QUORUM SENSING

Stewart's wilt disease of maize is caused by the plant pathogenic bacterium Pantoea stewartii subsp. stewartii (Erwinia stewartii). The pathogen colonizes the xylem of the maize host, where it synthesizes large quantities of an expolysaccharide (EPS) (stewartan) virulence factor. This results in plant wilt. The synthesis of this EPS is cell density-regulated by the esaI/esaR quorum sensing (QS) regulatory system. The EsaI gene product is an acyl homoserine lactone synthase (AHL). The EsaR gene product is a LuxR-type transcriptional regulator. An AHL-deficient mutant strain, ESN51, is AHL-deficient, blocked for EPS synthesis, and hence, avirulent. Mutations in the esaR gene lead to an excessive EPS production as a result of deregulated, constitutive synthesis of stewartan EPS.  These observations suggest an alternative model for QS regulation in P. stewartii, namely regulation by gene repression and AHL-mediated derepression (1, 2).
fig 1

EsaR autoregulates its own expression by repression and AHL-mediated derepression.  The location of the esaR box in the esaR promoter suggests a repressor autoregulatory role. We confirmed this prediction, showing that EsaR dimerizes and binds the esaR box target DNA sequence in absence of AHL signal (3, 4).  The EsaR DNA binding potential diminishes in the presence of AHL. In vivo expression studies show that a PesaR::lacZ gene fusion is repressed when EsaR is expressed in trans under AHL-limiting conditions, and becomes subject to rapid derepression upon exposure to AHL. 

EsaR represses the expression of the rcsA gene.  EPS synthesis in P. stewartii is governed by the Rcs phosphorelay signal transduction system, similar to colanic acid biosynthesis in E. coli (5, 6). EsaR does not regulate the primary promoter of the cps gene system, which encodes the functions required for stewartan EPS synthesis. Instead, EsaR controls the expression of the rcsA gene, which is an essential co-activator necessary for cps gene activation. The rcsA gene features two promoters in the 5’ untranslated region, a proximal constitutive promoter, and a distal RcsAB regulated promoter. DNA binding studies identified an EsaR-specific DNA binding site positioned between the two promoters.  EsaR binding at this site blocks transcription initiation from the distal promoter, with no consequence to the proximal constitutive promoter. Under AHL inducing conditions, EsaR repression is neutralized allowing the expression from the upstream RcsAB inducible promoter. This creates a positive feedback regulatory mechanism, for RcsAB are required for the activated expression of rcsA (7, 8).

fig 2

 

The cps Gene System -  Earlier genetic studies laid the foundation for further analysis of the stewartan cps gene system (10, 11).  More detailed studies in our lab identified at least three promoters associated with this 12 kb gene system. The primary promoter located upstream of the wceG1 (cpsA) gene, the first gene of the locus, features an RcsAB binding box. A highly related homolog gene, is
 
fig 3

wceG2, is located elsewhere on the genome. The wceG2 gene expresses at low constitutive levels, while wceG1 expresses primarily under RcsAB activating conditions. A mutation in wceG1 gives an intermediary EPS phenotype; a mutation in the wceG2 gene has a more severe effect on EPS synthesis The two encoded enzymes are predicted to facilitate the transfer of D-UDP-galactose to the isoprenoid lipid carrier, the first step in Stewartan EPS subunit synthesis (11).

A second promoter is located upstream of wza (cpsB) gene, which allows basal level expression of wza, wzb (cpsI), and wzc (cpsC). Wza is an outer membrane lipoprotein that forms a channel for polymer translocation and surface assembly. Wzb is a inner membrane associated protein tyrosine phosphatase, while Wzc is a tyrosine autokinase. The activity of Wzc depends on the cycling of its phosphorylation state, which involves the dephosphorylation activity of Wzb (11).  All three enzymes together participate in high-level EPS polymerization.

A third promoter is upstream of the wceB (cpsE) gene. This gene together with wceM (cpsF), wceN (cpsG), and wceK (cpsK) encode glycosyltransferases involved in Stewartan subunit synthesis (see above). The wzx1 (cpsM) gene located at the 3’end of the locus encodes a predicted “flippase” enzyme involved in subunit translocation across the inner membrane (11). Note nomenclature used for the cps gene cluster (wceG-wzx1) adheres to the recommendation by Reeves et al., 1996 (12).
 

References

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