Strain-Specific Gifsy-1 Prophage Genes Are Determinants for Expression of the RNA Repair Operon during the SOS Response in Salmonella enterica Serovar Typhimurium.

The adaptation of Salmonella enterica serovar Typhimurium to stress conditions involves expression of genes within the regulon of the alternative sigma factor RpoN (σ54). RpoN-dependent transcription requires an activated bacterial enhancer binding protein (bEBP) that hydrolyzes ATP to remodel the RpoN-holoenzyme-promoter complex for transcription initiation. The bEBP RtcR in S. Typhimurium strain 14028s is activated by genotoxic stress to direct RpoN-dependent expression of the RNA repair operon rsr-yrlBA-rtcBA. The molecular signal for RtcR activation is an oligoribonucleotide with a 3'-terminal 2',3'-cyclic phosphate. We show in S. Typhimurium 14028s that the molecular signal is not a direct product of nucleic acid damage, but signal generation is dependent on a RecA-controlled SOS-response pathway, specifically, induction of prophage Gifsy-1. A genome-wide mutant screen and utilization of Gifsy prophage-cured strains indicated that the nucleoid-associated protein Fis and the Gifsy-1 prophage significantly impact RtcR activation. Directed-deletion analysis and genetic mapping by transduction demonstrated that a three-gene region (STM14_3218-3220) in Gifsy-1, which is variable between S. Typhimurium strains, is required for RtcR activation in strain 14028s and that the absence of STM14_3218-3220 in the Gifsy-1 prophages of S. Typhimurium strains LT2 and 4/74, which renders these strains unable to activate RtcR during genotoxic stress, can be rescued by complementation in cis by the region encompassing STM14_3218-3220. Thus, even though RtcR and the RNA repair operon are highly conserved in Salmonella enterica serovars, RtcR-dependent expression of the RNA repair operon in S. Typhimurium is controlled by a variable region of a prophage present in only some strains. IMPORTANCE The transcriptional activator RtcR and the RNA repair proteins whose expression it regulates, RtcA and RtcB, are widely conserved in Proteobacteria. In Salmonella Typhimurium 14028s, genotoxic stress activates RtcR to direct RpoN-dependent expression of the rsr-yrlBA-rtcBA operon. This work identifies key elements of a RecA-dependent pathway that generates the signal for RtcR activation in strain 14028s. This signaling pathway requires the presence of a specific region within the prophage Gifsy-1, yet this region is absent in most other wild-type Salmonella strains. Thus, we show that the activity of a widely conserved regulatory protein can be controlled by prophages with narrow phylogenetic distributions. This work highlights an underappreciated phenomenon where bacterial physiological functions are altered due to genetic rearrangement of prophages.

Arginine Metabolism Powers Salmonella Resistance to Oxidative Stress.

Salmonella invades host cells and replicates inside acidified, remodeled vacuoles that are exposed to reactive oxygen species (ROS) generated by the innate immune response. Oxidative products of the phagocyte NADPH oxidase mediate antimicrobial activity, in part, by collapsing the ΔpH of intracellular Salmonella. Given the role of arginine in bacterial resistance to acidic pH, we screened a library of 54 single-gene mutants in Salmonella that are each involved in, but do not entirely block, arginine metabolism. We identified several mutants that affected Salmonella virulence in mice. The triple mutant ΔargCBH, which is deficient in arginine biosynthesis, was attenuated in immunocompetent mice, but recovered virulence in phagocyte NADPH oxidase deficient Cybb-/- mice. Furthermore, ΔargCBH Salmonella was profoundly susceptible to the bacteriostatic and bactericidal effects of hydrogen peroxide. Peroxide stress led to a larger collapse of the ΔpH in ΔargCBH mutants than occurred in wild-type Salmonella. The addition of exogenous arginine rescued ΔargCBH Salmonella from peroxide-induced ΔpH collapse and killing. Combined, these observations suggest that arginine metabolism is a hitherto unknown determinant of virulence that contributes to the antioxidant defenses of Salmonella by preserving pH homeostasis. In the absence of phagocyte NADPH oxidase-produced ROS, host cell-derived l-arginine appears to satisfy the needs of intracellular Salmonella. However, under oxidative stress, Salmonella must additionally rely on de novo biosynthesis to maintain full virulence.

