The CorA Mg2+ channel is required for the virulence of Salmonella enterica serovar typhimurium

CorA is the primary Mg²⁺ channel in Salmonella enterica serovar Typhimurium. A corA mutant is attenuated in mice and defective for invasion of and replication within epithelial cells. Microarray studies show that several virulence effectors are repressed in a corA mutant strain, which ultimately manifests itself as a decrease in virulence.     

The wetting agent required for swarming in Salmonella enterica serovar typhimurium is not a surfactant

We compared the abilities of media from agar plates surrounding swarming and nonswarming cells of Salmonella enterica serovar Typhimurium to wet a nonpolar surface by measuring the contact angles of small drops. The swarming cells were wild type for chemotaxis, and the nonswarming cells were nonchemotactic mutants with motor biases that were counterclockwise (cheY) or clockwise (cheZ). The latter strains have been shown to be defective for swarming because the agar remains dry (Q. Wang, A. Suzuki, S. Mariconda, S. Porwollik, and R. M. Harshey, EMBO J. 24:2034-2042, 2005). We found no differences in the abilities of the media surrounding these cells, either wild type or mutant, to wet a low-energy surface (freshly prepared polydimethylsiloxane); although, their contact angles were smaller than that of the medium harvested from the underlying agar. So the agent that promotes wetness produced by wild-type cells is not a surfactant; it is an osmotic agent.     

Aspartic peptide hydrolases in Salmonella enterica serovar typhimurium

Two well-characterized enzymes in Salmonella enterica serovar Typhimurium and Escherichia coli are able to hydrolyze N-terminal aspartyl (Asp) dipeptides: peptidase B, a broad-specificity aminopeptidase, and peptidase E, an Asp-specific dipeptidase. A serovar Typhimurium strain lacking both of these enzymes, however, can still utilize most N-terminal Asp dipeptides as sources of amino acids, and extracts of such a strain contain additional enzymatic activities able to hydrolyze Asp dipeptides. Here we report two such activities from extracts of pepB pepE mutant strains of serovar Typhimurium identified by their ability to hydrolyze Asp-Leu. Although each of these activities hydrolyzes Asp-Leu at a measurable rate, the preferred substrates for both are N-terminal isoAsp peptides. One of the activities is a previously characterized isoAsp dipeptidase from E. coli, the product of the iadA gene. The other is the product of the serovar Typhimurium homolog of E. coli ybiK, a gene of previously unknown function. This gene product is a member of the N-terminal nucleophile structural family of amidohydrolases. Like most other members of this family, the mature enzyme is generated from a precursor protein by proteolytic cleavage and the active enzyme is a heterotetramer. Based on its ability to hydrolyze an N-terminal isoAsp tripeptide as well as isoAsp dipeptides, the enzyme appears to be an isoAsp aminopeptidase, and we propose that the gene encoding it be designated iaaA (isoAsp aminopeptidase). A strain lacking both IadA and IaaA in addition to peptidase B and peptidase E has been constructed. This strain utilizes Asp-Leu as a leucine source, and extracts of this strain contain at least one additional, as-yet-uncharacterized, peptidase able to cleave Asp dipeptides.     

The GATC-binding protein SeqA is required for bile resistance and virulence in Salmonella enterica serovar typhimurium

Disruption of the seqA gene of Salmonella enterica serovar Typhimurium causes defects similar to those described in E. coli: filament formation, aberrant nucleoid segregation, induction of the SOS response, envelope instability, and increased sensitivity to membrane-damaging agents. Differences between SeqA⁻ mutants of E. coli and S. enterica, however, are found. SeqA⁻ mutants of S. enterica form normal colonies and do not exhibit alterations in phage plaquing morphology. Lack of SeqA causes attenuation of S. enterica virulence by the oral route but not by the intraperitoneal route, suggesting a virulence defect in the intestinal stage of infection. However, SeqA⁻ mutants are fully proficient in the invasion of epithelial cells. We hypothesize that attenuation of SeqA⁻ mutants by the oral route may be caused by bile sensitivity, which in turn may be a consequence of envelope instability.     

