IGEM:MIT/2005/*ToxR* info, etc.

• ToxR/lambda phage
  • Read the intro to this article (not just the abstract). Its VERY informative --> good for beginners.
• Co-regulation of cholera toxin genes
  • ToxR’s role is signal transduction through the membrane. It’s mechanism of action is thought to be the dimerization of ToxR (DiRita & Mekalanos, 1991). This mechanism of an extracellular protein causing a transmembrane protein to complex and activate transcription is fairly novel among bacterial transcriptional regulation systems.
  • Summary:
    • ToxR found in regulation of ctx operon (normally in Vibrio cholerae), which includes a gene for the toxin itself (ctx)
    • 3 regulator proteins are involved in the pathway:
      • ToxS: TM protein, only in periplasm
      • ToxR: TM protein, 1/3rd in periplasm, 2/3rds in cytoplasm
      • ToxT: inside cell (cytoplasm)
    • ToxS detects external environment (temp, pH, O2, osmolarity) via unknown mechanism. ToxR also does this in a limited way.
    • When activated, ToxS helps dimerize ToxR (mechanism does NOT include phosphorylation). ToxR's TM region winds up around each monomer (like a zipper)
    • When dimerized, ToxR's DNA binding domain causes transcription of ctx operon and also of ToxT gene
    • ToxT gene (along with ToxR) activates transcription of other proteins.
    • Structure of ToxR:
      • 294 aa (mw: 32,527 Da --> Miller et al. 1987)
      • Carboxy terminus: periplasm
      • Amino terminus: cytoplasm
      • TM region: Leu 183-Glu 198 (surrounded by Arg 182 on N-terminus and 3 uncharged aa on C terminus
      • Binding site known (Pfau & Taylor, 1996): TTTTGAT is tandemly repeated 8 times at -56 and once at -20 --> but these sequences are NOT enough! lacZ with these sequences will not produce B-gal.

• Synthesis of cholera toxin is positively regulated at the transcriptional level by toxR.
  • 1984 paper, mostly stuff we know, but in being sensitive to manipulating cells that can have negative human affects: "Southern blot analysis suggests
  • that all V. chokrae, including nontoxinogenic strains, have the toxR gene. Moreover, nontoxinogenic strains not only lack the structural genes for cholera toxin but also sequences associated with the larger 7-kilobase ctx genetic element." No, We Don't Have to Work With Virulent Cholera To Use ToxR.
• Invivo evidence for TonB dimerization with ToxR
  • I've only read the abstract, but TonB is a protein on the surface of E.Coli
  • Dimerization of ToxR causes ctx to be produced. But in E.Coli, this dimerization is studied by placing a lacZ (or other reporter gene) under the ctx promoter. So we don't need to be concerned about virulence factors.
• Co-ordinate expression of virulence genes by ToxR in Vibrio cholerae.
  • take home message: ToxR directly promotes some stuff (which includes production of ToxT). ToxT then directly promotes other stuff. ToxR can be a stand alone transcriptional factor.
• Changes in the periplasmic linker and in the expression level affect the activity of ToxR and lambda-ToxR fusion proteins in Escherichia coli.
  • Only read the abstract
  • These guys used ToxR-lambda phage repressor as a dimerization reporter
  • they constructed plasmids for various transmembrane fusion proteins.
  • Current status: reading it to summarize
  • Uh oh, problem: this article says: "the authors suggested that dimerization is not a requirement for ToxR activity in E. coli 10. Someone should get on the referenced paper right now and find out why this is a problem.
    • best I can tell, the reference claims that the ToxR periplasmic domain could be replaced with a few other dimers and a toxR-like monomer. Further analysis showed that this substitution only sort of worked. The "correct" ToxR conformation may be more important for function says the paper. This point is worth remembering, but likely not a game-breaker. It may be necassary to eliminate the possible substitutions in lab, but it would be better to know if this study manipulated the toxR itself significantly to use these substitutions. I'm inclined to think they did manipulate the protein significantly...