The multi-drug efflux system AcrABZ-TolC is essential for infection of Salmonella Typhimurium by the flagellum-dependent bacteriophage Chi.

Bacteriophages are the most abundant biological entities in the biosphere. Due to their host specificity and ability to kill bacteria rapidly, bacteriophages have many potential healthcare applications, including therapy against antibiotic-resistant bacteria. Infection by flagellotropic bacteriophages requires a properly rotating bacterial flagellar filament. The flagella-dependent phage χ (Chi) infects serovars of the pathogenic enterobacterium Salmonella enterica However, cell surface receptors and proteins involved in other stages of χ infection have not been discovered to date. We screened a multi-gene deletion library of S. enterica serovar Typhimurium by spotting mutants on soft agar plates seeded with bacteriophage χ and monitoring their ability to grow and form a swim ring, a characteristic of bacteriophage-resistant motile mutants. Those multi-gene deletion regions identified to be important for χ infectivity were further investigated by characterizing the phenotypes of corresponding single-gene deletion mutants. This way, we identified motile mutants with varying degrees of resistance to χ. Deletions in individual genes encoding the AcrABZ-TolC multi-drug efflux system drastically reduced infection by bacteriophage χ. Furthermore, an acrABtolC triple deletion strain was fully resistant to χ. Infection was severely reduced but not entirely blocked by the deletion of the gene tig encoding the molecular chaperone trigger factor. Finally, deletion in genes encoding enzymes involved in the synthesis of the antioxidants glutathione (GSH) and uric acid resulted in reduced infectivity. Our findings begin to elucidate poorly understood processes involved in later stages of flagellotropic bacteriophage infection and informs research aimed at the use of bacteriophages to combat antibiotic-resistant bacterial infections.IMPORTANCEAntimicrobial resistance is a large concern in the healthcare field. With more multi-drug resistant bacterial pathogens emerging, other techniques for eliminating bacterial infections are being explored. Among these is phage therapy, where combinations of specific phages are used to treat infections. Generally, phages utilize cell appendages and surface receptors for the initial attachment to their host. Phages that are flagellotropic are of particular interest because flagella are often important in bacterial virulence, making resistance to attachment of these phages harder to achieve without reducing virulence. This study discovered the importance of a multi-drug efflux pump for the infection of Salmonella enterica by a flagellotropic phage. In theory, if a bacterial pathogen develops phage resistance by altering expression of the efflux pump then the pathogen would simultaneously become more susceptible to the antibiotic substrates of the pump. Thus, co-administering antibiotics and flagellotropic phage may be a particularly potent antibacterial therapy.

Zinc-dependent substrate-level phosphorylation powers Salmonella growth under nitrosative stress of the innate host response.

The metabolic processes that enable the replication of intracellular Salmonella under nitrosative stress conditions engendered in the innate response of macrophages are poorly understood. A screen of Salmonella transposon mutants identified the ABC-type high-affinity zinc uptake system ZnuABC as a critical determinant of the adaptation of Salmonella to the nitrosative stress generated by the enzymatic activity of inducible nitric oxide (NO) synthase of mononuclear phagocytic cells. NO limits the virulence of a znuB mutant in an acute murine model of salmonellosis. The ZnuABC transporter is crucial for the glycolytic function of fructose bisphosphate aldolase, thereby fueling growth of Salmonella during nitrosative stress produced in the innate response of macrophages. Our investigations demonstrate that glycolysis mediates resistance of Salmonella to the antimicrobial activity of NO produced in an acute model of infection. The ATP synthesized by substrate-level phosphorylation at the payoff phase of glycolysis and acetate fermentation powers the replication of Salmonella experiencing high levels of nitrosative stress. In contrast, despite its high potential for ATP synthesis, oxidative phosphorylation is a major target of inhibition by NO and contributes little to the antinitrosative defenses of intracellular Salmonella. Our investigations have uncovered a previously unsuspected conjunction between zinc homeostasis, glucose metabolism and cellular energetics in the adaptation of intracellular Salmonella to the reactive nitrogen species synthesized in the innate host response.

