A Salmonella locus that controls resistance to microbicidal proteins from phagocytic cells.

Facultative intracellular pathogens pose an important health problem because they circumvent a primary defense mechanism of the host: killing and degradation by professional phagocytic cells. A gene of the intracellular pathogen Salmonella typhimurium that is required for virulence and intracellular survival was identified and shown to have a role in resistance to defensins and possibly to other microbicidal mechanisms of the phagocyte. This gene may prove to be a regulatory element in the expression of virulence functions.

Salmonella virulence: new clues to intramacrophage survival.

Salmonella are capable of survival in macrophages (cells which have evolved specific mechanisms to kill pathogenic bacteria). One mechanism involves the bactericidal peptides called defensins which insert into phospholipid bilayers to generate transmembrane pores. Synthesis of the Salmonella gene products which determine resistance to defensins has been found to be under the control of a transcriptional regulatory protein termed PhoP.

How bacteria resist killing by host-defense peptides.

Small cationic peptides with antimicrobial properties are part of the innate immune response of a wide variety of animal species, including insects, amphibians and mammals. Bacterial pathogens have evolved distinct mechanisms to avoid, inactivate or resist the killing effects of these peptides. Determinants necessary for resistance to peptides often contribute to the virulence properties of recognized pathogens.

How to become a pathogen.

For most bacterial species, virulence is viewed as a derived state, whereby pathogens acquire certain loci and are rendered virulent. The majority of virulence genes in Salmonella are present in closely related nonpathogenic species, and most genes known to be confined to the salmonellae are not essential for virulence. Alternative evolutionary scenarios may explain the origins of pathogenicity in Salmonella.

How Salmonella became a pathogen.

In many pathogens, virulence can be conferred by a single region of the genome. In contrast, the facultative intracellular lifestyle of Salmonella demands a large number of genes distributed around the chromosome. The evolution of Salmonella has been marked by the acquisition of several 'pathogenicity islands', each contributing to the unique virulence properties of this microorganism.

Acetyl phosphate-dependent activation of a mutant PhoP response regulator that functions independently of its cognate sensor kinase.

The two-component system is a signal communication network generally consisting of a sensor kinase that receives inputs from the environment and modifies the phosphorylated state of a response regulator that executes an adaptive behavior. PhoP is a response regulator that controls virulence gene expression in Salmonella enterica. Transcription of PhoP-regulated genes is modulated by the Mg(2+) levels detected by the sensor PhoQ. Here, we describe a PhoP mutant protein, PhoP*, that functions in the absence of its cognate sensor, thereby allowing transcription of PhoP-activated genes independently of the Mg(2+ )concentration in the environment. The PhoP* protein harbors a S93N substitution in the response regulator receiver domain. PhoP*-mediated transcription is abolished by either mutation of the aspartate residue that is conserved among response regulators as the site of phosphorylation or inactivation of the pta-encoded phosphotransacetylase. This enzyme mediates the production of acetyl phosphate, which has been shown to serve as a low molecular mass phosphate donor for certain response regulators. The purified PhoP* protein autophosphorylated from acetyl phosphate more efficiently than the wild-type PhoP protein in vitro. The PhoP* protein retained the capacity to interact with the PhoQ protein, which promoted phosphorylation of the PhoP* protein in vitro and abolished PhoP*-mediated transcription under high Mg(2+ )concentrations in vivo. Cumulatively, our results uncover a role of PhoQ in transcriptional repression during growth in millimolar Mg(2+ )and define a mutant response regulator form with an increased capacity to be phosphorylated by acetyl phosphate.

High frequency generalized transduction by miniMu plasmid phage.

Deletion derivatives of phage Mu which replicate as multicopy plasmids, and also transpose and package like Mu, have been developed for the in vivo cloning of bacterial genes. We show here that these miniMu plasmid phage are also efficient at generalized transduction and that both in vivo cloning and generalized transduction of a given gene can be accomplished in a single experiment.

In vivo DNA cloning with a mini-Mu replicon cosmid and a helper lambda phage.

