The Salmonella typhimurium PhoP-PhoQ two-component regulatory system controls the expression of several genes, some of which are necessary for virulence. During a screening for PhoP-regulated genes, we identified the phoPQ operon as a PhoP-activated locus. ␤-Galactosidase activity originating from phoPQ-lac transcriptional fusions required the presence of both the transcriptional regulator PhoP and its cognate sensor-kinase PhoQ. At low concentrations, PhoQ stimulated expression of phoPQ-lac transcriptional fusions. However, larger amounts of PhoQ protein without a concomitant increase in PhoP failed to activate phoPQ-lac fusions. Two different transcripts are produced from the phoPQ operon during exponential growth. These transcripts define two promoters: phoPp 1 , which requires both PhoP and PhoQ for activity and which is environmentally regulated, and phoPp 2 , which remains active in the absence of PhoP and PhoQ but which is slightly stimulated by these proteins. The pattern of transcriptional autoregulation was also observed at the protein level with anti-PhoP antibodies. In sum, autoregulation of the phoPQ operon provides several levels of control for the PhoP-PhoQ regulon. First, environmental signals would stimulate PhoQ to phosphorylate the PhoP protein that is produced at basal levels from the PhoP-PhoQ-independent promoter. Then, phospho-PhoP would activate transcription of phoPp 1 , resulting in larger amounts of PhoP and PhoQ and increased expression of PhoP-activated genes. A return to basal levels could be mediated by a posttranscriptional mechanism by which translation of the mRNA produced from phoPp 1 is inhibited.Salmonellae are facultative intracellular pathogens responsible for several disease syndromes in a wide variety of animal species. In humans, they have been implicated in four pathological conditions: typhoid fever, gastroenteritis (food poisoning), bacteremia, and the asymptomatic carrier state (14). Certain Salmonella serotypes have a very narrow host range, while others are poorly host adapted and cause distinct diseases in different hosts. For example, typhoid fever is primarily caused by the human-adapted Salmonella typhi, while Salmonella typhimurium, the leading serotype associated with gastroenteritis in humans, causes a typhoid-like disease in susceptible mice (16). The ability to genetically manipulate S. typhimurium and the availability of excellent models of infection have allowed the identification of many of the virulence determinants that enable Salmonella spp. to adapt and prosper within different host environments.In S. typhimurium, virulence is controlled at the transcriptional level by several proteins, including the RpoS sigma factor (7), the cyclic AMP-binding protein CRP (4), and the two-component regulatory systems OmpR-EnvZ (6) and PhoPPhoQ (8,11,13,28). The PhoP-PhoQ system was originally identified as a virulence determinant by the intramacrophage survival defect of S. typhimurium phoP mutants (8, 9). A virulence role for phoP was independently demonstrated b...

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.

Serratia marcescens is a Gram-negative bacterium that thrives in a wide variety of ambient niches and interacts with an ample range of hosts. As an opportunistic human pathogen, it has increased its clinical incidence in recent years, being responsible for life-threatening nosocomial infections. S. marcescens produces numerous exoproteins with toxic effects, including the ShlA pore-forming toxin, which has been catalogued as its most potent cytotoxin. However, the regulatory mechanisms that govern ShlA expression, as well as its action toward the host, have remained unclear. We have shown that S. marcescens elicits an autophagic response in host nonphagocytic cells. In this work, we determine that the expression of ShlA is responsible for the autophagic response that is promoted prior to bacterial internalization in epithelial cells. We show that a strain unable to express ShlA is no longer able to induce this autophagic mechanism, while heterologous expression of ShlA/ShlB suffices to confer on noninvasive Escherichia coli the capacity to trigger autophagy. We also demonstrate that shlBA harbors a binding motif for the RcsB regulator in its promoter region. RcsB-dependent control of shlBA constitutes a feed-forward regulatory mechanism that allows interplay with flagellar-biogenesis regulation. At the top of the circuit, activated RcsB downregulates expression of flagella by binding to the flhDC promoter region, preventing FliA-activated transcription of shlBA. Simultaneously, RcsB interaction within the shlBA promoter represses ShlA expression. This circuit offers multiple access points to fine-tune ShlA production. These findings also strengthen the case for an RcsB role in orchestrating the expression of Serratia virulence factors. Serratia marcescens is a highly ubiquitous Gram-negative enteric bacterium that can be isolated from most abiotic environmental sources (air, soil, and water), as well as from plants, insects, and nematodes. In the clinical setting, together with the Serratia liquefaciens complex (Serratia liquefaciens, Serratia proteamaculans, and Serratia grimesii), S. marcescens causes the majority of human Serratia infections and is the cause of urinary tract, respiratory, wound, ocular, cardiac, bloodstream, and surgical infections, mostly affecting intensive care unit patients (1). Its capacity to adhere and persist attached to hospital instrumentation and prostheses (2); its resistance to disinfection procedures; and the increasingly reported acquisition of resistance to lactams, cephalosporins, and aminoglycosides (1, 3) make S. marcescens a current health threat worldwide. However, no clear picture of the mechanisms that allow Serratia to succeed in the infected host has yet emerged.In our previous work, we have demonstrated that S. marcescens is able to be internalized by nonphagocytic cells. We showed that, once inside the cell, Serratia is able to inhabit and proliferate inside large membrane-bound compartments. These vesicles exhibit autophagic-like features, as they acquire markers t...