Genes affecting progression of bacteriophage P22 infection in Salmonella identified by transposon and single gene deletion screens.

Bacteriophages rely on their hosts for replication, and many host genes critically determine either viral progeny production or host success via phage resistance. A random insertion transposon library of 240,000 mutants in Salmonella enterica serovar Typhimurium was used to monitor effects of individual bacterial gene disruptions on bacteriophage P22 lytic infection. These experiments revealed candidate host genes that alter the timing of phage P22 propagation. Using a False Discovery Rate of < 0.1, mutations in 235 host genes either blocked or delayed progression of P22 lytic infection, including many genes for which this role was previously unknown. Mutations in 77 genes reduced the survival time of host DNA after infection, including mutations in genes for enterobacterial common antigen (ECA) synthesis and osmoregulated periplasmic glucan (OPG). We also screened over 2000 Salmonella single gene deletion mutants to identify genes that impacted either plaque formation or culture growth rates. The gene encoding the periplasmic membrane protein YajC was newly found to be essential for P22 infection. Targeted mutagenesis of yajC shows that an essentially full-length protein is required for function, and potassium efflux measurements demonstrated that YajC is critical for phage DNA ejection across the cytoplasmic membrane.

Interactions of Salmonella enterica Serovar Typhimurium and Pectobacterium carotovorum within a Tomato Soft Rot.

Salmonella spp. are remarkably adaptable pathogens, and this adaptability allows these bacteria to thrive in a variety of environments and hosts. The mechanisms with which these pathogens establish within a niche amid the native microbiota remain poorly understood. Here, we aimed to uncover the mechanisms that enable Salmonella enterica serovar Typhimurium strain ATCC 14028 to benefit from the degradation of plant tissue by a soft rot plant pathogen, Pectobacterium carotovorum The hypothesis that in the soft rot, the liberation of starch (not utilized by P. carotovorum) makes this polymer available to Salmonella spp., thus allowing it to colonize soft rots, was tested first and proven null. To identify the functions involved in Salmonella soft rot colonization, we carried out transposon insertion sequencing coupled with the phenotypic characterization of the mutants. The data indicate that Salmonella spp. experience a metabolic shift in response to the changes in the environment brought on by Pectobacterium spp. and likely coordinated by the csrBC small regulatory RNA. While csrBC and flhD appear to be of importance in the soft rot, the global two-component system encoded by barA sirA (which controls csrBC and flhDC under laboratory conditions) does not appear to be necessary for the observed phenotype. Motility and the synthesis of nucleotides and amino acids play critical roles in the growth of Salmonella spp. in the soft rot.IMPORTANCE Outbreaks of produce-associated illness continue to be a food safety concern. Earlier studies demonstrated that the presence of phytopathogens on produce was a significant risk factor associated with increased Salmonella carriage on fruits and vegetables. Here, we genetically characterize some of the requirements for interactions between Salmonella and phytobacteria that allow Salmonella spp. to establish a niche within an alternate host (tomato). Pathways necessary for nucleotide synthesis, amino acid synthesis, and motility are identified as contributors to the persistence of Salmonella spp. in soft rots.

Novel DNA Binding and Regulatory Activities for σ54 (RpoN) in Salmonella enterica Serovar Typhimurium 14028s.

