Salmonella enterica Serovar Typhimurium 14028s Genomic Regions Required for Colonization of Lettuce Leaves.

Contamination of edible produce leaves with human bacterial pathogens has been associated with serious disease outbreaks and has become a major public health concern affecting all aspects of the market, from farmers to consumers. While pathogen populations residing on the surface of ready-to-eat produce can be potentially removed through thorough washing, there is no disinfection technology available that effectively eliminates internal bacterial populations. By screening 303 multi-gene deletion (MGD) mutants of Salmonella enterica serovar Typhimurium (STm) 14028s, we were able to identify ten genomic regions that play a role in opening the stomatal pore of lettuce leaves. The major metabolic functions of the deleted regions are associated with sensing the environment, bacterium movement, transport through the bacterial membrane, and biosynthesis of surface appendages. Interestingly, at 21 days post inoculation, seven of these mutants showed increased population titers inside the leaf, two mutants showed similar titers as the wild type bacterium, whereas one mutant with a large deletion that includes the Salmonella pathogenicity island 2 (SPI-2) showed significantly impaired persistence in the leaf apoplast. These findings suggest that not all the genomic regions required for initiation of leaf colonization (i.e., epiphytic behavior and tissue penetration) are essential for continuing bacterial survival as an endophyte. We also observed that mutants lacking either SPI-1 (Mut3) or SPI-2 (Mut9) induce callose deposition levels comparable to those of the wild type STm 14028s; therefore, these islands do not seem to affect this lettuce defense mechanism. However, the growth of Mut9, but not Mut3, was significantly impaired in the leaf apoplastic wash fluid (AWF) suggesting that the STm persistence in the apoplast may be linked to nutrient acquisition capabilities or overall bacterial fitness in this niche, which are dependent on the gene(s) deleted in the Mut9 strain. The genetic basis of STm colonization of leaves investigated in this study provides a foundation from which to develop mitigation tactics to enhance food safety

Glycolytic reprograming in Salmonella counters NOX2-mediated dissipation of ΔpH.

The microbial adaptations to the respiratory burst remain poorly understood, and establishing how the NADPH oxidase (NOX2) kills microbes has proven elusive. Here we demonstrate that NOX2 collapses the ΔpH of intracellular Salmonella Typhimurium. The depolarization experienced by Salmonella undergoing oxidative stress impairs folding of periplasmic proteins. Depolarization in respiring Salmonella mediates intense bactericidal activity of reactive oxygen species (ROS). Salmonella adapts to the challenges oxidative stress imposes on membrane bioenergetics by shifting redox balance to glycolysis and fermentation, thereby diminishing electron flow through the membrane, meeting energetic requirements and anaplerotically generating tricarboxylic acid intermediates. By diverting electrons away from the respiratory chain, glycolysis also enables thiol/disulfide exchange-mediated folding of bacterial cell envelope proteins during periods of oxidative stress. Thus, primordial metabolic pathways, already present in bacteria before aerobic respiration evolved, offer a solution to the stress ROS exert on molecular targets at the bacterial cell envelope

The Fur regulon in anaerobically grown Salmonella enterica sv. Typhimurium: identification of new Fur targets

