[Abstract unavailable

Ions are not traditionally thought to act as first messengers in signal transduction cascades. However, while searching for genes regulated by the PhoP/PhoQ virulence regulatory system of Salmonella typhimurium, we recovered two loci whose expression is controlled by the concentration of Mg2+. To determine whether Mg2+ is the signal modulating the whole PhoP/PhoQ system, we evaluated the gene expression pattern of six PhoP-activated genes. Growth in physiological concentrations of divalent cations repressed transcription of PhoP-activated genes and rendered wild-type Salmonella phenotypically PhoP-. Mg2+ changed the conformation of the periplasmic domain of PhoQ, identifying this protein as a Mg2+ sensor. A mutation in the sensing domain of PhoQ altered the set point for Mg2+ and rendered Salmonella avirulent.

The PhoP-PhoQ two-component system is essential for virulence in Salmonella typhimurium. This system controls expression of some 40 different proteins, yet most PhoP-regulated genes remain unknown. To identify PhoP-regulated genes, we isolated a library of 50,000 independent lac gene transcriptional fusion strains and investigated whether production of ␤-galactosidase was regulated by PhoP. We recovered 47 lac gene fusions that were activated and 7 that were repressed when PhoP was expressed. Analysis of 40 such fusions defined some 30 loci, including mgtA and mgtCB, which encode two of the three Mg 2؉ uptake systems of S. typhimurium; ugd, encoding UDP-glucose dehydrogenase; phoP, indicative that the phoPQ operon is autoregulated; and an open reading frame encoding a protein with sequence similarity to VanX, a dipeptidase required for resistance to vancomycin. Transcription of PhoP-activated genes was regulated by the levels of Mg 2؉ in a PhoP-dependent manner. Strains with mutations in phoP or phoQ were defective for growth in low-Mg 2؉ media. The mgtA and mgtCB mutants reached lower optical densities than the wild-type strain in low-Mg 2؉ liquid media but displayed normal growth on low-Mg 2؉ solid media. Six PhoP-activated genes were identified as essential to form colonies on low-Mg 2؉ solid media. Cumulatively, our experiments establish that the PhoP-PhoQ system governs the adaptation to magnesium-limiting environments.

The PhoP/PhoQ two-component system controls the extracellular magnesium deprivation response in Salmonella enterica. In addition, several virulence-associated genes that are mainly required for intramacrophage survival during the infection process are under the control of its transcriptional regulation. Despite shared Mg 2؉ modulation of the expression of the PhoP-activated genes, no consensus sequence common to all of them could be detected in their promoter regions. We have investigated the transcriptional regulation and the interaction of the response regulator PhoP with the promoter regions of the PhoP-activated loci phoPQ, mgtA, slyB, pmrD, pcgL, phoN, pagC, and mgtCB. A direct repeat of the heptanucleotide sequence (G/T)GTTTA(A/T) was identified as the conserved motif recognized by PhoP to directly control the gene expression of the first five loci, among which the first four are ancestral to enterobacteria. On the other hand, no direct interaction of the response regulator with the promoter of phoN, pagC, or mgtCB was apparent by either in vitro or in vivo assays. These loci are Salmonella specific and were probably acquired by horizontal DNA transfer. Besides, sequence analysis of pag promoters revealed the presence of a conserved PhoP box in 6 out of the 12 genes analyzed. Our results strongly suggest that the expression of a set of Mg 2؉ -controlled genes is driven by PhoP via unknown intermediate regulatory mechanisms that could also involve ancillary factors.

Extracellular Mg(2+) controls the activation of the Salmonella PhoP/PhoQ regulatory system. One of the adaptive responses driven by PhoP/PhoQ includes the transcriptional induction of mgtA and mgtCB, which encode two P-type Mg(2+) transporters. Mg(2+) also controls mgtA expression by a riboswitch located in its 5'-untranslated region (5'UTR). In this work, it was shown that the 5'UTR of both mgtA and mgtCB is responsible for a fine-tuned Mg(2+)-dependent regulation of these genes. Evidence was also provided that the Mg(2+) riboswitch targets the mgtA transcript for degradation by RNase E when cells are grown in high Mg(2+) environments.

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.

Serratia marcescens is an opportunistic human pathogen that represents a growing problem for public health, particularly in hospitalized or immunocompromised patients. However, little is known about factors and mechanisms that contribute to S. marcescens pathogenesis within its host. In this work, we explore the invasion process of this opportunistic pathogen to epithelial cells. We demonstrate that once internalized, Serratia is able not only to persist but also to multiply inside a large membrane-bound compartment. This structure displays autophagic-like features, acquiring LC3 and Rab7, markers described to be recruited throughout the progression of antibacterial autophagy. The majority of the autophagic-like vacuoles in which Serratia resides and proliferates are non-acidic and have no degradative properties, indicating that the bacteria are capable to either delay or prevent fusion with lysosomal compartments, altering the expected progression of autophagosome maturation. In addition, our results demonstrate that Serratia triggers a non-canonical autophagic process before internalization. These findings reveal that S. marcescens is able to manipulate the autophagic traffic, generating a suitable niche for survival and proliferation inside the host cell.

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 ...