Fernando C. Soncini

Mg2+ as an Extracellular Signal: Environmental Regulation of Salmonella Virulence

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.

A Signal Transduction System that Responds to Extracellular Iron

Iron is essential for all organisms but can be toxic in excess. Iron homeostasis is typically regulated by cytoplasmic iron binding proteins, but here we describe a signal transduction system (PmrA/PmrB) that responds to extracytoplasmic ferric iron. Iron promoted transcription of PmrA-activated genes and resistance to the antibiotic polymyxin in Salmonella. The PmrB protein bound iron via its periplasmic domain which harbors two copies of the sequence ExxE, a motif present in the Saccharomyces FTR1 iron transporter and in mammalian ferritin light chain. A pmrA mutant was hypersensitive to killing by iron but displayed wild-type resistance to a variety of oxidants, suggesting PmrA/PmrB controls a novel pathway mediating the avoidance of iron toxicity.

Molecular Characterization of the Mg2+-Responsive PhoP-PhoQ Regulon in Salmonella enterica

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.

The Phosphatase Activity Is the Target for Mg2+Regulation of the Sensor Protein PhoQ in Salmonella

The PhoP/PhoQ two-component system controls the expression of essential virulence traits in the pathogenic bacterium Salmonella enterica serovar Typhimurium. Environmental deprivation of Mg2+ activates the PhoP/PhoQ signal transduction cascade, which results in an increased expression of genes necessary for survival inside the host. It was previously demonstrated that the interaction of Mg2+ with the periplasmic domain of PhoQ promotes a conformational change in the sensor protein that leads to the down-regulation of PhoP-activated genes. We have now examined the regulatory effect of Mg2+ on the putative activities of the membrane-bound PhoQ. We demonstrated that Mg2+ promotes a phospho-PhoP phosphatase activity in the sensor protein. This activity depends on the intactness of the conserved His-277, suggesting that the phosphatase active site overlaps the H box. The integrity of the N-terminal domain of PhoQ was essential for the induction of the phosphatase activity, because Mg2+ did not stimulate the release of inorganic phosphate from phospho-PhoP in a fusion protein that lacks this sensing domain. These findings reveal that the sensor PhoQ harbors a phospho-PhoP phosphatase activity, and that this phosphatase activity is the target of the extracellular Mg2+-triggered regulation of the PhoP/PhoQ system.

Bacterial sensing of and resistance to gold salts

The MerR family is a group of bacterial transcriptional regulators that respond to different environmental stimuli, such as heavy metals, oxidative stress or antibiotics. Here we characterize a new member of this family that is highly selective for Au ions. We show that this Salmonella regulator, named GolS, directly controls the expression of at least two transcriptional units specifically required for Au resistance. By chromosomal mutagenesis, we demonstrated that Au‐selectivity is accomplished by a metal‐binding motif in GolS. Among the monovalent metal‐ion sensing MerR regulators GolS clusters in a branch distant from enterobacterial CueR orthologues. We propose that GolS and its homologues evolved to cope with toxic concentration of Au ion, allowing microorganisms to withstand contaminated environments.

The role of the PhoP/PhoQ regulon in Salmonella virulence

Salmonella typhimurium is a facultative intracellular pathogen that is able to survive in a wide variety of inhibitory and nutritionally deprived host environments. The ability to survive under such hostile conditions, which are often encountered during the course of infection, contributes to its pathogenic properties. Some of the virulence determinants of S. typhimurium are under the transcriptional control of the PhoPQ two-component regulatory system. Several virulence phenotypes have been associated with mutations in the phoPQ operon including the inability to survive within macrophages and increased susceptibility to antimicrobial peptides and acid pH. Only 25% of PhoP-modulated genes are involved in virulence and the phoPQ operon is present in both pathogenic and non-pathogenic microbes. These data suggest that PhoP is not exclusively involved in virulence and that it is required for the physiological control of activities common to other bacteria.

GolS controls the response to gold by the hierarchical induction of Salmonella-specific genes that include a CBA efflux-coding operon.

Salmonella employs a specific set of proteins that allows it to detect the presence of gold salts in the environment and to mount the appropriate resistance response. This includes a P‐type ATPase, GolT, and a small cytoplasmic metal binding protein, GolB. Their expression is controlled by a MerR‐like sensor, GolS, which is highly selective for Au ions. Here, we identify a new GolS‐controlled operon named gesABC which codes for a CBA efflux system, and establish its role in Au resistance. GesABC can also mediate drug resistance when induced by Au in a GolS‐dependent manner, in a strain deleted in the main drug transporter acrAB. The GolS‐controlled transcription of gesABC differs from the other GolS‐regulated loci. It is activated by gold, but not induced by copper, even in a strain deleted of the main Cu transporter gene copA, which triggers a substantial GolS‐dependent induction of golTS and golB. We demonstrate that the Au‐dependent induction of gesABC transcription requires higher GolS levels than for the other members of the gol regulon. This correlates with a divergent GolS operator in the gesABC promoter. We propose that the hierarchical induction within the gol regulon allows Salmonella to cope with Au‐contaminated environments.

Alternative periplasmic copper‐resistance mechanisms in Gram negative bacteria

Bacteria have evolved different systems to tightly control both cytosolic and envelope copper concentration to fulfil their requirements and at the same time, avoid copper toxicity. We have previously demonstrated that, as in Escherichia coli, the Salmonella cue system protects the cytosol from copper excess. On the other hand, and even though Salmonella lacks the CusCFBA periplasmic copper efflux system, it can support higher copper concentrations than E. coli under anaerobic conditions. Here we show that the Salmonella cue regulon is also responsible for the control of copper toxicity in anaerobiosis. We establish that resistance in this condition requires a novel CueR‐controlled gene named cueP. A ΔcueP mutant is highly susceptible to copper in the absence of oxygen, but shows a faint phenotype in aerobic conditions unless other copper‐resistance genes are also deleted, resembling the E. coli CusCFBA behaviour. Species that contain a cueP homologue under CueR regulation have no functional CusR/CusS‐dependent Cus‐coding operon. Conversely, species that carry a CusR/CusS‐regulated cus operon have no cueP homologues. Even more, we show that the CueR‐controlled cueP expression increases copper resistance of a Δcus E. coli. We posit that CueP can functionally replace the Cus complex for periplasmic copper resistance, in particular under anaerobic conditions.

PhoP Can Activate Its Target Genes in a PhoQ-Independent Manner

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.

Phosphorylated PmrA interacts with the promoter region of ugd in Salmonella enterica serovar typhimurium.

The Salmonella PmrA-PmrB system controls the expression of genes necessary for polymyxin B resistance. Four loci were previously identified as part of the regulon, and interaction of PmrA with the promoter region of three of them was observed. Here we characterized the interaction of PmrA with the promoter region of ugd, previously suggested to be regulated indirectly by PmrA. Our results indicate that PmrA controls the expression of ugd by interacting with a specific sequence in the promoter region of this gene.