The Virulence of aDickeya dadantii3937 Mutant Devoid of Osmoregulated Periplasmic Glucans Is Restored by Inactivation of the RcsCD-RcsB Phosphorelay

Abstract

Dickeya dadantii is a pectinolytic phytopathogen enterobacterium that causes soft rot disease on a wide range of plant species. The virulence of D. dadantii involves several factors, including the osmoregulated periplasmic glucans (OPGs) that are general constituents of the envelope of proteobacteria. In addition to the loss of virulence, opg-negative mutants display a pleiotropic phenotype, including decreased motility and increased exopolysaccharide synthesis. A nitrosoguanidine-induced mutagenesis was performed on the opgG strain, and restoration of motility was used as a screen. The phenotype of the opg mutant echoes that of the Rcs system: high level activation of the RcsCD-RcsB phosphorelay is needed to activate exopolysaccharide synthesis and to repress motility, while low level activation is required for virulence in enterobacteria. Here, we show that mutations in the RcsCDB phosphorelay system restored virulence and motility in a D. dadantii opg-negative strain, indicating a relationship between the Rcs phosphorelay and OPGs.Osmoregulated periplasmic glucans (OPGs) are general periplasmic constituents of the envelope of most proteobacteria. Their common features are that glucose is the sole constituent sugar, and their abundance in the periplasm increases as the osmolarity of the medium decreases. In Enterobacteriaceae and related bacteria, the glucose backbone synthesis is catalyzed by both products of the opgGH operon (5). Studies of several bacterial pathogens, including Dickeya dadantii, showed the importance of OPGs for virulence (4,5,18,25,26).Dickeya dadantii is a member of the pectinolytic erwiniae causing soft rot disease in a wide range of plant species (33). The virulence of D. dadantii is associated with the synthesis and the secretion of a set of plant cell wall-degrading enzymes (pectinases, cellulases, and proteases) causing maceration of the plant tissues (22). D. dadantii synthesize OPGs containing 5 to 12 glucose units joined by ␤,1-2 linkages and branched by ␤,1-6 linkages that are substituted with succinyl and acetyl residues (11). The opgG or opgH mutants unable to synthesize OPGs show a pleiotropic phenotype. They are nonvirulent on chicory leaves and potato tubers, and synthesis and secretion of pectate-lyases, cellulases, and proteases are reduced (32). Motility is severely reduced, while exopolysaccharide secretion is increased (mucoid phenotype) (32). Data suggest that the opg mutants are impaired in perception of the environment, which prevents D. dadantii from recognizing host cells, suggesting a possible dysfunction of phosphorelay signaling pathways, major systems required for environmental perception in bacteria (6). In these systems, upon stimuli, a kinase/phosphatase sensor autophosphorylates and transfers the phosphate group to a cytoplasmic regulator which modulates expression of target genes.Here, we show that mutations in the rcsC and rcsB genes, encoding, respectively, the sensor and the cognate regulator of the RcsCD-RcsB phosphorelay, suppress several phenotypes o...

Inactivation of pecS restores the virulence of mutants devoid of osmoregulated periplasmic glucans in the phytopathogenic bacterium Dickeya dadantii

“…By contrast, the other known suppressors of the Opg phenotype, i.e. the rcsC gene (Bouchart et al, 2010), the kdgR and the pecT genes (this study), allowed restoration only on potato tubers. All these suppressors restore pectinase synthesis and secretion, and the data presented here show that this restoration is sufficient to restore virulence in potato tubers in strains devoid of OPGs.…”

“…The role of OPGs remains unclear and is probably indirect. In a previous study, the relationship between OPGs and the phosphorelay RcsCDB was demonstrated (Bouchart et al, 2010). Direct interaction between the RcsC and/or RcsD sensor proteins and OPGs cannot be excluded, as the periplasmic domain of both proteins may interact with these periplasmic glucans.…”

“…In strains of D. dadantii devoid of OPGs, inactivation of the RcsCDB phosphorelay regulatory system restored PCWDE synthesis and secretion, motility, normal exopolysaccharide synthesis and virulence in potato tubers, indicating that OPGs control the activation level of this phosphorelay (Bontemps-Gallo et al, 2013;Bouchart et al, 2010). The relationship between this phosphorelay and OPGs highlights the role of these glucans as intermediates in the perception of environmental changes, given that phosphorelays are the main systems used by bacteria to sense and adapt to environmental variations (Clarke, 2010).…”

Increased phosphorylation of the RcsB regulator of the RcsCDB phosphorelay in strains of Dickeya dadantii devoid of osmoregulated periplasmic glucans revealed by Phos-tag gel analysis

“…More recently, we showed that the three main repressors of virulence of D. dadantii (namely: KdgR, PecT and PecS) cannot be derepressed in opg deficient mutant strains (BontempsGallo et al, 2014). Loss of OPGs affects the perception of the environment of bacteria in such a way that bacteria were unable to recognize the environment properly, including its various plant hosts (Bouchart et al, 2010;Bontemps-Gallo et al, 2013, 2014.…”

“…In these strains, the secondary mutation encoded a mutated RcsC2 sensor with an increased phosphatase activity, no RcsC sensor or a non-phosphorylatable RcsB D56N regulator, respectively (Bouchart et al, 2010;Bontemps-Gallo et al, 2013). Phosphorylation of the RcsB D56N protein is impossible since its phosphorylatable aspartic acid residue at position 56 was changed to an asparagine residue.…”