediated surface movement arises from the retraction of an extended pilus. Many individual T4P are involved in this process, allowing a bacterium to twitch in, many different directions.

  
• Twitching assays quantify the ability of piliated bacteria to move across a plastic surface covered by solid media. Twitching assays are a phenotypic read-out but don't inform on molecular details.

  
• Western blot analysis can be used to monitor covalent modification of proteins. PilJ methylation status of twitching bacteria can be assayed using western blotting

  
• Che system is a two-component system that controls flagella-based motility. CheA is the HK, and CheY and CheB are the response regulators
• Chp (pil) system is a two-component system that controls pilus motility. ChpA is the HKm and PilG, PilH, and ChpB are the response regulator.