The variable sigma (σ) subunit of the bacterial RNA polymerase (RNAP) holoenzyme, which is responsible for promoter specificity and open complex formation, plays a strategic role in the response to environmental changes. Salmonella enterica serovar Typhimurium utilizes the housekeeping σ70 and five alternative sigma factors, including σ54 The σ54-RNAP differs from other σ-RNAP holoenzymes in that it forms a stable closed complex with the promoter and requires ATP hydrolysis by an activated cognate bacterial enhancer binding protein (bEBP) to transition to an open complex and initiate transcription. In S. Typhimurium, σ54-dependent promoters normally respond to one of 13 different bEBPs, each of which is activated under a specific growth condition. Here, we utilized a constitutively active, promiscuous bEBP to perform a genome-wide identification of σ54-RNAP DNA binding sites and the transcriptome of the σ54 regulon of S. Typhimurium. The position and context of many of the identified σ54 RNAP DNA binding sites suggest regulatory roles for σ54-RNAP that connect the σ54 regulon to regulons of other σ factors to provide a dynamic response to rapidly changing environmental conditions.IMPORTANCE The alternative sigma factor σ54 (RpoN) is required for expression of genes involved in processes with significance in agriculture, bioenergy production, bioremediation, and host-microbe interactions. The characterization of the σ54 regulon of the versatile pathogen S. Typhimurium has expanded our understanding of the scope of the σ54 regulon and how it links to other σ regulons within the complex regulatory network for gene expression in bacteria.

Contribution of Asparagine Catabolism to Salmonella Virulence.

Salmonellae are pathogenic bacteria that cause significant morbidity and mortality in humans worldwide. Salmonellae establish infection and avoid clearance by the immune system by mechanisms that are not well understood. We previously showed that l-asparaginase II produced by Salmonella enterica serovar Typhimurium (S Typhimurium) inhibits T cell responses and mediates virulence. In addition, we previously showed that asparagine deprivation such as that mediated by l-asparaginase II of S Typhimurium causes suppression of activation-induced T cell metabolic reprogramming. Here, we report that STM3997, which encodes a homolog of disulfide bond protein A (dsbA) of Escherichia coli, is required for l-asparaginase II stability and function. Furthermore, we report that l-asparaginase II localizes primarily to the periplasm and acts together with l-asparaginase I to provide S Typhimurium the ability to catabolize asparagine and assimilate nitrogen. Importantly, we determined that, in a murine model of infection, S Typhimurium lacking both l-asparaginase I and II genes competes poorly with wild-type S Typhimurium for colonization of target tissues. Collectively, these results indicate that asparagine catabolism contributes to S Typhimurium virulence, providing new insights into the competition for nutrients at the host-pathogen interface.

Salmonella Persistence in Tomatoes Requires a Distinct Set of Metabolic Functions Identified by Transposon Insertion Sequencing.

Human enteric pathogens, such as Salmonella spp. and verotoxigenic Escherichia coli, are increasingly recognized as causes of gastroenteritis outbreaks associated with the consumption of fruits and vegetables. Persistence in plants represents an important part of the life cycle of these pathogens. The identification of the full complement of Salmonella genes involved in the colonization of the model plant (tomato) was carried out using transposon insertion sequencing analysis. With this approach, 230,000 transposon insertions were screened in tomato pericarps to identify loci with reduction in fitness, followed by validation of the screen results using competition assays of the isogenic mutants against the wild type. A comparison with studies in animals revealed a distinct plant-associated set of genes, which only partially overlaps with the genes required to elicit disease in animals. De novo biosynthesis of amino acids was critical to persistence within tomatoes, while amino acid scavenging was prevalent in animal infections. Fitness reduction of the Salmonella amino acid synthesis mutants was generally more severe in the tomato rin mutant, which hyperaccumulates certain amino acids, suggesting that these nutrients remain unavailable to Salmonella spp. within plants. Salmonella lipopolysaccharide (LPS) was required for persistence in both animals and plants, exemplifying some shared pathogenesis-related mechanisms in animal and plant hosts. Similarly to phytopathogens, Salmonella spp. required biosynthesis of amino acids, LPS, and nucleotides to colonize tomatoes. Overall, however, it appears that while Salmonella shares some strategies with phytopathogens and taps into its animal virulence-related functions, colonization of tomatoes represents a distinct strategy, highlighting this pathogen's flexible metabolism.IMPORTANCE Outbreaks of gastroenteritis caused by human pathogens have been increasingly associated with foods of plant origin, with tomatoes being one of the common culprits. Recent studies also suggest that these human pathogens can use plants as alternate hosts as a part of their life cycle. While dual (animal/plant) lifestyles of other members of the Enterobacteriaceae family are well known, the strategies with which Salmonella colonizes plants are only partially understood. Therefore, we undertook a high-throughput characterization of the functions required for Salmonella persistence within tomatoes. The results of this study were compared with what is known about genes required for Salmonella virulence in animals and interactions of plant pathogens with their hosts to determine whether Salmonella repurposes its virulence repertoire inside plants or whether it behaves more as a phytopathogen during plant colonization. Even though Salmonella utilized some of its virulence-related genes in tomatoes, plant colonization required a distinct set of functions.

