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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 a phosphorelay system. In response to membrane stress, RcsB is phosphorylated into its DNA binding form. In this state, it is able to regulate ~5% of the E.coli genome 4.
The Rcs Phosphorelay System:
In this system, RcsC encodes a membrane bound sensor kinase, while RcsB functions as the response regulator. The protein RcsA is an auxiliary activator protein that heterodimerises with RcsB to induce a mucoid phenotype. Levels of RcsA are controlled by the ATP dependent protease Lon.
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.
yfbR
- dCMP phosphohydrolase
Membrane Proteins:
bdm (biofilm-dependent modulation protein)
OsmB
- OsmB encodes an outer membrane lipoprotein.
OsmY
Hyperosmotically inducible periplasmic protein
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.]
4) [http://www.ncbi.nlm.nih.gov/pubmed/16153174?ordinalpos=5&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_DefaultReportPanel.Pubmed_RVDocSum The Rcs phosphorelay: a complex signal transduction system.]
