NYMU Taipei/Materials/Two component system for glucose sensing in E. coli/The Rcs phosphorelay

From 2007.igem.org

Research in Microbiology 157 (2006) 206–212.

  • The Rcs phosphorelay in E. coli was originally described as a regulator of the expression of the cps operon, encoding the proteins required for the production of capsular polysaccharide colanic acid.
  • The major gene cluster involved in colanic acid synthesis is located at 45.8 min on the canonical E. coli K-12 genetic map. 
    The 5'-proximal gene cluster consists of the wza, wzb, and wzc genes, which are followed by the wca operon consisting of about 20 genes.
  • The RcsCDB His-Asp phosphorelay system, initially identified as a positive regulator of the capsular exopolysaccharide (EPS) biosynthesis gene cluster (wza-wca) in E. coli, is conserved in several gamma-proteobacteria, including animal and plant pathogens.
  • Activation of the RcsCDB pathway, usually observed by monitoring the expression of the wza-wca gene cluster and the development of a mucoid phenotype
    occurs under some environmental conditions, such as dehydration and osmotic shock.
  • the involvement of the Rcs system in the development of biofilms? 
  • <a href="http://igem.ym.edu.tw/private_2007/images/a/a0/Rcs_phosphorelay.jpg">The Rcs phosphorelay</a> is unique in E. coli as it is composed of 3 separate proteins containing 4 signalling domains.
    • The sensor kinase of this phosphorelay, <a href="http://www.genome.ad.jp/dbget-bin/www_bget?eco+b2218">RcsC</a>, is a hybrid kinase, as the protein contains both a transmitter domain (H1) and a receiver domain (D1).
      The conserved histidine and aspartate residues in both domains have been shown to be involved in the phosphorelay.
    • RcsB is the response regulator of the Rcs phosphorelay and RcsB contains a conserved receiver domain (D2).
    • In addition to RcsC and RcsB, a third protein, RcsD (previously called YojN), which contains a histidine phosphotransfer (HPt) domain, has been shown to be required for the Rcs phosphorelay.
  • After RcsC senses an environmental signal, the conserved His in H1 is phosphorylated by an autophosphorylation event and the phosphoryl group is transferred to the conserved Asp in the D1 domain of RcsC. The phosphoryl group is then transferred to the His residue in the HPt domain of RcsD and finally to the D2 domain of RcsB. 
    Once phosphorylated, phospho-RcsB is able to bind DNA and to regulate the transcription of its target genes.
  • the phosphorylated RcsB functions as a DNA-binding transcriptional regulator, which together with RcsA transcribes the capsular polysaccharide synthesis (cps) genes.
  • Additional control of this regulatory network is provided by the dependence on the alternate sigma factor, RpoN, for the synthesis of RcsB.
  • RcsC is a transmembrane protein with a periplasmic N-terminal domain and cytoplasmic C-terminal domain.
  • Targets regulated by the Rcs system are of two types, depending on responsiveness to the RcsB-cofactor RcsA.
    • For the RcsA-independent class of RcsB targets, the site required for RcsB activity (the RcsB box) is located next to the -35 sequence, centered at -41/-42.
    • For the RcsA-dependent class, this site (the RcsAB box) is located either further upstream or downstream from the promoter.
  • The RcsA protein is not a component of the phosphorelay but functions as a coregulator of the cps transcription by forming a heterodimer with phosphorylated RcsB.
  • the RcsB homodimer is a likely transcriptional regulator 
  • RcsC and RcsD are periplasmic membrane proteins that heterodimerize to function as a ‘modified hybrid sensor kinase’ and RcsB is a cytoplasmic response regulator. 
  • In E. coli, the Rcs system is required for recovery from chlorpromazine-induced stress. 
    It is also involved in multidrug resistance and participates in biofilm development.
  • the framework of the Rcs signaling system formulated: RcsF (outer membrane protein)/RcsC (inner membrane protein) → RcsD → RcsB/RcsA → Rcs regulon (target genes).
  • In this Rcs signaling system, the rcsF gene product (a putative outer membrane-located lipoprotein) was also an essential signaling component.

(JOURNAL OF BACTERIOLOGY, June 2006, p. 4264–4270) 
The overproduction of two proteins is able to activate RcsC

  • one is DjlA, an inner membrane protein which belongs to the DnaJ family;
  • the other one is RcsF. RcsF was first identified as a regulator gene for exopolysaccharide synthesis.
  • activation of this pathway by overproduction of the membrane chaperone-like protein DjlA does not require RcsF. 
    Conversely, activation of the pathway by RcsF overproduction does not require DjlA either, 
    indicating the existence of two independent signaling pathways toward RcsC.
  • activation by overproduction of RcsF was indeed dependent upon the presence of RcsC. (therefore, we can overproduce RcsF to see if our RcsC chimeric protein works.)
    colonic acid production was stimulated by a combination of low temperature (20°C), zinc, and glucose, and that this stimulation was abolished in an rcsF mutant.
  • RcsF is an outer membrane lipoprotein oriented towards the periplasm.
  • <a href="http://www.genome.ad.jp/dbget-bin/www_bget?eco+b0196">RcsF interacts with RcsB?</a>