Jason T. Noel

Specific Responses of Salmonella enterica to Tomato Varieties and Fruit Ripeness Identified by In Vivo Expression Technology

Background Recent outbreaks of vegetable-associated gastroenteritis suggest that enteric pathogens colonize, multiply and persist in plants for extended periods of time, eventually infecting people. Genetic and physiological pathways, by which enterics colonize plants, are still poorly understood. Methodology/Principal Findings To better understand interactions between Salmonella enterica sv. Typhimurium and tomatoes, a gfp-tagged Salmonella promoter library was screened inside red ripe fruits. Fifty-one unique constructs that were potentially differentially regulated in tomato relative to in vitro growth were identified. The expression of a subset of these promoters was tested in planta using recombinase-based in vivo expression technology (RIVET) and fitness of the corresponding mutants was tested. Gene expression in Salmonella was affected by fruit maturity and tomato cultivar. A putative fadH promoter was upregulated most strongly in immature tomatoes. Expression of the fadH construct depended on the presence of linoleic acid, which is consistent with the reduced accumulation of this compound in mature tomato fruits. The cysB construct was activated in the fruit of cv. Hawaii 7997 (resistant to a race of Ralstonia solanacearum) more strongly than in the universally susceptible tomato cv. Bonny Best. Known Salmonella motility and animal virulence genes (hilA, flhDC, fliF and those encoded on the pSLT virulence plasmid) did not contribute significantly to fitness of the bacteria inside tomatoes, even though deletions of sirA and motA modestly increased fitness of Salmonella inside tomatoes. Conclusions/Significance This study reveals the genetic basis of the interactions of Salmonella with plant hosts. Salmonella relies on a distinct set of metabolic and regulatory genes, which are differentially regulated in planta in response to host genotype and fruit maturity. This enteric pathogen colonizes tissues of tomatoes differently than plant pathogens, and relies little on its animal virulence genes for persistence within the fruit.

Salmonella SdiA Recognizes N-acyl Homoserine Lactone Signals from Pectobacterium carotovorum in Vitro, but Not in a Bacterial Soft Rot

Genomes of Salmonella enterica isolates, including those linked to outbreaks of produce-associated gastroenteritis, contain sdiA, which encodes a receptor of N-acyl homoserine lactones (AHL). AHL are the quorum-sensing signals used by bacteria to coordinately regulate gene expression within -their populations. Because S. enterica does not produce its own AHL, SdiA is hypothesized to function in the interspecies cross-talk with AHL-producing bacteria. Under laboratory conditions, S. enterica responded to AHL from phytobacteria by upregulating expression of srgE. AHL-dependent expression of srgE required a functional sdiA. Essentially, no sdiA-dependent resolution of the srgE recombinase-based (RIVET) reporter was observed inside a soft rot formed on a tomato by an AHL-producing strain of Pectobacterium carotovorum. The results of the control experiments suggest that sdiA is not expressed inside tomato, pepper, green onion, or carrot affected by the soft rot, and the lack of sdiA expression in planta prevents Salmonella spp. from responding to AHL. Despite its inability to detect and respond to AHL during colonization of soft rots, S. enterica reached higher final cell numbers inside a tomato soft rot compared with its growth in intact tomato fruit. The synergistic effect was the strongest under the conditions that are typical for the Florida fall/winter production season.

Factors That Affect Proliferation of Salmonella in Tomatoes Post-Harvest: The Roles of Seasonal Effects, Irrigation Regime, Crop and Pathogen Genotype

Main Objectives Fresh fruits and vegetables become increasingly recognized as vehicles of human salmonellosis. Physiological, ecological, and environmental factors are all thought to contribute to the ability of Salmonella to colonize fruits and vegetables pre- and post-harvest. The goal of this study was to test how irrigation levels, fruit water congestion, crop and pathogen genotypes affect the ability of Salmonella to multiply in tomatoes post-harvest. Experimental Design Fruits from three tomato varieties, grown over three production seasons in two Florida locations, were infected with seven strains of Salmonella and their ability to multiply post-harvest in field-grown tomatoes was tested. The field experiments were set up as a two-factor factorial split plot experiment, with the whole-plot treatments arranged in a randomized complete-block design. The irrigation treatment (at three levels) was the whole-plot factor, and the split-plot factor was tomato variety, with three levels. The significance of the main, two-way, and three-way interaction effects was tested using the (type III) F-tests for fixed effects. Mean separation for each significant fixed effect in the model was performed using Tukey’s multiple comparison testing procedure. Most Important Discoveries and Significance The irrigation regime per se did not affect susceptibility of the crop to post-harvest proliferation of Salmonella. However, Salmonella grew significantly better in water-congested tissues of green tomatoes. Tomato maturity and genotype, Salmonella genotype, and inter-seasonal differences were the strongest factors affecting proliferation. Red ripe tomatoes were significantly and consistently more conducive to proliferation of Salmonella. Tomatoes harvested in the driest, sunniest season were the most conducive to post-harvest proliferation of the pathogen. Statistically significant interactions between production conditions affected post-harvest susceptibility of the crop to the pathogen. UV irradiation of tomatoes post-harvest promoted Salmonella growth.

