Mutations of the cytoplasmic regulatory protease, Lon, often cause a mucoid phenotype due to the overproduction of exopolysaccharide capsules. This observation allowed identification of the regulatory components of the Rcs regulon. Individual rcs genes, rcsA, rcsB and rcsC were identified using lac
fusions of the capsular polysaccharides synthesis genes, cps/man, responsible
for synthesis of colonic acid capsules, in Lon-positive vs Lon-negative backgrounds (Gottesman et al., 1985). Sequence homology to known 2-
component regulators suggested the sensor kinase and effector roles for RcsC and RcsB, respectively, and later identified RcsD as a phosphorelay protein
transmitting the activating phosphate from the C-terminal domain of RcsC to the N-terminal domain of RcsB (Stout & Gottesman, 1990, Takeda et al., 2001).
Upon activation by phosphorylation, RcsB forms a homodimer (RcsB2) and heterodimer with RcsA (RcsAB) (Figure 6) (Stout et al., 1991). The presence of
C-terminal helix-turn-helix motifs in both RcsA and RcsB indicated these proteins bind DNA. The accumulation of RcsA, but not RcsB, in lon– cells
revealed RcsA as the target for degradation by the Lon protease (Torrescabassa & Gottesman, 1987). Further characterisation of RcsA and RcsB determined that RcsB was the primary DNA-binding dual transcriptional regulator of the regulon, with RcsA acting as an ancillary protein, amplifying the potency of transcriptional regulation by RcsB at RcsA-dependent promoters, which are characterised by a conserved RcsAB box 50-100 nt upstream of the - 35 promoter region. Conversely, RcsA-independent promoters, require binding of RcsB2 to a conserved box immediately upstream of the -35 promoter region for expression (Brill et al., 1988, Francez-Charlot et al., 2003, Wehland &
Figure 6: Regulation of the Rcs phosphorelay.
(1) OM protein RcsF transmits inducing signals from the OM to the RcsC sensor kinase in the
IM. Peptidoglycan damage may also activate the pathway by directly stimulating RcsC. (2)
RcsC transfers phosphate to intermediate protein RcsD which activates RcsB by
phosphorylation (3). RcsB may then form a homodimer to activate the RcsA-independent (4)
branch of the regulon, or complex with RcsA to activate expression of rcsA, and regulate the
RcsA-dependent branch of the regulon (5). Both RcsB2 and RcsAB act as dual transcriptional
Identifying suppressors of the ftsZ84 division mutation lead to the discovery of
the OM protein, RcsF, that induces a mucoid phenotype in an RcsB-dependent manner, by transducing the Rcs-inducing signals detected at the OM to RcsC (Gervais & Drapeau, 1992). However, RcsF is dispensable for induction of capsule synthesis and the Rcs regulons can be induced by peptidoglycan damage, thus the Rcs-phosphorelay is sensitive to both OM and periplasmic stressors (Laubacher & Ades, 2008).
The Rcs-regulon can be described as having two branches, defined by the dependence on RcsA for full expression from the Rcs-regulated promoters (Figure 6). Typically, interaction of RcsAB at RcsA-dependent promoters induce the expression of capsular polysaccharide synthesis operons (Ferrieres et al.,
2007, Wehland & Bernhard, 2000), but RcsAB has also been shown to autoregulate the expression of rcsA (Ebel & Trempy, 1999, Wehland &
Bernhard, 2000) and repress motility by inhibiting the expression of the motility master regulator pseudogene flhDC. The expression of curli pilin is similarly
repressed by the RcsAB dimer (Francez-Charlot et al., 2003, Vianney et al.,
2005). Conversely, RcsB2 binds RcsA-independent promoters and controls the expression of a surprisingly wide variety of genes, ranging from effectors of osmotic shock and acid resistance, to regulators of cell division, and an alternative sigma factor, RpoS/σ38. It has been suggested the Rcs regulons directly and indirectly affect the expression of as much as 2.5% of the E. coli
Figure 7: Regulation of the sigma-E, Cpx and EnvZ/OmpR responses.
(A) In the absence of stress, the IM-embedded anti-sigma factor, RseA, sequesters sigma-E
(σE), preventing expression from sigma-E dependent promoters. (1) Overproduction of OM-
proteins (OMP), formation of aggregates and/or misfolded proteins, stimulate a conformational
shift in RseB, allowing binding of protease, DegS, to RseA. (2) The PDZ domain of DegS blocks
access to RseA, preventing cleavage of the RseAB complex. In addition to overproduced, misfolded or aggregated OMPs, the accumulation of LPS intermediates within the periplasm promotes conformational shift in the PDZ domain of DegS, allowing access to RseA, and eventual release of sigma-E. The removal of the RseAB complex causes a conformational shift in the cytoplasmic portion of RseA, allowing an IM protease, RseP, to cleave the RseA-sigma-E
complex, releasing it from the membrane. (3) This complex is then further processed by
cytoplasmic proteases (Ssp/ClpPX), which degrade the cytoplasmic domain of RseA, freeing
sigma-E to allow expression of sigma-E dependent promoters. (B) Similar to the sigma-E
response, Cpx response is induced by aggregated and/or misfolded proteins, and is specifically induced by the over-production of lipoprotein NlpE. The signal activates the CpxA cytoplasmic ATPase domain, activating the Cpx response regulator (CpxR) by phosphorylation.
Phosphorylated CpxR activates the expression of the cpx operon, and regulates the expression
of regulon members. (4) CpxP can interact with misfolded/aggregated proteins and
consequently becomes targeted for destruction by DegP, freeing CpxA kinase-activity from
CpxP-mediated inhibition (C) The EnvZ/OmpR 2-component signal transduction pathway reacts
to changes in osmolarity. Conformational changes in EnvZ result in the activation of the response regulator, OmpR by phosphorylation. OmpR-P can then regulate the expression of OmpR-dependent promoters. In low osmolarity environments, the phosphatase activity of EnvZ dominates, dephosphorylating OmpR and shutting down the response, a process also requiring ATP.