Part:BBa_K200000

Colanic acid global regulator -> RcsB

Regulation for Capsular Synthesis (RCS) Phosphorelay System

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.

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.

RcsB as a transcription factor

Together, the RcsB~P homodimer and the RcsB~P+RcsA heterodimer are thought to regulate ~5% of the E.coli genome 4. It is important to note that RcsB is a global regulator found in many [http://en.wikipedia.org/wiki/Enterobacteria Enterobacteria]. In Salmonella, up to 38 genes were found to be up-regulated by RcsB including ftsZ which affects the rate of cell division. 1 The majority of upregulated genes are associated with enhancing structural integrety and acid resistance. 22 genes were found to be downregualted by RcsB in the same microarray experiment.3

Colanic Acid Biosynthesis:

The RcsB~P+RcsA heterodimer acts as a positive regulator for genes responsible for colanic acid production. 2 This is via the activation of the ugd/cps operon which is required for capsule synthesis.
Colanic acid is a heteropolymer containing glucose, galactose, fucose, and glucuronic acid. 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.

Usage and Biology

RcsB was used alongside YgiV and Rfal by the Imperial iGEM 2009 [http://2009.igem.org/Team:Imperial_College_London The E.ncapsulator] team to upregulate colanic acid production as part of the auto-encapsulation mechanism.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]

Characterisation

As you can see from the diagram, the control cells have a smooth cell membrane.
However, for those cells where RcsB (BBa_K200000) is induced, there is an obvious outer coating of colanic acid that forms on the outside of the cells, encapsulating the cell fully.

We have in addition conducted a functionality test to see if colanic acid encapsulation protects the cell from acidic environments.

Overnight cultures were incubated in pH 2 buffer solutions for 4 hours. Cells were plated before and after the experiment to determine the colony forming units. From the graph, it shows that cells encapsulated with colanic acid have a much improved cell viability, with colony forming units almost 2 orders of magnitude higher than control cells. This provides a clear demonstration of the protective effects of colanic acid expression via RcsB.

With the additional genes YgiV and RfaI, we expect the cell viability to increase further as an even more complete protective capsule is being produced.

References

<biblio>

1. 1 pmid=1597415
2. 2 pmid=2404948
3. 3 pmid=19124574

</biblio>