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Team:Imperial College London/M2/genes
From 2009.igem.org
Through the course of evolution, E.coli have equipped themselves with a multitude of defences to enable colonisation of the intestine. We are using two global transcription factors (RcsB & YgiV) to hijack this natural process in a way that maximises acid resitance. We have additionally upregulated a third enzyme (rfal) to enhance the encapsulation of single cells (over and above colony encapsulation). Finally, the two biosynthetic genes (OtsA & OtsB) code for enzymes responsible for the production of trehalose. Our manipulation of endogenous pathways reduces virulence while enchancing pill functionality.
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RcsB
Background:
RcsB is a transcription factor that forms part of the phosphorelay system. In response to membrane stress, RcsB is phosphorylated into its DNA binding form. In this state, it is able to both upregulate and downregulate a large number of genes.
RcsB upregulates the following genes:
Colanic Acid Biosynthesis:
wcaF
- Putative colanic acid biosynthesis acetyltransferase 2
gmd
Codes for GDP-mannose 4,6-dehydratase which is a subunit of GDP-mannose 4,6-dehydratase. This enzyme is involved in colanic acid biosynthesis.
wcaD
- Predicted colanic acid polymerase
wza
- This codes for a lipoprotein that is required for colanic acid translocation through the outer membrane. The lipoprotein is a subunit of the capsular polysaccharide export apparatus.
fcl
- Codes for a subunit of GDP-fucose synthase. This enzyme is involved in colanic acid biosynthesis.
General Protective:
ivy (Inhibitor of Vertebrate lysozyme)
- Discovered in 2001 as the first bacterial lysozyme inhibitor. This Type-C lysozyme inhibitor resides in the periplasm.1
MilC (Membrane-bound lysozyme inhibitor of Type C lysozyme)
- This is a lipoprotein that resides in the membrane. 1
OsmC (Osmotically-inducible protein C)
- This protein provides defense against oxidative stress caused by exposure to organic hydroperoxides. 2 3
KatE
- Codes for a subunit of heme d synthase / hydroperoxidase. This bacterial catalase protects against osmotic stress.
Global Regulators:
Sra (stationary-phase-induced ribosome-associated)
Sra is a sub-stoichiometric component of the 30S ribosomal subunit that is more abundant at stationary phase than during log phase growth.
rprA
rprA is a gene encoding a small RNA molecule that is involved in the post-transcriptional control of the alternative sigma factor, ("sigma S").
FtsA
FtsA is an essential cell division protein which colocalizes with FtsZ to the septal ring structure.
FtsZ
Essential cell division protein.
Membrane Proteins:
bdm (biofilm-dependent modulation protein)
OsmB
- OsmB encodes an outer membrane lipoprotein.
PhnQ
ycfJ
- Uncharacterized putative inner membrane protein.
ygaC
spy
ymgD
- Putative periplasmic protein.
ypeC
- Unknown function
ymgG
YbaY
Predicted outer membrane lipoprotein
References
1) [http://www.ncbi.nlm.nih.gov/pubmed/19136591 The Rcs two-component system regulates expression of lysozyme inhibitors and is induced by exposure to lysozyme]
2) [http://www.ncbi.nlm.nih.gov/pubmed/19346340 Gene Expression Induced in Escherichia coli O157:H7 upon Exposure to Model Apple Juice]
3) [http://www.ncbi.nlm.nih.gov/pubmed/14627744 Structural and functional features of the Escherichia coli hydroperoxide resistance protein OsmC.]
YgiV
Background:
In nature, the colanic acid synthesis phase occurs prior to biofilm formation. The latter process of biofilm formation is associated with the upregulation of a number of virulence factors. The transcription factor YgiV blocks the progression into biofilm formation by maintaining colanic acid production. Thus YgiV serves to increase acid resistance and decrease virulence.
Rfal
Background:
In the majority of E.coli, the enzyme Rfal joins the O-antigen to the membrane-bound lipid core molecule. Since the K-12 strain has an insertion mutation in the gene coding for O-antigen, the enzyme Rfal is free to join colanic acid to the lipid core.
