Selection for novel metabolic capabilities in Salmonella enterica

Bacteria are known to display extensive metabolic diversity and many studies have shown that they can use an extensive repertoire of small molecules as carbon‐ and energy sources. However, it is less clear to what extent a bacterium can expand its existing metabolic capabilities by acquiring mutations that, for example, rewire its metabolic pathways. To investigate this capability and potential for evolution of novel phenotypes, we sampled large populations of mutagenized Salmonella enterica to select very rare mutants that can grow on minimal media containing 124 low molecular weight compounds as sole carbon sources. We found mutants growing on 18 of these novel carbon sources, and identified the causal mutations that allowed growth for four of them. Mutations that relieve physiological constraints or increase expression of existing pathways were found to be important contributors to the novel phenotypes. For the remaining 14 novel phenotypes, whole genome sequencing of independent mutants and genetic analysis suggested that these novel metabolic phenotypes result from a combination of multiple mutations. This work, by virtue of identifying the genetic and mechanistic basis for new metabolic capabilities, sheds light on the properties of adaptive landscapes underlying the evolution of novel phenotypes.     

Intracellular Salmonella enterica redirect exocytic transport processes in a Salmonella pathogenicity island 2-dependent manner

During intracellular life, Salmonella enterica proliferate within a specialized membrane compartment, the Salmonella-containing vacuole (SCV), and interfere with the microtubule cytoskeleton and cellular transport. To characterize the interaction of intracellular Salmonella with host cell transport processes, we utilized various model systems to follow microtubule-dependent transport. The vesicular stomatitis virus glycoprotein (VSVG) is a commonly used marker to follow protein transport from the Golgi to the plasma membrane. Using a VSVG-GFP fusion protein, we observed that virulent intracellular Salmonella alter exocytotic transport and recruit exocytotic transport vesicles to the SCV. This virulence function was dependent on the function of the type III secretion system encoded by Salmonella Pathogenicity Island 2 (SPI2) and more specifically on a subset of SPI2 effector proteins. Furthermore, the Golgi to plasma membrane traffic of the shingolipid C(5)-ceramide was redirected to the SCV by virulent Salmonella. We propose that Salmonella modulates the biogenesis of the SCV by deviating this compartment from the default endocytic pathway to an organelle that interacts with the exocytic pathway. This observation might reveal a novel element of the intracellular survival and replication strategy of Salmonella.     

Cobalt stress in Escherichia coli and Salmonella enterica: molecular bases for toxicity and resistance

Cobalt (Co) is present in trace amounts in the environment but it can be toxic when it accumulates in cells. The question of how Co produces its toxic effects and how living organisms protect themselves from, and resist to, such a stress remains to be clarified. Studies pertaining to these issues were recently carried out in Escherichia coli and Salmonella enterica. Iron-sulfur proteins were identified as primary targets of Co ions. Perturbation of iron homeostasis, oxidative stress and possible effects on sulfur assimilation were noticed. Cells were found to respond by up-regulating genes involved in the biosynthesis of Fe-S clusters as well as genes involved in Co efflux. These data are summarized in this review article to provide a preliminary general view of Co toxicity mechanisms in these two bacterial models.     

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.     

Molecular fingerprinting of Salmonella enterica subsp. enterica Typhimurium and Salmonella enterica subsp. enterica derby isolated from tropical seafood in South India

Salmonella enterica subsp. enterica Typhimurium and Salmonella enterica subsp. enterica Derby strains isolated from different seafood were genotyped by PCR-ribotyping and ERIC-PCR assays. This study has ascertained the genetic relatedness among serovars prevalent in tropical seafood. PCR-ribotyping exhibited genetic variation in both Salmonella serovars, and ribotype profile (II) was most predominant, which was observed in 10/18 of Salmonella enterica subsp. enterica Typhimurium and 7/17 Salmonella enterica subsp. enterica Derby isolates. Cluster analysis of ERIC-PCR for Salmonella enterica subsp. enterica Typhimurium strains exhibited nine different banding patterns and four strains showed >95% genetic homology within the cluster pairs. ERIC-PCR produced more genetic variations in Salmonella enterica subsp. enterica Typhimurium; nevertheless, both methods were found to be comparable for Salmonella enterica subsp. enterica Derby isolates. Discrimination index of PCR-ribotyping for Salmonella enterica subsp. enterica Typhimurium isolates was obtained at 0.674 and index value 0.714 was observed for Salmonella enterica subsp. enterica Derby strains. Molecular fingerprinting investigation highlighted the hypothesis of diverse routes of Salmonella contamination in seafood as multiple clones of Salmonella enterica subsp. enterica Typhimurium and Salmonella enterica subsp. enterica Derby were detected in same or different seafood throughout the study period.     