<p>Abstract</p> <p>Background</p> <p>The Ferric uptake regulator (Fur) is a transcriptional regulator that controls iron homeostasis in bacteria. Although the regulatory role of Fur in <it>Escherichia coli </it>is well characterized, most of the studies were conducted under routine culture conditions, i.e., in ambient oxygen concentration. To reveal potentially novel aspects of the Fur regulon in <it>Salmonella enterica </it>serovar Typhimurium under oxygen conditions similar to that encountered in the host, we compared the transcriptional profiles of the virulent wild-type strain (ATCC 14028s) and its isogenic Δ<it>fur </it>strain under anaerobic conditions.</p> <p>Results</p> <p>Microarray analysis of anaerobically grown Δ<it>fur S</it>. Typhimurium identified 298 differentially expressed genes. Expression of several genes controlled by Fnr and NsrR appeared to be also dependent on Fur. Furthermore, Fur was required for the activity of the cytoplasmic superoxide disumutases (MnSOD and FeSOD). The regulation of FeSOD gene, <it>sodB</it>, occurred via small RNAs (i.e., the <it>ryhB </it>homologs, <it>rfrA </it>and <it>rfrB</it>) with the aid of the RNA chaperone Hfq. The transcription of <it>sodA </it>was increased in Δ<it>fur; </it>however, the enzyme was inactive due to the incorporation of iron instead of manganese in SodA. Additionally, in Δ<it>fur</it>, the expression of the gene coding for the ferritin-like protein (<it>ftnB</it>) was down-regulated, while the transcription of the gene coding for the nitric oxide (NO^·) detoxifying flavohemoglobin (<it>hmpA</it>) was up-regulated. The promoters of <it>ftnB </it>and <it>hmpA </it>do not contain recognized Fur binding motifs, which indicated their probable indirect regulation by Fur. However, Fur activation of <it>ftnB </it>was independent of Fnr. In addition, the expression of the gene coding for the histone-like protein, H-NS (<it>hns</it>) was increased in Δ<it>fur</it>. This may explain the observed down-regulation of the <it>tdc </it>operon, responsible for the anaerobic degradation of threonine, and <it>ftnB </it>in Δ<it>fur</it>.</p> <p>Conclusions</p> <p>This study determined that Fur is a positive factor in <it>ftnB </it>regulation, while serving to repress the expression of <it>hmpA</it>. Furthermore, Fur is required for the proper expression and activation of the antioxidant enzymes, FeSOD and MnSOD. Finally, this work identified twenty-six new targets of Fur regulation, and demonstrates that H-NS repressed genes are down-regulated in Δ<it>fur</it>.</p

SpoT Induces Intracellular Salmonella Virulence Programs in the Phagosome.

Guanosine tetraphosphate (ppGpp) and guanosine pentaphosphate (pppGpp), together named (p)ppGpp, regulate diverse aspects of Salmonella pathogenesis, including synthesis of nutrients, resistance to inflammatory mediators, and expression of secretion systems. In Salmonella, these nucleotide alarmones are generated by the synthetase activities of RelA and SpoT proteins. In addition, the (p)ppGpp hydrolase activity of the bifunctional SpoT protein is essential to preserve cell viability. The contribution of SpoT to physiology and pathogenesis has proven elusive in organisms such as Salmonella, because the hydrolytic activity of this RelA and SpoT homologue (RSH) is vital to prevent inhibitory effects of (p)ppGpp produced by a functional RelA. Here, we describe the biochemical and functional characterization of a spoT-Δctd mutant Salmonella strain encoding a SpoT protein that lacks the C-terminal regulatory elements collectively referred to as "ctd." Salmonella expressing the spoT-Δctd variant hydrolyzes (p)ppGpp with similar kinetics to those of wild-type bacteria, but it is defective at synthesizing (p)ppGpp in response to acidic pH. Salmonella spoT-Δctd mutants have virtually normal adaptations to nutritional, nitrosative, and oxidative stresses, but poorly induce metal cation uptake systems and Salmonella pathogenicity island 2 (SPI-2) genes in response to the acidic pH of the phagosome. Importantly, spoT-Δctd mutant Salmonella replicates poorly intracellularly and is attenuated in a murine model of acute salmonellosis. Collectively, these investigations indicate that (p)ppGpp synthesized by SpoT serves a unique function in the adaptation of Salmonella to the intracellular environment of host phagocytes that cannot be compensated by the presence of a functional RelA.IMPORTANCE Pathogenic bacteria experience nutritional challenges during colonization and infection of mammalian hosts. Binding of the alarmone nucleotide guanosine tetraphosphate (ppGpp) to RNA polymerase coordinates metabolic adaptations and virulence gene transcription, increasing the fitness of diverse Gram-positive and Gram-negative bacteria as well as that of actinomycetes. Gammaproteobacteria such as Salmonella synthesize ppGpp by the combined activities of the closely related RelA and SpoT synthetases. Due to its profound inhibitory effects on growth, ppGpp must be removed; in Salmonella, this process is catalyzed by the vital hydrolytic activity of the bifunctional SpoT protein. Because SpoT hydrolase activity is essential in cells expressing a functional RelA, we have a very limited understanding of unique roles these two synthetases may assume during interactions of bacterial pathogens with their hosts. We describe here a SpoT truncation mutant that lacks ppGpp synthetase activity and all C-terminal regulatory domains but retains excellent hydrolase activity. Our studies of this mutant reveal that SpoT uniquely senses the acidification of phagosomes, inducing virulence programs that increase Salmonella fitness in an acute model of infection. Our investigations indicate that the coexistence of RelA/SpoT homologues in a bacterial cell is driven by the need to mount a stringent response to a myriad of physiological and host-specific signatures