A simplified multiplex PCR-based typing method for common Salmonella enterica serovars supported by online server-based detection system.

BACKGROUND & OBJECTIVES: A rapid and simple alternative method is needed to replace the laborious, time-consuming Salmonella serotyping. The objective of the present study was to improve and simplify a previously reported multiplex polymerase chain reaction (PCR)-based method and to create an online server to enable rapid determination of serovars. METHODS: A method of multiplex PCR-based genome typing (MPGT) was standardized using 59 Salmonella isolates of 31 serovars. Several previously reported primers were modified to obtain a more accurate performance. The screen was separated into four different multiplex reactions distinguishable on standard electrophoresis. A blind study was subsequently performed with 81 isolates of 10 serovars most prevalent in India. Whole genome information from 440 Salmonella isolates was used to confirm the usefulness of this method and concurrence of in silico predictions and PCR results were investigated. A public server (http://www.mpgt-salmonella.res.in) was established for data storage and determination of closest previously observed Salmonella isolates based on obtained MPGT patterns. RESULTS: The 16 target genes amplified showed variability in their presence in strains from different serotypes. Hence, identical amplification patterns suggested genetic relatedness of strains and usually identical serological behaviour. The observed absence/presence patterns of genes were converted to an MPGT code. Altogether, 83 different codes were predicted in silico based on the whole genome information of 440 strains. Results confirmed that major serovars usually displayed unique MPGT codes. INTERPRETATION & CONCLUSIONS: The multiplex PCR assay resulted in specific binary codes for isolates from each of the 31 Salmonella serovars tested. The online server allowed the user to compare obtained PCR results with stored previous patterns. Simplicity, speed and cost-effectiveness make this tool useful for quick outbreak management.

Genetic Determinants of Salmonella enterica Serovar Typhimurium Proliferation in the Cytosol of Epithelial Cells.

Intestinal epithelial cells provide an important colonization niche for Salmonella enterica serovar Typhimurium during gastrointestinal infections. In infected epithelial cells, a subpopulation of S Typhimurium bacteria damage their internalization vacuole, leading to escape from the Salmonella-containing vacuole (SCV) and extensive proliferation in the cytosol. Little is known about the bacterial determinants of nascent SCV lysis and subsequent survival and replication of Salmonella in the cytosol. To pinpoint S Typhimurium virulence factors responsible for these steps in the intracellular infectious cycle, we screened a S Typhimurium multigene deletion library in Caco-2 C2Bbe1 and HeLa epithelial cells for mutants that had an altered proportion of cytosolic bacteria compared to the wild type. We used a gentamicin protection assay in combination with a chloroquine resistance assay to quantify total and cytosolic bacteria, respectively, for each strain. Mutants of three S Typhimurium genes, STM1461 (ydgT), STM2829 (recA), and STM3952 (corA), had reduced cytosolic proliferation compared to wild-type bacteria, and one gene, STM2120 (asmA), displayed increased cytosolic replication. None of the mutants were affected for lysis of the nascent SCV or vacuolar replication in epithelial cells, indicating that these genes are specifically required for survival and proliferation of S Typhimurium in the epithelial cell cytosol. These are the first genes identified to contribute to this step of the S Typhimurium infectious cycle.

Novel Two-Step Hierarchical Screening of Mutant Pools Reveals Mutants under Selection in Chicks.