Genome-Wide Methylation Patterns in Salmonella enterica Subsp. enterica Serovars

The methylation of DNA bases plays an important role in numerous biological processes including development, gene expression, and DNA replication. Salmonella is an important foodborne pathogen, and methylation in Salmonella is implicated in virulence. Using single molecule real-time (SMRT) DNA-sequencing, we sequenced and assembled the complete genomes of eleven Salmonella enterica isolates from nine different serovars, and analysed the whole-genome methylation patterns of each genome. We describe 16 distinct N⁶-methyladenine (m6A) methylated motifs, one N⁴-methylcytosine (m4C) motif, and one combined m6A-m4C motif. Eight of these motifs are novel, i.e., they have not been previously described. We also identified the methyltransferases (MTases) associated with 13 of the motifs. Some motifs are conserved across all Salmonella serovars tested, while others were found only in a subset of serovars. Eight of the nine serovars contained a unique methylated motif that was not found in any other serovar (most of these motifs were part of Type I restriction modification systems), indicating the high diversity of methylation patterns present in Salmonella.     

L‐2,3‐diaminopropionate generates diverse metabolic stresses in Salmonella enterica

Unchecked amino acid accumulation in living cells has the potential to cause stress by disrupting normal metabolic processes. Thus, many organisms have evolved degradation strategies that prevent endogenous accumulation of amino acids. L‐2,3‐diaminopropionate (Dap) is a non‐protein amino acid produced in nature where it serves as a precursor to siderophores, neurotoxins and antibiotics. Dap accumulation in Salmonella enterica was previously shown to inhibit growth by unknown mechanisms. The production of diaminopropionate ammonia‐lyase (DpaL) alleviated Dap toxicity in S. enterica by catalyzing the degradation of Dap to pyruvate and ammonia. Here, we demonstrate that Dap accumulation in S. enterica elicits a proline requirement for growth and specifically inhibits coenzyme A and isoleucine biosynthesis. Additionally, we establish that the DpaL‐dependent degradation of Dap to pyruvate proceeds through an unbound 2‐aminoacrylate (2AA) intermediate, thus contributing to 2AA stress inside the cell. The reactive intermediate deaminase, RidA, is shown to prevent 2AA damage caused by DpaL‐dependent Dap degradation by enhancing the rate of 2AA hydrolysis. The results presented herein inform our understanding of the effects Dap has on metabolism in S. enterica, and likely other organisms, and highlight the critical role played by RidA in preventing 2AA stress stemming from Dap detoxification.     

Plasmid-mediated antimicrobial resistance in Salmonella enterica

The selective pressure imposed by the use of antimicrobials in both human and veterinary medicine promotes the spread of multiple antimicrobial resistance. The dissemination of antimicrobial resistance in Salmonella enterica strains, causing severe enteritis in human, has been reported worldwide and is largely attributed to conjugative DNA exchange. In the present review, the relevance of plasmids to the dissemination of antimicrobial resistance in S. enterica is discussed. Recent examples of plasmid-mediated resistance to expanded-spectrum cephalosporins are reported to illustrate the severity of current situation in enteric pathogens. The exchanges between plasmid(s) and the bacterial chromosome and the integration of resistance genes into specialised genetic elements, called integrons, play a major role in acquisition and dissemination of resistance genes. The evolution of a plasmid through the acquisition of integrons is reported, describing novel mechanisms for short-term accumulation of resistance determinants in plasmids circulating in Salmonella.     

Bile-induced DNA damage in Salmonella enterica

In the absence of DNA adenine methylase, growth of Salmonella enterica serovar Typhimurium is inhibited by bile. Mutations in any of the mutH, mutL, and mutS genes suppress bile sensitivity in a Dam⁻ background, indicating that an active MutHLS system renders Dam⁻ mutants bile sensitive. However, inactivation of the MutHLS system does not cause bile sensitivity. An analogy with Escherichia coli, in which the MutHLS system sensitizes Dam⁻ mutants to DNA-injuring agents, suggested that bile might cause DNA damage. In support of this hypothesis, we show that bile induces the SOS response in S. enterica and increases the frequency of point mutations and chromosomal rearrangements. Mutations in mutH, mutL, or mutS cause partial relief of virulence attenuation in a Dam⁻ background (50- to 100-fold by the oral route and 10-fold intraperitoneally), suggesting that an active MutHLS system reduces the ability of Salmonella Dam⁻ mutants to cope with DNA-damaging agents (bile and others) encountered during the infection process. The DNA-damaging ability of bile under laboratory conditions raises the possibility that the phenomenon may be relevant in vivo, since high bile concentrations are found in the gallbladder, the niche for chronic Salmonella infections.     

Recombination and population structure in Salmonella enterica

Salmonella enterica is a bacterial pathogen that causes enteric fever and gastroenteritis in humans and animals. Although its population structure was long described as clonal, based on high linkage disequilibrium between loci typed by enzyme electrophoresis, recent examination of gene sequences has revealed that recombination plays an important evolutionary role. We sequenced around 10% of the core genome of 114 isolates of enterica using a resequencing microarray. Application of two different analysis methods (Structure and ClonalFrame) to our genomic data allowed us to define five clear lineages within S. enterica subspecies enterica, one of which is five times older than the other four and two thirds of the age of the whole subspecies. We show that some of these lineages display more evidence of recombination than others. We also demonstrate that some level of sexual isolation exists between the lineages, so that recombination has occurred predominantly between members of the same lineage. This pattern of recombination is compatible with expectations from the previously described ecological structuring of the enterica population as well as mechanistic barriers to recombination observed in laboratory experiments. In spite of their relatively low level of genetic differentiation, these lineages might therefore represent incipient species.     

