The PhoP/PhoQ two-component system controls the expression of essential virulence traits in the pathogenic bacterium Salmonella enterica serovar Typhimurium. Reversible protein phosphorylation is one of the most conspicuous mechanisms that regulate biological processes in cells, including modulation of enzymatic activities, proteinprotein and protein-DNA interactions, and extracellular signal transduction. In prokaryotes and in lower eukaryotes and plants (1), the most widespread and efficient sensory-response devices rely on protein phosphotransfer. They are the so-called two-component regulatory systems that enable bacteria to monitor changes in their environment and adjust their structure and physiology accordingly to survive.

Outer membrane vesicles (OMVs) have been identified in a wide range of bacteria, yet little is known of their biogenesis. It has been proposed that OMVs can act as long-range toxin delivery vectors and as a novel stress response. We have found that the formation of OMVs in the Gram-negative opportunistic pathogen Serratia marcescens is thermoregulated, with a significant amount of OMVs produced at 22 or 30°C and negligible quantities formed at 37°C under laboratory conditions. Inactivation of the synthesis of the enterobacterial common antigen (ECA) resulted in a hypervesiculation phenotype, supporting the hypothesis that OMVs are produced in response to stress. We demonstrate that the phenotype can be reversed to wild-type (WT) levels upon the loss of the Rcs phosphorelay response regulator RcsB, but not RcsA, suggesting a role for the Rcs phosphorelay in the production of OMVs. MS fingerprinting of the OMVs provided evidence of cargo selection within wild-type cells, suggesting a possible role for Serratia OMVs in toxin delivery. In addition, OMV-associated cargo proved toxic upon injection into the haemocoel of Galleria mellonella larvae. These experiments demonstrate that OMVs are the result of a regulated process in Serratia and suggest that OMVs could play a role in virulence.

The PhoP/PhoQ two-component system controls the extracellular magnesium depletion response in Salmonella enterica. Previous studies have shown that PhoP is unable to up-regulate its target genes in the absence of PhoQ function. In this work, we demonstrate that PhoP overexpression can substitute for PhoQ-and phosphorylation-dependent activation. Either a high concentration of PhoP or activation via phosphorylation stimulates PhoP self-association.

The enterobacterial common antigen (ECA) is a highly conserved exopolysaccharide in Gram-negative bacteria whose role remains largely uncharacterized. In a previous work, we have demonstrated that disrupting the integrity of the ECA biosynthetic pathway imposed severe deficiencies to the Serratia marcescens motile (swimming and swarming) capacity. In this work, we show that alterations in the ECA structure activate the Rcs phosphorelay, which results in the repression of the flagellar biogenesis regulatory cascade. In addition, a detailed analysis of wec cluster mutant strains, which provoke the disruption of the ECA biosynthesis at different levels of the pathway, suggests that the absence of the periplasmic ECA cyclic structure could constitute a potential signal detected by the RcsF-RcsCDB phosphorelay. We also identify SMA1167 as a member of the S. marcescens Rcs regulon and show that high osmolarity induces Rcs activity in this bacterium. These results provide a new perspective from which to understand the phylogenetic conservation of ECA among enterobacteria and the basis for the virulence attenuation detected in wec mutant strains in other pathogenic bacteria.Serratia marcescens is a Gram-negative enteric bacterium that acts as a pathogen with a remarkably wide host range; it has been isolated from plants, insects, vertebrates, and humans (35). In humans, S. marcescens is an opportunistic pathogen causing infections in patients who are often immunocompromised or preventively treated with broad-spectrum antibiotics and subjected to diverse instrumentation. Serratia can produce urinary and respiratory tract infections, surgical wound infections, and septicemia or local infections, such as osteomyelitis and ocular or skin infections (35). The incidence of Serratia infections has increased in recent years mainly due to the acquisition of multiple-antibiotic resistance (19,40,59,76).Despite the clinical emergence of Serratia, the virulence mechanisms (adherence, invasion, dissemination, preferred niches) of this pathogen are unresolved. S. marcescens produces numerous exoproteins (phospholipase PhlA, DNases, chitinases, esterases, hemolysin, lipase, metalloproteases, and S-layer protein) which are predicted to play a role as virulence determinants (8,33,37,44,46). However, the regulatory strategies that govern the expression of these potential virulence factors remain poorly characterized.Protein secretion plays a central role in modulating the interactions of pathogenic bacteria with host organisms. Gramnegative bacteria have evolved different secretion systems to transport proteins across their membranes. At least six general classes of protein secretion systems showing considerable diversity have been described (9, 28). Bioinformatic searches indicate that Serratia lacks a specialized type III secretion system (TTSS), which has been shown to be required for virulence in many Gram-negative pathogenic bacteria (18). The flagellar appendage has a tight phylogenetic and structural relatedness with the TTSS and ...