Contaminated chicken/egg products are major sources of human salmonellosis, yet the strategies used by Salmonella to colonize chickens are poorly understood. We applied a novel two-step hierarchical procedure to identify new genes important for colonization and persistence of Salmonella enterica serotype Typhimurium in chickens. A library of 182 S. Typhimurium mutants each containing a targeted deletion of a group of contiguous genes (for a total of 2,069 genes deleted) was used to identify regions under selection at 1, 3, and 9 days postinfection in chicks. Mutants in 11 regions were under selection at all assayed times (colonization mutants), and mutants in 15 regions were under selection only at day 9 (persistence mutants). We assembled a pool of 92 mutants, each deleted for a single gene, representing nearly all genes in nine regions under selection. Twelve single gene deletion mutants were under selection in this assay, and we confirmed 6 of 9 of these candidate mutants via competitive infections and complementation analysis in chicks. STM0580, STM1295, STM1297, STM3612, STM3615, and STM3734 are needed for Salmonella to colonize and persist in chicks and were not previously associated with this ability. One of these key genes, STM1297 (selD), is required for anaerobic growth and supports the ability to utilize formate under these conditions, suggesting that metabolism of formate is important during infection. We report a hierarchical screening strategy to interrogate large portions of the genome during infection of animals using pools of mutants of low complexity. Using this strategy, we identified six genes not previously known to be needed during infection in chicks, and one of these (STM1297) suggests an important role for formate metabolism during infection.

Persistent Infections by Nontyphoidal Salmonella in Humans: Epidemiology and Genetics.

BACKGROUND: Although chronic infections by typhoidal Salmonella are well-known, prolonged human infections by nontyphoidal Salmonella (NTS) are poorly characterized. METHODS: We retrospectively analyzed 48 345 culture-confirmed NTS infections that occurred in Israel 1995-2012. A case-control study was performed to identify risk factors associated with persistent infections. Whole-genome-sequencing, pulsed-field gel electrophoresis (PFGE), and a mouse infection model were used to study genetic and phenotypic differences between same-patient persistent, recurring isolates. RESULTS: In total, 1047 cases of persistent NTS infections, comprising 2.2% of all reported cases of salmonellosis, were identified. The persistence periods ranged between 30 days to 8.3 years. The majority (93%) of the persistently infected patients were immunocompetent, and 65% were symptomatic with relapsing diarrhea, indicating a distinct clinical manifestation from the asymptomatic carriage of typhoidal Salmonella. Four NTS serovars (Mbandaka, Bredeney, Infantis and Virchow) were found to be significantly more frequently associated with persistence than others. Comparative genomics between early and later isolates obtained from the same patients confirmed clonal infection and showed 0 to 10 SNPs between persistent isolates. A different composition of mobile genetic elements (plasmids and phages) or amino acid substitutions in global regulators was identified in multiple cases. These changes resulted in differences in phenotype and virulence between early and later same-patient isolates. CONCLUSIONS: These results illuminate the overlooked clinical manifestation of persistent salmonellosis that can serve as a human reservoir for NTS infections. Additionally, we demonstrate mechanisms of in-host microevolution and exhibit their potential to shape Salmonella pathogenicity, antimicrobial resistance and host-pathogen interactions.

The 4-cysteine zinc-finger motif of the RNA polymerase regulator DksA serves as a thiol switch for sensing oxidative and nitrosative stress.

We show that thiols in the 4-cysteine zinc-finger motif of DksA, an RNA polymerase accessory protein known to regulate the stringent response, sense oxidative and nitrosative stress. Hydrogen peroxide- or nitric oxide (NO)-mediated modifications of thiols in the DksA 4-cysteine zinc-finger motif release the metal cofactor and drive reversible changes in the α-helicity of the protein. Wild-type and relA spoT mutant Salmonella, but not isogenic dksA-deficient bacteria, experience the downregulation of r-protein and amino acid transport expression after NO treatment, suggesting that DksA can regulate gene expression in response to NO congeners independently of the ppGpp alarmone. Oxidative stress enhances the DksA-dependent repression of rpsM, while preventing the activation of livJ and hisG gene transcription that is supported by reduced, zinc-bound DksA. The inhibitory effects of oxidized DksA on transcription are reversible with dithiothreitol. Our investigations indicate that sensing of reactive species by DksA redox active thiols fine-tunes the expression of translational machinery and amino acid assimilation and biosynthesis in accord with the metabolic stress imposed by oxidative and nitrosative stress. Given the conservation of Cys(114) , and neighbouring hydrophobic and charged amino acids in DksA orthologues, phylogenetically diverse microorganisms may use the DksA thiol switch to regulate transcriptional responses to oxidative and nitrosative stress.

rpoS-Regulated core genes involved in the competitive fitness of Salmonella enterica Serovar Kentucky in the intestines of chickens.

