Week 3-4

• Hanks-type Ser/Thr kinases (HSTKs) are present in Eukarya, bacteria, and archaea most likely due to a common ancestor. They are membrane or cytoplasmic proteins.

  
• HSTKs play a role in various regulatory networks in a transcription-factor independent manner. It is often associated with bacterial physiology such as cell division/replication

  
• HSTKs contain auxiliary domains that are required for activation. They bind extracellular stimuli for example. They have associated protein phosphatase that can modulate their kinase activity

  
• HSTKs: it has an N-terminal lobe that binds and orients ATP in the active site cleft. It also contains a C-terminal lobe that binds to the protein target substrate and transfers the phosphate group. Autophosphorylation is proposed to occur through a dimerization of kinase domains

  
• Ser/Thr protein phosphatases (STPs): a conserved catalytic domain that contains 9-11 signature motifs. They are Metal-dependent usually using Mg or Mn. Usually removes phosphate of cognate kinase to inactivate the signal cascade

  
• Bacterial sporulation: Many Gram-positive bacteria form endospores in response to stress or nutrient limitation. Spores are morphologically distinct cells that are highly resistant to heat, chemicals, and radiation.

  
• Spores are dormant cells that can reinitiate growth rapidly in response to environmental signals like amino acids or cell-wall muropeptides released by growing cells. These processes are well regulated and orchestrated by a series of protein phosphorylation events and changes in gene expressions controlled by sigma factors

  
• Spore formation only occurs for gram-positive bacteria
• there are 5 related histidine kinases that are capable of phosphorylating the RR SpoOF: KinA, KinB, KinC, KinD, KinE

  
• KinA: senses NAD/NADH in the cytosol

  
• KinB: senses low oxygen levels and high iron livels

  
• KinC: senses potassium efflux from cell due to membrane damage (surfactants)

  
• KinD: Senses glycerol, manganese, and malic acid

  
• SpoOf is an internal response regulator or receiver domain.

  
• SpoOB is a phosphotransferase

  
• SpoOA is a response regulator that acts as a transcription factor ont he spoII operons
• Signal cascade occurs during starvation and environmental stress to activate sporulation.

  
• Sporulation is an analog signal, with multiple fates along the signalling pathway. the 3 different fates are the early assessment, competence window, and final commitment. As phosphorylated SpoOA increases, the further it goes along the sporulation process.

  
• RapA is a SpoOF phosphatase that is activated by the competence TCS. Competence is induced by quorum sensing, which allows for rapid DNA uptake during stress.

  
• SpoOE is a SpoOA phosphatase that is activated by sigma b, which is induced under the general stress response

  
• sporulation starts off with the activation of Kin HKs which leads to SpoOA phosphorylation. Then it leads to the activation of YabT that turns on RecA by phosphorylation, this leads to the activation of the SOS-DNA damage response system. Lastly, YabT activates EF-Tu by phosphorylation to stop protein polymerization.

  
• HSTKs play a role in various biological processes in a transcription-factor independent manner. They usually have an associated phosphatase that can inactivate it.
• Gram-positive bacteria form endospores in response to stress or nutrient limitation- They are extremely resistant and can revive into viable bacteria when in a beneficial environment
• modulation of SpoOA levels allows for different outcomes along the sporulation pathway --> early assessment, competence window, final commitment
• YabT is a membrane bound HSTKs that contains a juxtamembrane DNA-binding domain. YabT impacts sporulation through RecA, which is an activator of the SOS-DNA damage response system.

  
• RecA activation stimulates DNA repair and signals an additional system to inactivate SpoOA transcription to allow for DNA repair before sporulation.

  
• YabT can recognize damaged parts of the DNA. The DNA will in the linker region of YabT. This is activated when the DNA is damaged. It will autophosphorylate and hand the phosphate group to RecA. RecA will inhibit SpoOA activity and activate the SOS-DNA damage response system

  
• activation of RecA gives the bacteria enough time to turn on the DNA response system before committing to sporulation.
• When the DNA is repaired, SpoOA stops being inhibited. SpoOA then turns on spoIIE genes that will make SpoIIE phosphatase. This phosphatase will dephosphorylate RecA and turn it off completely

  
• In the absence of DNA, YabT can phosphorylate EF-Tu. Phosphorylation of EF-Tu inactivates the protein.

  
• EF-Tu is essential for protein synthesis by binding to aa-tRNA molecules in a GTP dependent manner. TheEF-Tu-GTP-aa-tRNA complex associates with the ribosome and is required for translation

  
• before binding to aa-tRNA, EF-Tu needs to bind to GTP first. This complex will then move to the A-site of the ribosome (entrance). When the complex moves to the P-site, GTP hydrolysis and Ef-Tu dissociation occur.