A mini-Mu bacteriophage, containing the cohesive-end packaging site (cos) from a lambda-phi 80 hybrid phage, a high-copy-number plasmid replicon, and a kanamycin-resistance gene for independent selection, was constructed to clone genes in vivo. This mini-Mu element can be derepressed to transpose at a high frequency. DNA segments that become flanked by copies of this mini-Mu element in the same orientation can be packaged by a helper lambda phage. The resulting lambda lysate can be used to infect recipient cells where the injected DNA can circularize by annealing at the cos termini. Drug-resistant transductants obtained carry the mini-Mu-replicon cosmid element with inserts of different nucleotide sequences. These are analogous to recombinant DNA clones generated in vitro with restriction endonuclease cutting and ligase joining reactions replaced by the Mu transposition process. Clones of particular genes were isolated by their ability to complement specific mutations. Both recA+ and recA- recipient cells can be used with equal efficiency. Clones obtained with a helper lambda phage require the presence of the cos site in the mini-Mu replicon. They carry larger inserts than those isolated with the same mini-Mu element and Mu as a helper phage. The mini-Mu replicon-cosmid bacteriophage contains a lac-gene fusing segment for isolating fusions of lac operon DNA to gene control regions in the cloned sequences. Independent clones of a particular gene can be used to prepare a restriction map of the gene and its flanking regions.

Genome mapping and protein coding region identification using bacteriophage Mu.

Transposons such as bacteriophage Mu provide a means to clone bacterial genes as alternatives to using standard recombinant DNA technologies. A DNA-cloning and gene-expressing system has been developed with a bacteriophage Mu (DNA capacity of 38 kb) vector that combines the Mu transposition capabilities and a specialized promoter from bacteriophage T7. Genes cloned with this vector can be identified by transcription in vivo with T7 RNA polymerase and subsequent host translation. This system, illustrated with the characterization of a 35-kb region of the Escherichia coli K-12 chromosome, is applicable to other Enterobacteriaceae, which are hosts for Mu phage, and is potentially applicable to other bacteria, including Pseudomonas aeruginosa, which have Mu-like phage, and to other organisms for which high-frequency transposons are available.

The origin and evolution of species differences in Escherichia coli and Salmonella typhimurium.

Since diverging from a common ancestor some 120 million years, Escherichia coli and Salmonella typhimurium have accumulated numerous phenotypic characteristics which have traditionally been used to distinguish these enteric species. While most of the genetic differences between these species are due to the accumulation of point mutations, the majority of the observed variation in phenotypic characters is attributable to segments of the genome confined to only one of the species. We have analyzed the map positions, G+C contents, nucleotide sequences and functions of regions unique to the Salmonella chromosome in an attempt to determine the ancestry of species-specific sequences. Some of the Salmonella-specific regions had uncharacteristically low base compositions and contained open reading frames of atypical codon usage patterns suggesting that portions of the genome were acquired by horizontal transfer from distantly-related bacterial species. The role of these species-specific sequences was assayed by constructing mutant strains harboring deletions in the corresponding regions of the genome. Several functions were ascribed to these unique portions of the Salmonella chromosome, including one encoding proteins involved in virulence and invasion of host epithelial cells.

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.

Sequence and evolution of the FruR protein of Salmonella typhimurium: a pleiotropic transcriptional regulatory protein possessing both activator and repressor functions which is homologous to the periplasmic ribose-binding protein.

The repressor of the fructose (fru) operon of Salmonella typhimurium (FruR) has been implicated in the transcriptional regulation of dozens of genes concerned with central metabolic pathways of carbon utilization. We here report the nucleotide sequence of the gene encoding FruR and analyse both its operator-promoter region and its deduced amino acyl sequence. The FruR protein was overexpressed and was shown to have a molecular weight of about 36 kDa in agreement with the molecular weight deduced from the gene sequence. Sequence analyses revealed that FruR is homologous to 9 distinct bacterial DNA-binding proteins, most of which recognize sugar inducers and all of which possess helix-turn-helix motifs within their N-terminal regions and exhibit sequence identity throughout most of their lengths. FruR is also homologous to the periplasmic ribose-binding protein which serves as a constituent of the ribose transport/chemoreception system. The ribose-binding protein is in turn homologous to binding proteins specific for arabinose and galactose. The periplasmic binding proteins, the structures of some of which have been elucidated in three dimensions, lack the N-terminal helix-turn-helix region, but instead possess N-terminal hydrophobic signal sequences which target them to the periplasm. A phylogenetic tree for the more closely related proteins of this superfamily was constructed, and a signature motif was identified which should facilitate future detection of additional transcriptional regulatory proteins belonging to this family.