Salmonella enterica serovar Kentucky has become the most frequently isolated serovar from poultry in the United States over the past decade. Despite its prevalence in poultry, it causes few human illnesses in the United States. The dominance of S. Kentucky in poultry does not appear to be due to single introduction of a clonal strain, and its reduced virulence appears to correlate with the absence of virulence genes grvA, sseI, sopE, and sodC1. S. Kentucky's prevalence in poultry is possibly attributable to its metabolic adaptation to the chicken cecum. While there were no difference in the growth rate of S. Kentucky and S. Typhimurium grown microaerophilically in cecal contents, S. Kentucky persisted longer when chickens were coinfected with S. Typhimurium. The in vivo advantage that S. Kentucky has over S. Typhimurium appears to be due to differential regulation of core Salmonella genes via the stationary-phase sigma factor rpoS. Microarray analysis of Salmonella grown in cecal contents in vitro identified several metabolic genes and motility and adherence genes that are differentially activated in S. Kentucky. The contributions of four of these operons (mgl, prp, nar, and csg) to Salmonella colonization in chickens were assessed. Deletion of mgl and csg reduced S. Kentucky persistence in competition studies in chickens infected with wild-type or mutant strains. Subtle mutations affecting differential regulation of core Salmonella genes appear to be important in Salmonella's adaptation to its animal host and especially for S. Kentucky's emergence as the dominant serovar in poultry.

Genomic epidemiology of Salmonella enterica serotype Enteritidis based on population structure of prevalent lineages.

Salmonella enterica serotype Enteritidis is one of the most commonly reported causes of human salmonellosis. Its low genetic diversity, measured by fingerprinting methods, has made subtyping a challenge. We used whole-genome sequencing to characterize 125 S. enterica Enteritidis and 3 S. enterica serotype Nitra strains. Single-nucleotide polymorphisms were filtered to identify 4,887 reliable loci that distinguished all isolates from each other. Our whole-genome single-nucleotide polymorphism typing approach was robust for S. enterica Enteritidis subtyping with combined data for different strains from 2 different sequencing platforms. Five major genetic lineages were recognized, which revealed possible patterns of geographic and epidemiologic distribution. Analyses on the population dynamics and evolutionary history estimated that major lineages emerged during the 17th-18th centuries and diversified during the 1920s and 1950s.

Identification of a Salmonella ancillary copper detoxification mechanism by a comparative analysis of the genome-wide transcriptional response to copper and zinc excess.

Copper and zinc are essential metal ions, but toxic in excess. Bacteria have evolved different strategies to control their intracellular concentrations, ensuring proper supply while avoiding toxicity, including the induction of metal-specific as well as non-specific mechanisms. We compared the transcriptional profiles of Salmonella Typhimurium after exposure to either copper or zinc ions in both rich and minimal media. Besides metal-specific regulatory networks many global stress-response pathways react to an excess of either of these metal ions. Copper excess affects both zinc and iron homeostasis by inducing transcription of these metal-specific regulons. In addition to the control of zinc-specific regulons, zinc excess affects the Cpx regulon and the σ(E) envelope-stress responses. Finally, novel metal-specific upregulated genes were detected including a new copper-detoxification pathway that involves the siderophore enterobactin and the outer-membrane protein TolC. This work sheds light onto the transcriptional landscape of Salmonella after copper or zinc overload, and discloses a new mechanism of copper detoxification.

A genome-wide collection of barcoded single-gene deletion mutants in Salmonella enterica serovar Typhimurium.