Hypochlorous acid and hydrogen peroxide-induced negative regulation of Salmonella enterica serovar Typhimurium ompW by the response regulator ArcA

<p>Abstract</p> <p>Background</p> <p>Hydrogen peroxide (H₂O₂) and hypochlorous acid (HOCl) are reactive oxygen species that are part of the oxidative burst encountered by <it>Salmonella enterica</it> serovar Typhimurium (<it>S</it>. Typhimurium) upon internalization by phagocytic cells. In order to survive, bacteria must sense these signals and modulate gene expression. Growing evidence indicates that the ArcAB two component system plays a role in the resistance to reactive oxygen species. We investigated the influx of H₂O₂ and HOCl through OmpW and the role of ArcAB in modulating its expression after exposure to both toxic compounds in <it>S.</it> Typhimurium.</p> <p>Results</p> <p>H₂O₂ and HOCl influx was determined both <it>in vitro</it> and <it>in vivo</it>. A <it>S</it>. Typhimurium <it>ompW</it> mutant strain (∆<it>ompW</it>) exposed to sub-lethal levels of H₂O₂ and HOCl showed a decreased influx of both compounds as compared to a wild type strain. Further evidence of H₂O₂ and HOCl diffusion through OmpW was obtained by using reconstituted proteoliposomes. We hypothesized that <it>ompW</it> expression should be negatively regulated upon exposure to H₂O₂ and HOCl to better exclude these compounds from the cell. As expected, qRT-PCR showed a negative regulation in a wild type strain treated with sub-lethal concentrations of these compounds. A bioinformatic analysis in search for potential negative regulators predicted the presence of three ArcA binding sites at the <it>ompW</it> promoter region. By electrophoretic mobility shift assay (EMSA) and using transcriptional fusions we demonstrated an interaction between ArcA and one site at the <it>ompW</it> promoter region. Moreover, qRT-PCR showed that the negative regulation observed in the wild type strain was lost in an <it>arcA</it> and in <it>arcB</it> mutant strains.</p> <p>Conclusions</p> <p>OmpW allows the influx of H₂O₂ and HOCl and is negatively regulated by ArcA by direct interaction with the <it>ompW</it> promoter region upon exposure to both toxic compounds.</p

Genome-wide analysis of the PreA/PreB (QseB/QseC) regulon of Salmonella enterica serovar Typhimurium

<p>Abstract</p> <p>Background</p> <p>The <it>Salmonella </it>PreA/PreB two-component system (TCS) is an ortholog of the QseBC TCS of <it>Escherichia coli</it>. In both <it>Salmonella </it>and <it>E. coli</it>, this system has been shown to affect motility and virulence in response to quorum-sensing and hormonal signals, and to affect the transcription of the <it>Salmonella enterica </it>serovar Typhimurium (<it>S</it>. Typhimurium) <it>pmrAB </it>operon, which encodes an important virulence-associated TCS.</p> <p>Results</p> <p>To determine the PreA/PreB regulon in <it>S</it>. Typhimurium, we performed DNA microarrays comparing the wild type strain and various <it>preA </it>and/or <it>preB </it>mutants in the presence of ectopically expressed <it>preA </it>(<it>qseB</it>). These data confirmed our previous findings of the negative effect of PreB on PreA gene regulation and identified candidate PreA-regulated genes. A proportion of the activated loci were previously identified as PmrA-activated genes (<it>yibD</it>, <it>pmrAB</it>, <it>cptA</it>, etc.) or were genes located in the local region around <it>preA</it>, including the <it>preAB </it>operon. The transcriptional units were defined in this local region by RT-PCR, suggesting three PreA activated operons composed of <it>preA-preB</it>, <it>mdaB-ygiN</it>, and <it>ygiW</it>-STM3175. Several putative virulence-related phenotypes were examined for <it>preAB </it>mutants, resulting in the observation of a host cell invasion and slight virulence defect of a <it>preAB </it>mutant. Contrary to previous reports on this TCS, we were unable to show a PreA/PreB-dependent effect of the quorum-sensing signal AI-2 or of epinephrine on <it>S</it>. Typhimurium with regard to bacterial motility.</p> <p>Conclusion</p> <p>This work further characterizes this unorthadox OmpR/EnvZ class TCS and provides novel candidate regulated genes for further study. This first in-depth study of the PreA/PreB regulatory system phenotypes and regulation suggests significant comparative differences to the reported function of the orthologous QseB/QseC in <it>E. coli</it>.</p