A Mg2+-responding RNA that controls the expression of a Mg2+ transporter.

Mg2+ is the most abundant divalent cation in biological systems. It is required for ATP-mediated enzymatic reactions and as a stabilizer of ribosomes and membranes. The enteric bacterium Salmonella enterica serovar Typhimurium harbors three Mg2+ transporters and a regulatory system-termed PhoP/PhoQ-whose activity is regulated by the extracytoplasmic levels of Mg2+. We have determined that expression of the PhoP-activated Mg2+ transporter MgtA is also controlled by its 5'-untranslated region (5'UTR). The 5'UTR of the mgtA gene can adopt different stem-loop structures depending on the Mg2+ levels, which determine whether transcription reads through into the mgtA-coding region or stops within the 5'UTR. This makes the mgtA 5'UTR the first example of a cation-responding riboswitch. The initiation of mgtA transcription responds to extracytoplasmic Mg2+, and its elongation into the coding region to cytoplasmic Mg2+, which provides a singular example where the same ligand is sensed in different cellular compartments to regulate disparate steps in gene transcription. The PhoP-activated Mg2+ transporter mgtB is also regulated by Mg2+ in a strain lacking the Mg2+ sensor PhoQ, suggesting the presence of additional Mg2+-responding devices.

The ins and outs of virulence gene expression: Mg2+ as a regulatory signal.

The facultative intracellular pathogen Salmonella enterica serovar Typhimurium faces multiple environments during infection, including different cell types as well as extracellular fluids. We propose that Salmonella ascertains its cellular location by assessing the Mg2+ concentration of its milieu. A signal transduction system, PhoP/PhoQ, signals Salmonella its presence in a intracellular (low Mg2+) or extracellular (high Mg2+) environment, thereby promoting transcription of genes required for survival within or entry into host cells. The PhoP/PhoQ system is high in a regulatory hierarchy that controls other signal transduction systems that respond to different host cues, enabling the microorganism to determine its precise tissue and cellular location.

Mini-mu bacteriophage with plasmid replicons for in vivo cloning and lac gene fusing.

New mini-Mu transposons with plasmid replicons were constructed with additional features for in vivo DNA cloning and lac gene fusing in Escherichia coli. These mini-Mu replicons can be used to clone DNA by growing them with a complementing Mu bacteriophage and by using the resulting lysate to transduce Mu-lysogenic cells. These mini-Mu phage have selectable genes for resistance to kanamycin, chloramphenicol, and spectinomycin-streptomycin, and replicons from the high-copy-number plasmids pMB1 and P15A and the low-copy, broad-host-range plasmid pSa. The most efficient of these elements can be used to clone genes 100 times more frequently than with the previously described mini-Mu replicon Mu dII4042, such that complete gene banks can be made with as little as 1 microliter of a lysate containing 10(6) helper phage. The 39-kilobase-pair Mu headful DNA packaging mechanism limits the size of the clones formed. The smallest of the mini-Mu elements is only 7.9 kilobase pairs long, allowing the cloning of DNA fragments of up to 31.1 kilobase pairs, and the largest of them is 21.7 kilobase pairs, requiring that clones carry insertions of less than 17.3 kilobase pairs. Elements have been constructed to form both transcriptional and translational types of lac gene fusions to promoters present in the cloned fragment. Two of these elements also contain the origin-of-transfer sequence oriT from the plasmid RK2, so that clones obtained with these mini-Mu bacteriophage can be efficiently mobilized by conjugation.