Team:Imperial College London/M2/genes

From 2009.igem.org

RcsB is a transcription factor that forms part of a phosphorelay system. As the name suggests, a phosphorelay system consists of a series of nodes down which a phosphate group is passed. The transfer of this phosphate group down along the Rcs relay system is initiated by membrane stress. Once the phosphate group has been passed to RcsB, it can bind to DNA and act as a transcription factor. Phosphorylated RcsB is thought to regulate ~5% of the E.coli genome ⁴.

The Rcs phosphorelay is composed of two membrane-anchored proteins, RcsC and RcsD, and a cytoplasmic protein, RcsB. In response to a signal, the sensor kinase RcsC autophosphorylates and the phosphoryl group is transferred to RcsB, via RcsD. RcsB can both homodimerise and heterodimerise with RcsA, these two complexes serve to regulate different operons.

Signal Amplification:

RcsB is able to heterodimerise with the protein RcsA. The RcsB+RcsA complex acts as a transcription factor that induces the expression of a number of genes. One of these genes codes for RcsA, this initiates a positive feedback loop.

Colanic Acid Biosynthesis:

The overexpression of RcsB is akin to fitting a stethoscope to the end of a megaphone. In essence, the sensitivity of the RCS Phosphorelay system becomes such that the smallest of membrane perturbations becomes sufficient to trigger colanic acid production. Thus, the background noise switches the RCS Phosphorelay system on permanently.

There are a total of 19 genes involved in colonic acid biosynthesis, the best characterised of which are shown below.

Other genes involved in colanic acid biosynthesis are listed below.

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

fcl

Codes for a subunit of GDP-fucose synthase. This enzyme is involved in colanic acid biosynthesis.

General Protective:

The following proteins are thought play a role in membrane fortification to confer protection against the rigours of dessication, acid exposure and osmotic stress.

Other RcsB induced genes coding for protective proteins are listed below.

KatE

Codes for a subunit of heme d synthase / hydroperoxidase. This bacterial catalase protects against osmotic stress.

Host Defence:

Humans synthesise a number of enzymes that are able to burst bacterial cells. These enzymes are called lysosymes. RcsB is able to induce a number of proteins that act as lysosyme inhibitors that will prevent the host-mediated lysis of our chassis.

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

Global Regulators:

Repression:

RcsB is also is able to act as a repressor, preventing the expression of both curli and fimbriae. This reduces cell motility and plays a role in biofilm formation.

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.]