Genetic screening of pools of mutants can reveal genetic determinants involved in complex biological interactions, processes, and systems. We previously constructed two single-gene deletion resources for Salmonella enterica serovar Typhimurium 14028s in which kanamycin (KanR) and chloramphenicol (CamR) cassettes were used to replace non-essential genes. We have now used lambda-red recombination to convert the antibiotic cassettes in these resources into a tetracycline-resistant (TetR) version where each mutant contains a different 21-base barcode flanked by Illumina Read1 and Read2 primer sequences. A motility assay of a pool of the entire library, followed by a single-tube processing of the bacterial pellet, PCR, and sequencing, was used to verify the performance of the barcoded TetR collection. The new resource is useful for experiments with defined subsets of barcoded mutant strains where biological bottlenecks preclude high numbers of founder bacteria, such as in animal infections. The TetR version of the library will also facilitate the construction of triple mutants by transduction. The resource of 6197 mutants covering 3490 genes is deposited at Biological and Emerging Infections Resources (beiresources.org).

Intraspecies competition among Salmonella enterica isolates in the lettuce leaf apoplast.

Multiple Salmonella enterica serovars and strains have been reported to be able to persist inside the foliar tissue of lettuce (Lactuca sativa L.), potentially resisting washing steps and reaching the consumer. Intraspecies variation of the bacterial pathogen and of the plant host can both significantly affect the outcome of foliar colonization. However, current understanding of the mechanisms underlying this phenomenon is still very limited. In this study, we evaluated the foliar fitness of 14 genetically barcoded S. enterica isolates from 10 different serovars, collected from plant and animal sources. The S. enterica isolates were vacuum-infiltrated individually or in pools into the leaves of three- to four-week-old lettuce plants. To estimate the survival capacity of individual isolates, we enumerated the bacterial populations at 0- and 10- days post-inoculation (DPI) and calculated their net growth. The competition of isolates in the lettuce apoplast was assessed through the determination of the relative abundance change of barcode counts of each isolate within pools during the 10 DPI experimental period. Isolates exhibiting varying apoplast fitness phenotypes were used to evaluate their capacity to grow in metabolites extracted from the lettuce apoplast and to elicit the reactive oxygen species burst immune response. Our study revealed that strains of S. enterica can substantially differ in their ability to survive and compete in a co-inhabited lettuce leaf apoplast. The differential foliar fitness observed among these S. enterica isolates might be explained, in part, by their ability to utilize nutrients available in the apoplast and to evade plant immune responses in this niche.

Peeling back the many layers of competitive exclusion.

Baby chicks administered a fecal transplant from adult chickens are resistant to Salmonella colonization by competitive exclusion. A two-pronged approach was used to investigate the mechanism of this process. First, Salmonella response to an exclusive (Salmonella competitive exclusion product, Aviguard®) or permissive microbial community (chicken cecal contents from colonized birds containing 7.85 Log10Salmonella genomes/gram) was assessed ex vivo using a S. typhimurium reporter strain with fluorescent YFP and CFP gene fusions to rrn and hilA operon, respectively. Second, cecal transcriptome analysis was used to assess the cecal communities' response to Salmonella in chickens with low (≤5.85 Log10 genomes/g) or high (≥6.00 Log10 genomes/g) Salmonella colonization. The ex vivo experiment revealed a reduction in Salmonella growth and hilA expression following co-culture with the exclusive community. The exclusive community also repressed Salmonella's SPI-1 virulence genes and LPS modification, while the anti-virulence/inflammatory gene avrA was upregulated. Salmonella transcriptome analysis revealed significant metabolic disparities in Salmonella grown with the two different communities. Propanediol utilization and vitamin B12 synthesis were central to Salmonella metabolism co-cultured with either community, and mutations in propanediol and vitamin B12 metabolism altered Salmonella growth in the exclusive community. There were significant differences in the cecal community's stress response to Salmonella colonization. Cecal community transcripts indicated that antimicrobials were central to the type of stress response detected in the low Salmonella abundance community, suggesting antagonism involved in Salmonella exclusion. This study indicates complex community interactions that modulate Salmonella metabolism and pathogenic behavior and reduce growth through antagonism may be key to exclusion.