Bistability and phase variation in Salmonella enterica

Cell-to-cell differences in bacterial gene expression can merely reflect the occurrence of noise. In certain cases, however, heterogeneous gene expression is a programmed event that results in bistable expression. If bistability is heritable, bacterial lineages are formed. When programmed bistability is reversible, the phenomenon is known as phase variation. In certain cases, bistability is controlled by genetic mechanisms (e. g., DNA rearrangement). In other cases, bistability has epigenetic origin. A robust epigenetic mechanism for the formation of bacterial lineages is the formation of heritable DNA methylation patterns. However, bistability can also arise upon propagation of gene expression patterns by feedback loops that are stable upon cell division. This review describes examples of bistability and phase variation in Salmonella enterica and discusses their adaptive value, sometimes in a speculative manner.     

Novel haemolysins of Salmonella enterica spp. enterica serovar Gallinarum

Haemolysins of Salmonella are important due to their probable role in pathogenesis of systemic salmonellosis and use in sub-serovar level typing. The present study was undertaken to determine haemolytic potential of Salmonella Gallinarum strains through phenotypic and genotypic methods. Amplification of haemolysin gene (clyA) and cytolysin gene (slyA) was attempted in order to determine their role in haemolysin production. Study on 94 strains of S. Gallinarum revealed the production of two types of haemolysis viz., beneath the colony haemolysis (BCH) or contact haemolysis and clear zone haemolysis (CZH). Haemolysis was observed on blood agar prepared with blood of cattle, buffalo, sheep, goat, horse, rabbit, guinea pig, fowl, and human blood group A, B, AB and O. Although, haemolysis was also observed on blood agar prepared with whole blood, clarity of zone was more evident on blood agar made from washed erythrocytes. Clear zone haemolysis was best observed on blood agar prepared with washed erythrocytes of goat and a total of 12% (11 of 94) S. Gallinarum strains under study produced CZH on it. The clyA gene could not be detected in any of the 94 strains under study, while slyA gene could be amplified uniformly irrespective of haemolytic potential (CZH) and haemolytic pattern (BCH) of the strains. The study suggested that the two types of haemolysis (CZH and BCH) observed among S. Gallinarum strains may not be due to either slyA or clyA gene products and thus there may be some other gene responsible for haemolytic trait in Gallinarum serovar. Different haemolytic patterns of strains under study indicated multiplicity of haemolysins in S. Gallinarum.     

Detection of hilA gene sequences in serovars of Salmonella enterica subspecies enterica

hilA gene promoter, component of the Salmonella Pathogenicity Island 1, has been found in Salmonella serovar Typhimurium, being important for the regulation of type III secretion apparatus genes. We detected hilA gene sequences in Salmonella serovars Typhi, Enteritidis, Choleraesuis, Paratyphi A and B, and Pullorum, by polymerase chain reaction (PCR) and hybridization techniques. The primers to carry out PCR were designed according to hilA sequence. A low stringency hybridization with the probe pVV441 (hilA open-reading-frame plasmid) was carried out. To find hilA gene sequences in other Salmonella sp. suggest that these serovars could have similar sequences of this kind of virulence genes.     

Cloth-based hybridization array system for the detection of multiple antibiotic resistance genes in Salmonella enterica subsp. enterica serotype Typhimurium DT104

AIMS: A simple DNA macroarray system was developed for detection of antibiotic resistance and other marker genes associated with the multidrug-resistant food pathogen Salmonella enterica subsp. enterica serotype Typhimurium DT104. METHODS AND RESULTS: A multiplex polymerase chain reaction (PCR) incorporating digoxigenin-dUTP was used to simultaneously amplify seven marker sequences, with subsequent rapid detection of the amplicons by hybridization with an array of probes immobilized on polyester cloth and immunoenzymatic assay of the bound label. This system provided sensitive detection of the different genetic markers in the S. Typhimurium DT104 genome, giving positive reactions with as few as 10 CFU, and the hybridizations were highly specific, with no reactions of amplicons with heterologous probes on the array. CONCLUSIONS: This cloth-based hybridization array system (CHAS) provides a simple, cost-effective tool for monitoring S. Typhimurium DT104 in foods and their production environment. SIGNIFICANCE AND IMPACT OF THE STUDY: The CHAS is a simple and cost-effective tool for the simultaneous detection of amplicons generated in a multiplex PCR, and the concept is broadly applicable to the detection and characterization of food pathogens.     

Substructure within Salmonella enterica subsp. enterica isolates from Australian wildlife

Multilocus sequence typing of 56 Salmonella enterica subsp. enterica strains isolated from Australian wildlife hosts was performed. The results of population assignment algorithms revealed that the 56 strains could be subdivided into two distinct clades. Strains belonging to the two clades were further distinguished phenotypically, genotypically, and with respect to host distribution.