Gene set analyses for interpreting microarray experiments on prokaryotic organisms

Background Despite the widespread usage of DNA microarrays, questions remain about how best to interpret the wealth of gene-by-gene transcriptional levels that they measure. Recently, methods have been proposed which use biologically defined sets of genes in interpretation, instead of examining results gene-by-gene. Despite a serious limitation, a method based on Fisher\u27s exact test remains one of the few plausible options for gene set analysis when an experiment has few replicates, as is typically the case for prokaryotes. Results We extend five methods of gene set analysis from use on experiments with multiple replicates, for use on experiments with few replicates. We then use simulated and real data to compare these methods with each other and with the Fisher\u27s exact test (FET) method. As a result of the simulation we find that a method named MAXMEAN-NR, maintains the nominal rate of false positive findings (type I error rate) while offering good statistical power and robustness to a variety of gene set distributions for set sizes of at least 10. Other methods (ABSSUM-NR or SUM-NR) are shown to be powerful for set sizes less than 10. Analysis of three sets of experimental data shows similar results. Furthermore, the MAXMEAN-NR method is shown to be able to detect biologically relevant sets as significant, when other methods (including FET) cannot. We also find that the popular GSEA-NR method performs poorly when compared to MAXMEAN-NR. Conclusion MAXMEAN-NR is a method of gene set analysis for experiments with few replicates, as is common for prokaryotes. Results of simulation and real data analysis suggest that the MAXMEAN-NR method offers increased robustness and biological relevance of findings as compared to FET and other methods, while maintaining the nominal type I error rate

Analysis of the ArcA regulon in anaerobically grown Salmonella enterica sv. Typhimurium

<p>Abstract</p> <p>Background</p> <p><it>Salmonella enterica </it>serovar Typhimurium (<it>S</it>. Typhimurium) is a Gram-negative pathogen that must successfully adapt to the broad fluctuations in the concentration of dissolved dioxygen encountered in the host. In <it>Escherichia coli</it>, ArcA (Aerobic Respiratory Control) helps the cells to sense and respond to the presence of dioxygen. The global role of ArcA in <it>E. coli </it>is well characterized; however, little is known about its role in anaerobically grown <it>S</it>. Typhimurium.</p> <p>Results</p> <p>We compared the transcriptional profiles of the virulent wild-type (WT) strain (ATCC 14028s) and its isogenic <it>arcA </it>mutant grown under anaerobic conditions. We found that ArcA directly or indirectly regulates 392 genes (8.5% of the genome); of these, 138 genes are poorly characterized. Regulation by ArcA in <it>S</it>. Typhimurium is similar, but distinct from that in <it>E</it>. <it>coli</it>. Thus, genes/operons involved in core metabolic pathways (e.g., succinyl-CoA, fatty acid degradation, cytochrome oxidase complexes, flagellar biosynthesis, motility, and chemotaxis) were regulated similarly in the two organisms. However, genes/operons present in both organisms, but regulated differently by ArcA in <it>S</it>. Typhimurium included those coding for ethanolamine utilization, lactate transport and metabolism, and succinate dehydrogenases. <it>Salmonella</it>-specific genes/operons regulated by ArcA included those required for propanediol utilization, flagellar genes (<it>mcpAC</it>, <it>cheV</it>), Gifsy-1 prophage genes, and three SPI-3 genes (<it>mgtBC</it>, <it>slsA</it>, STM3784). In agreement with our microarray data, the <it>arcA </it>mutant was non-motile, lacked flagella, and was as virulent in mice as the WT. Additionally, we identified a set of 120 genes whose regulation was shared with the anaerobic redox regulator, Fnr.</p> <p>Conclusion(s)</p> <p>We have identified the ArcA regulon in anaerobically grown <it>S</it>. Typhimurium. Our results demonstrated that in <it>S</it>. Typhimurium, ArcA serves as a transcriptional regulator coordinating cellular metabolism, flagella biosynthesis, and motility. Furthermore, ArcA and Fnr share in the regulation of 120 <it>S</it>. Typhimurium genes.</p