rpoS mutants in archival cultures of Salmonella enterica serovar typhimurium

Long-term survival under limited growth conditions presents bacterial populations with unique environmental challenges. The existence of Salmonella enterica serovar Typhimurium cultures undisturbed in sealed nutrient agar stab vials for 34 to 45 years offered a unique opportunity to examine genetic variability under natural conditions. We have initiated a study of genetic changes in these archival cultures. We chose to start with examination of the rpoS gene since, among gram-negative bacteria, many genes needed for survival are regulated by RpoS, the stationary-phase sigma factor. In each of 27 vials examined, cells had the rpoS start codon UUG instead of the expected AUG of Salmonella and Escherichia coli strains recorded in GenBank. Ten of the 27 had additional mutations in the rpoS gene compared with the X77752 wild-type strain currently recorded in GenBank. The rpoS mutations in the 10 strains included two deletions as well as point mutations that altered amino acid sequences substantially. Since these stored strains were derived from ancestral cells inoculated decades ago and remained undisturbed, it is assumed that the 10 rpoS mutations occurred during storage. Since the remaining 17 sequences were wild type (other than in the start codon), it is obvious that rpoS remained relatively stable during decades of sealed storage.     

The effect of methylation on DNA replication in Salmonella enterica serovar typhimurium

The DNA adenine methylase of Salmonella typhimurium methylates adenine at GATC sequences. Strains deficient in this methylase are not well transformed by methylated plasmids, but unmethylated plasmids transform them at high frequencies. Hemimethylated daughter molecules accumulate after the transformation of dam(-) strains with fully methylated plasmids, suggesting that hemimethylation prevents DNA replication. It will also be shown that plasmids isolated from dam(-) bacteria are hemimethylated by restriction enzyme digestion. These results may explain why newly formed daughter molecules are not substrates for immediate reinitiation of DNA replication in dam(-) bacteria.     

Survival and filamentation of Salmonella enterica serovar enteritidis PT4 and Salmonella enterica serovar typhimurium DT104 at low water activity

In this study we investigated the long-term survival of and morphological changes in Salmonella strains at low water activity (a(w)). Salmonella enterica serovar Enteritidis PT4 and Salmonella enterica serovar Typhimurium DT104 survived at low a(w) for long periods, but minimum humectant concentrations of 8% NaCl (a(w), 0. 95), 96% sucrose (a(w), 0.94), and 32% glycerol (a(w), 0.92) were bactericidal under most conditions. Salmonella rpoS mutants were usually more sensitive to bactericidal levels of NaCl, sucrose, and glycerol. At a lethal a(w), incubation at 37 degrees C resulted in more rapid loss of viability than incubation at 21 degrees C. At a(w) values of 0.93 to 0.98, strains of S. enterica serovar Enteritidis and S. enterica serovar Typhimurium formed filaments, some of which were at least 200 microm long. Filamentation was independent of rpoS expression. When the preparations were returned to high-a(w) conditions, the filaments formed septa, and division was complete within approximately 2 to 3 h. The variable survival of Salmonella strains at low a(w) highlights the importance of strain choice when researchers produce modelling data to simulate worst-case scenarios or conduct risk assessments based on laboratory data. The continued increase in Salmonella biomass at low a(w) (without a concomitant increase in microbial count) would not have been detected by traditional microbiological enumeration tests if the tests had been performed immediately after low-a(w) storage. If Salmonella strains form filaments in food products that have low a(w) values (0.92 to 0.98), there are significant implications for public health and for designing methods for microbiological monitoring.     

Salmonella enterica serovar typhimurium peptidase B is a leucyl aminopeptidase with specificity for acidic amino acids

Peptidase B (PepB) of Salmonella enterica serovar Typhimurium is one of three broad-specificity aminopeptidases found in this organism. We have sequenced the pepB gene and found that it encodes a 427-amino-acid (46.36-kDa) protein, which can be unambiguously assigned to the leucyl aminopeptidase (LAP) structural family. PepB has been overexpressed and purified. The active enzyme shows many similarities to other members of the LAP family: it is a heat-stable (70 degrees C; 20 min) hexameric ( approximately 270-kDa) metallopeptidase with a pH optimum of 8.5 to 9.5. A detailed study of the substrate specificity of the purified protein shows that it differs from other members of the family in its ability to hydrolyze peptides with N-terminal acidic residues. The preferred substrates for PepB are peptides with N-terminal Asp or Glu residues. Comparison of the amino acid sequence of PepB with those of other LAPs leads to the conclusion that PepB is the prototype of a new LAP subfamily with representatives in several other eubacterial species and to the prediction that the members of this family share the ability to hydrolyze peptides with N-terminal acidic residues. Site-directed mutagenesis has been used to show that this specificity appears to be determined by a single Lys residue present in a sequence motif conserved in all members of the subfamily.     

