Team:Newcastle/End of crack & signalling system

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Subtilin

  • A type 1 antimicrobial peptide [AMP] or lantibiotic produced by B. subtilis
    • NB. Lantibiotic = peptide-derived antibiotics with high antimicrobial activity against various Gram-positive bacteria, including pathogenic bacteria such as propionibacteria, staphylococci, clostridia, enterococci and streptococci.
    • NB. Subtilin not same as Subtilisin!!!!!
  • Production of subtilin involves the spa gene cluster encompassing 10 genes, spaBTCSIFEGRK

NCL - Biobrick for Subtilin Sensor.PNG

The construct above contains:

  • spaRK promotor
  • spaR (subtilin peptide antibiotic Regulation) - the 220 amino acid product of this gene usually regulates the downstream production of subtilin antibiotic. It has an N-terminal domain that can be phosphorylated and a C-terminal domian that has DNA binding properties [http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6T0M-4D4XNMM-4&_user=224739&_coverDate=09%2F01%2F2004&_rdoc=1&_fmt=high&_orig=search&_sort=d&_docanchor=&view=c&_acct=C000014659&_version=1&_urlVersion=0&_userid=224739&md5=1f421f180a48e2d68b86da579cc7f920|1]
  • spaK (subtilin peptide antibiotic Kinase) - this gene codes for a 325 amino acid histadine kinase peptide that phosphorylates the N-terminus of spaR [http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6T0M-4D5KTFV-1&_user=224739&_coverDate=09%2F01%2F2004&_rdoc=1&_fmt=high&_orig=search&_sort=d&_docanchor=&view=c&_acct=C000014659&_version=1&_urlVersion=0&_userid=224739&md5=8d450f455463638579798872811ae5c0|2]. This activates the DNA binding ability of the C-terminus of spaR, which in turn initiates transcription of the downstream gene. In the case of our construct, this gene is gfp.
  • rrnO transcriptional terminator
  • spaS promotor - a strong promotor inducible by upstream activation of spaRK. It can be placed in front any gene to regulate its activity.

Genes required for swarming

  1. comP: Histidine sensor kinase of ComX and phrC
  2. comA: Response regulator of Comp
  3. sfp: Active surfactin synthetase
  4. srfA,B and C: Surfactin synthetase
  5. swrA,B and C: Novel gene for swarming and not for swimming
  6. efp: elongation factor P
  7. yabR and ymfl: Contribute to proper coordinationamong cells of the swarming population but are not absolutely required for surface migration.
  8. cheC and D: chemotaxis protein
  9. sigD: transcription factor sigma D, which is required for the expression of genes activated in late phase of flagellum biosynthesis.