Differential Regulation of Salmonella Typhimurium Genes Involved in O-Antigen Capsule Production and Their Role in Persistence Within Tomato Fruit

Enteric pathogens, including non-typhoidal Salmonella spp. and enterovirulent Escherichia coli, are capable of persisting and multiplying within plants. Yet, little is still known about the mechanisms of these interactions. This study identified the Salmonella yihT gene (involved in synthesis of the O-antigen capsule) as contributing to persistence in immature tomato fruit. Deletion of yihT reduced competitive fitness of S. enterica sv. Typhimurium in green (but not ripe, regardless of color) tomato fruit by approximately 3 logs. The yihT recombinase-based in vivo expression technology (RIVET) reporter was strongly activated in unripe tomato fruit, and fitness of the mutant inversely correlated with the level of the yihT gene expression. Expression of yihT in mature tomato fruit was low, and yihT did not affect competitive fitness within mature fruit. To better understand the molecular basis of the phenotype, behaviors of the yihT RIVET reporter and the yihT mutant were tested in tomato fruit defective in ethylene signaling. These experiments suggest a role for functional ethylene-mediated signaling in the persistence of Salmonella spp. within tomato fruit. Furthermore, jasmonic acid and its precursors strongly reduced expression of yihT.

The diffusive influx and carrier efflux have a strong effect on the bistability of the lac operon in Escherichia coli

In the presence of gratuitous inducers, the lac operon of Escherichia coli exhibits bistability. Most models in the literature assume that the inducer enters the cell via the carrier (permease), and exits by a diffusion-like process. The diffusive influx and carrier efflux are neglected. However, analysis of the data shows that in non-induced cells, the diffusive influx is comparable to the carrier influx, and in induced cells, the carrier efflux is comparable to the diffusive efflux. Since bistability entails the coexistence of steady states corresponding to both non-induced and induced cells, neither one of these fluxes can be ignored. We present a model accounting for both fluxes, and show that: (1) The thresholds (i.e., the extracellular inducer levels at which transcription turns on or off) are profoundly affected by both fluxes. The diffusive influx reduces the on threshold, and eliminates irreversible bistability, a phenomenon that is inconsistent with data. The carrier efflux increases the off threshold, and abolishes bistability at large permease activities, a conclusion that can be tested experimentally. (2) The thresholds are well approximated by simple analytical expressions obtained by considering two limiting cases (no carrier efflux and no diffusive influx). (3) The simulations are in good agreement with the data for isopropyl thiogalactoside (IPTG), but somewhat discrepant with respect to the data for thiomethyl galactoside (TMG). We discuss the potential sources of the discrepancy.

Ethylene signalling affects susceptibility of tomatoes to Salmonella

Fresh fruits and vegetables are increasingly recognized as important reservoirs of human pathogens, and therefore, significant attention has been directed recently to understanding mechanisms of the interactions between plants and enterics, like Salmonella. A screen of tomato cultivars for their susceptibility to Salmonella revealed significant differences in the ability of this human pathogen to multiply within fruits; expression of the Salmonella genes (cysB, agfB, fadH) involved in the interactions with tomatoes depended on the tomato genotype and maturity stage. Proliferation of Salmonella was strongly reduced in the tomato mutants with defects in ethylene synthesis, perception and signal transduction. While mutation in the ripening‐related ethylene receptor Nr resulted only in a modest reduction in Salmonella numbers within tomatoes, strong inhibition of the Salmonella proliferation was observed in rin and nor tomato mutants. RIN and NOR are regulators of ethylene synthesis and ripening. A commercial tomato variety heterozygous for rin was less susceptible to Salmonella under the greenhouse conditions but not when tested in the field over three production seasons.

Effect of the irrigation regime on the susceptibility of pepper and tomato to post-harvest proliferation of Salmonella enterica.

Raw produce is increasingly recognized as a vehicle of human gastroenteritis. Non-typhoidal Salmonella, pathogenic Escherichia coli, and other human pathogens have been isolated from fruits and vegetables in the field and in the marketplace, which led to the hypothesis that these microbes can use plants as alternate hosts. However, environmental and physiological factors that facilitate persistence of these bacteria in the crop production environment and make produce more vulnerable to post-harvest contamination have not been fully delineated. This study tested the effect of irrigation regimes on the susceptibility of peppers and tomatoes to post-harvest proliferation of Salmonella. The experiments were carried out over three experimental seasons in two locations using seven strains of Salmonella. The irrigation regime per se did not affect susceptibility of tomatoes and peppers to post-harvest proliferation of Salmonella; however, in some of the seasons, irrigation regime-dependent differences were observed. Red peppers and tomatoes were more conducive to proliferation of Salmonella than green fruit in all seasons. Inter-seasonal differences were the strongest factors affecting proliferation of Salmonella in peppers.