Novel missense mutations that affect the transport function of MalK, the ATP-binding-cassette subunit of the Salmonella enterica serovar typhimurium maltose transport system

We report on novel mutations in the malK gene of Salmonella enterica serovar Typhimurium, encoding the ATPase subunit of the maltose transporter (MalFGK(2)). Biochemical analysis suggests that (i) L86 might be involved in a signaling step during substrate translocation and (ii) E306 may be critical for the structural integrity of the protein.     

Molecular markers with potential to replace phage typing for Salmonella enterica serovar typhimurium

Using Amplified Fragment Length Polymorphism (AFLP) analysis of isolates from 23 phage types, we isolated 11 molecular markers that are potentially useful for molecular typing of Salmonella enterica serovar typhimurium. We tested these and 11 previously studied markers for their ability to discriminate among isolates and for correlation of their distribution with phage types. The Simpson's index of discriminatory power for the molecular markers is 0.96. One hundred and twenty one isolates from 33 phage types tested were divided into 51 types which are further grouped into 24 patterns. Eight patterns can unambiguously identify 8 phage types and a further 12 correlated with phage type distribution, showing the usefulness of these markers for molecular phage typing.     

Complete genome sequence of Salmonella enterica serovar typhimurium bacteriophage SPN1S

To understand the interaction between the host of pathogenic Salmonella enterica serovar Typhimurium and its bacteriophage, we isolated the bacteriophage SPN1S. It is a lysogenic phage in the Podoviridae family and uses the O-antigen of lipopolysaccharides (LPS) as a host receptor. Comparative genomic analysis of phage SPN1S and the S. enterica serovar Anatum-specific phage ε15 revealed different host specificities, probably due to the low homology of host specificity-related genes. Here we report the complete circular genome sequence of S. Typhimurium-specific bacteriophage SPN1S and show the results of our analysis.     

Transduction of multiple drug resistance of Salmonella enterica serovar typhimurium DT104

Epidemic strain Salmonella typhimurium DT104 is characterized by various multiresistance patterns. At least some of the resistance genes are organized as integrons. Resistance genes of DT104 isolates can be efficiently transduced by P22-like phage ES18 and by phage PDT17 which is released by all DT104 isolates so far analyzed. Cotransduction tests demonstrate that the resistance genes, although not organized in a unique integron, are tightly clustered on the Salmonella chromosome. The spread of resistance genes in this strain by generalized transduction is discussed.     

OmpR regulates the stationary-phase acid tolerance response of Salmonella enterica serovar typhimurium

Tolerance to acidic environments is an important property of free-living and pathogenic enteric bacteria. Salmonella enterica serovar Typhimurium possesses two general forms of inducible acid tolerance. One is evident in exponentially growing cells exposed to a sudden acid shock. The other is induced when stationary-phase cells are subjected to a similar shock. These log-phase and stationary-phase acid tolerance responses (ATRs) are distinct in that genes identified as participating in log-phase ATR have little to no effect on the stationary-phase ATR (I. S. Lee, J. L. Slouczewski, and J. W. Foster, J. Bacteriol. 176:1422-1426, 1994). An insertion mutagenesis strategy designed to reveal genes associated with acid-inducible stationary-phase acid tolerance (stationary-phase ATR) yielded two insertions in the response regulator gene ompR. The ompR mutants were defective in stationary-phase ATR but not log-phase ATR. EnvZ, the known cognate sensor kinase, and the porin genes known to be controlled by OmpR, ompC and ompF, were not required for stationary-phase ATR. However, the alternate phosphodonor acetyl phosphate appears to play a crucial role in OmpR-mediated stationary-phase ATR and in the OmpR-dependent acid induction of ompC. This conclusion was based on finding that a mutant form of OmpR, which is active even though it cannot be phosphorylated, was able to suppress the acid-sensitive phenotype of an ack pta mutant lacking acetyl phosphate. The data also revealed that acid shock increases the level of ompR message and protein in stationary-phase cells. Thus, it appears that acid shock induces the production of OmpR, which in its phosphorylated state can trigger expression of genes needed for acid-induced stationary-phase acid tolerance.