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 ...

The efficient production of functional proteins in heterologous hosts is one of the major bases of modern biotechnology. Unfortunately, many genes are difficult to express outside their original context. Due to their apparent “silent” nature, synonymous codon substitutions have long been thought to be trivial. In recent years, this dogma has been refuted by evidence that codon replacement can have a significant impact on gene expression levels and protein folding. In the past decade, considerable advances in the speed and cost of gene synthesis have facilitated the complete redesign of entire gene sequences, dramatically improving the likelihood of high protein expression. This technology significantly impacts the economic feasibility of microbial-based biotechnological processes by, for example, increasing the volumetric productivities of recombinant proteins or facilitating the redesign of novel biosynthetic routes for the production of metabolites. This review discusses the current applications of this technology, particularly those regarding the production of small molecules and industrially relevant recombinant enzymes. Suggestions for future research and potential uses are provided as well.

Serratia marcescens strains are ubiquitous bacteria isolated from environmental niches, such as soil, water, and air, and also constitute emergent nosocomial opportunistic pathogens. Among the numerous extracellular factors that S. marcescens is able to produce, the PhlA phospholipase is the only described exoprotein secreted by the flagellar apparatus while simultaneously being a member of the flagellar regulon. To gain insight into the regulatory mechanism that couples PhlA and flagellar expression, we conducted a generalized insertional mutagenesis and screened for PhlA-deficient strains. We found that three independent mutations in the wec cluster, which impaired the assembly of enterobacterial common antigen (ECA), provoked the inhibition of PhlA expression. Swimming and swarming assays showed that in these strains, motility was severely affected. Microscopic examination and flagellin immunodetection demonstrated that a strong defect in flagellum expression was responsible for the reduced motility in the wec mutant strains. Furthermore, we determined that in the ECA-defective strains, the transcriptional cascade that controls flagellar assembly was turned off due to the down-regulation of flhDC expression. These findings provide a new perspective on the physiological role of the ECA, providing evidence that in S. marcescens, its biosynthesis conditions the expression of the flagellar regulon.Serratia marcescens is an opportunistic human pathogen associated with urinary and respiratory tract as well as wound and eye infections, endocarditis, osteomyelitis, meningitis, and septicemia. Immunocompromised people and newborns are the most affected hosts. The incidence of S. marcescens infection has increased over the last years, mainly due to the acquisition of multiple antibiotic resistance (19,20). S. marcescens produces numerous extracellular factors, including hemolysin, esterase, DNase, chloroperoxidase, S-layer, lipases, proteases, chitinases, and siderophohore (21,24,26,27,31,38,44,45). These factors are predicted to play a role in bacterial environmental adaptive capacity, in either nonhost or host environments, contributing to its ambient persistence and to its pathogenic potential. In addition, an extracellular phospholipase named PhlA in Serratia liquefaciens MG1 (recently reclassified as S. marcescens MG1) and PlaA in Serratia sp. strain MK1 has been described previously (16, 48). The phlAB (or plaAS) locus codes for the PhlA (PlaA) phospholipase and for an accessory protein (PhlB/PlaS) that prevents the intracellular phospholipase enzymatic action. Interestingly, it has been shown not only that the phlAB promoter region displays homology to class III promoters of the flagellar transcriptional cascade controlled by the FliA sigma factor (15) but also that PhlA secretion depends on the integrity of the type III flagellar system export apparatus (15-17).The flagellar structure has its dedicated type III protein export embedded apparatus that translocates proteins involved in the self-assembly process an...

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Serratia marcescens is able to invade, persist, and multiply inside nonphagocytic cells, residing in nonacidic, nondegradative, autophagosome-like vacuoles. In this work, we have examined the physiological role of the PhoP/PhoQ system and its function in the control of critical virulence phenotypes in S. marcescens . We have demonstrated the involvement of the PhoP/PhoQ system in the adaptation of this bacterium to growth on scarce environmental Mg 2+ , at acidic pH, and in the presence of polymyxin B. We have also shown that these environmental conditions constitute signals that activate the PhoP/PhoQ system. We have found that the two S. marcescens mgtE orthologs present a conserved PhoP-binding motif and demonstrated that mgtE1 expression is PhoP dependent, reinforcing the importance of PhoP control in magnesium homeostasis. Finally, we have demonstrated that phoP expression is activated intracellularly and that a phoP mutant strain is defective in survival inside epithelial cells. We have shown that the Serratia PhoP/PhoQ system is involved in prevention of the delivery to degradative/acidic compartments.