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 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.     

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.     

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.     

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.     

Requirement of Fur for the full induction of Dps expression in Salmonella enterica serovar typhimurium

The Dps protein, which is overexpressed in harsh environments, is known to play a critical role in the protection of DNA against oxidative stresses. In this study, the roles of Fur in the expression of the dps gene in Salmonella and the protection mechanisms against oxidative stress in Salmonella cells preexposed to iron-stress were investigated. Two putative Fur boxes were predicted within the promoter region o f th e S. typhimurium dps gene . The profile of dps expression performed by the LacZ reporter assay revealed growth-phase dependency regardless of iron-status under the culture conditions. Thefur mutant, chi4659, evidenced a reduced level of beta-galactosidase as compared to the wild-type strain. The results observed after the measurement of the Dps protein in various Salmonella regulatory mutants were consistent with the results acquired in the reporter assay. This evidence suggested that Fur performs a function as a subsidiary regulator in the expression of dps. The survival ability of Salmonella strains after exposure to oxidative stress demonstrated that the Dps protein performs a pivotal function in the survival of stationary-phase S. typhimurium against oxidative stress. Salmonella cells grown in iron-restricted condition required Dps for full protection against oxidative stress. The CK24 (Deltadps) cells grown in iron-replete condition survived at a rate similar to that observed in the wild-type strain, thereby suggesting the induction of an unknown protection mechanism(s) other than Dps in this condition.     

FliZ regulates expression of the Salmonella pathogenicity island 1 invasion locus by controlling HilD protein activity in Salmonella enterica serovar typhimurium

A prerequisite for Salmonella enterica to cause both intestinal and systemic disease is the direct injection of effector proteins into host intestinal epithelial cells via a type three secretion system (T3SS); the T3SS genes are carried on Salmonella pathogenicity island 1 (SPI1). These effector proteins induce inflammatory diarrhea and bacterial invasion. Expression of the SPI1 T3SS is tightly regulated in response to environmental signals through a variety of global regulatory systems. We have previously shown that three AraC-like regulators, HilD, HilC, and RtsA, act in a complex feed-forward regulatory loop to control the expression of the hilA gene, which encodes the direct regulator of the SPI1 structural genes. In this work, we characterize a major positive regulator of this system, the flagellar protein FliZ. Through genetic and biochemical analyses, we show that FliZ posttranslationally controls HilD to positively regulate hilA expression. This mechanism is independent of other flagellar components and is not mediated through the negative regulator HilE or through FliZ-mediated RpoS regulation. We demonstrate that FliZ controls HilD protein activity and not stability. FliZ regulates HilD in the absence of Lon protease, previously shown to degrade HilD. Indeed, it appears that FliZ, rather than HilD, is the most relevant target of Lon as it relates to SPI1 expression. Mutants lacking FliZ are significantly attenuated in their ability to colonize the intestine but are unaffected during systemic infection. The intestinal attenuation is partially dependent on SPI1, but FliZ has additional pleiotropic effects.     

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.