Part:BBa_K4359000

E.coli K-12 Cytolysin A (ClyA)

Cytolysin A (ClyA) is a 34-kDa protein coded for by the ClyA gene in E.coli genome. ClyA is also known as Hemolysin E (HlyE) or Silent Hemolysin A (SheA).

Biology

ClyA is a hemolysin that is present in E.coli and other Enterobacteriaceae. Most laboratory strains of E.coli harbour a chromosomal copy of the ClyA gene, however the coding sequence is often disrupted and produces a defective protein. ClyA also remains transcriptionally silenced by the heat-stable nucleoid-structuring (H-NS) protein, but expression can be derepressed in H-NS deficient strains. ClyA selectively localizes to the bacterial outer membrane, from where it is secreted to the extracellular medium via outer membrane vesicles (OMVs). ClyA lacks a canonical signal sequence and the mechanism by which it is transported to the outer membrane remains unknown.

ClyA exists in three known conformations: monomeric, protomeric and oligomeric. The monomer is the soluble form of the protein that undergoes a conformational change to the membrane-inserted protomeric form. The conformational change involves rearrangement of the beta-tongue motif to a helix-turn-helix motif, and the N-terminus alpha helix swings out to embed itself in the membrane. Upon reaching the target membrane, ClyA protomers oligomerize to for an annular 8-mer or 13-mer which inserts into the membrane to form a pore.

Usage

ClyA preferentially localizes to the outer membrane, and is suitable for heterologous protein display in gram negative bacterial systems. Additionally, ClyA is enriched in OMVs and can be used for of display recombinant proteins on OMVs. Localization to the outer membrane/OMVs is independent of the position of fusion, however C terminus fusions are preferred as N terminus fusion often result in inconsistent protein display. Some proteins (such as beta-galactosidase) are incompatible with ClyA-mediated display as they contain sequences which disrupt transportation. The addition of an intervening flexible peptide linker can improve display by extending the fusion partner into the extracellular medium.

Safety

Cloning and expression of ClyA under another promoter may result in the production of a toxin with hemolytic properties. Genetic fusions of ClyA with other proteins can abolish the pore-forming functionality of ClyA, possibly by interfering with oligomer assembly. For an attenuated version of ClyA, see ClyA (Y181F) BBa_K4359001

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal EcoRI site found at 1003
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal EcoRI site found at 1003
  Illegal NheI site found at 82
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal EcoRI site found at 1003
  Illegal BglII site found at 159
  Illegal BamHI site found at 997
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal EcoRI site found at 1003
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal EcoRI site found at 1003
  
• 1000
  COMPATIBLE WITH RFC[1000]