BackgroundRecent 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 FindingsTo 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/SignificanceThis 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.

Interactions within microbial communities associated with marine holobionts contribute importantly to the health of these symbiotic organisms formed by invertebrates, dinoflagellates and bacteria. However, mechanisms that control invertebrate-associated microbiota are not yet fully understood. Hydrophobic compounds that were isolated from surfaces of asymptomatic corals inhibited biofilm formation by the white pox pathogen Serratia marcescens PDL100, indicating that signals capable of affecting the associated microbiota are produced in situ. However, neither the origin nor structures of these signals are currently known. A functional survey of bacteria recovered from coral mucus and from cultures of the dinoflagellate Symbiodinium spp. revealed that they could alter swarming and biofilm formation in S. marcescens. As swarming and biofilm formation are inversely regulated, the ability of some native a-proteobacteria to affect both behaviors suggests that the a-proteobacterial signal(s) target a global regulatory switch controlling the behaviors in the pathogen. Isolates of Marinobacter sp. inhibited both biofilm formation and swarming in S. marcescens PDL100, without affecting growth of the coral pathogen, indicative of the production of multiple inhibitors, likely targeting lower level regulatory genes or functions. A multi-species cocktail containing these strains inhibited progression of a disease caused by S. marcescens in a model polyp Aiptasia pallida. An a-proteobacterial isolate 44B9 had a similar effect. Even though B4% of native holobiont-associated bacteria produced compounds capable of triggering responses in well-characterized N-acyl homoserine lactone (AHL) biosensors, there was no strong correlation between the production of AHL-like signals and disruption of biofilms or swarming in S. marcescens.

Orthologues of the Salmonella enterica serovar Typhimurium (S. typhimurium) BarA/SirA twocomponent system are important for biofilm formation and virulence in many c-Proteobacteria. In S. typhimurium, SirA activates the csrB and csrC carbon storage regulatory RNAs and the virulence gene regulators hilA and hilC. The regulatory RNAs antagonize the activity of the CsrA protein, allowing translation of those same virulence genes, and inhibiting the translation of flagellar genes. In this report, it was determined that SirA and the Csr system also control the fim operon that encodes type 1 fimbriae. sirA orthologues in other bacterial species, and the fim operon of S. typhimurium, are known to play a role in biofilm formation; therefore, all members of the S. typhimurium sirA regulon were tested for in vitro biofilm production. A sirA mutant, a csrB csrC double mutant, and a fimI mutant, were all defective in biofilm formation. Conversely, inactivation of flhDC increased biofilm formation. Therefore, SirA activates csrB, csrC and the fim operon to promote biofilm formation. In turn, csrB and csrC promote the translation of the fim operon, while at the same time inhibiting the translation of flagella, which are inhibitory to biofilm formation.

Extracts of several cyanobacterial species collected from different marine and estuarine locations predominately in Florida (USA), with one sample each from Belize and Oman, were screened for their ability to disrupt quorum sensing (QS) in the reporter strain Chromobacterium violaceum CV017. Inhibitory activities were detected in the ethyl acetate : methanol (1:1) extracts of several Lyngbya spp., and extracts of Lyngbya majuscula contained the strongest QS inhibitory activities. Extracts of L. majuscula from the Indian River Lagoon, FL, USA, were further purified by bioassay-guided fractionation. The antibiotic malyngolide (MAL) was identified as a QS inhibitor. Activity of MAL was investigated using N-acyl homoserine lactone (AHL) reporters based on the LasR receptor of Pseudomonas aeruginosa. MAL at concentrations ranging from 3.57 µM to 57 µM (EC50  = 12.2 ± 1.6 µM) inhibited responses of the LasR reporters without affecting bacterial growth. MAL inhibited (EC50  =  10.6 ± 1.8 µM) Las QS-dependent production of elastase by P. aeruginosa PAO1. We propose that this QS inhibitor plays a role in controlling interactions of heterotrophic bacteria associated with the cyanobacterium L. majuscula.

The outcome of the interactions between native commensal microorganisms and opportunistic pathogens is crucial to the health of the coral holobiont. During the establishment within the coral surface mucus layer, opportunistic pathogens, including a white pox pathogen Serratia marcescens PDL100, compete with native bacteria for available nutrients. Both commensals and pathogens employ glycosidases and N-acetyl-glucosaminidase to utilize components of coral mucus. This study tested the hypothesis that specific glycosidases were critical for the growth of S. marcescens on mucus and that their inhibition by native coral microbiota reduces fitness of the pathogen. Consistent with this hypothesis, a S. marcescens transposon mutant with reduced glycosidase and N-acetyl-glucosaminidase activities was unable to compete with the wild type on the mucus of the host coral Acropora palmata, although it was at least as competitive as the wild type on a minimal medium with glycerol and casamino acids. Virulence of the mutant was modestly reduced in the Aiptasia model. A survey revealed that B8% of culturable coral commensal bacteria have the ability to inhibit glycosidases in the pathogen. A small molecular weight, ethanol-soluble substance(s) produced by the coral commensal Exiguobacterium sp. was capable of the inhibition of the induction of catabolic enzymes in S. marcescens. This inhibition was in part responsible for the 10-100-fold reduction in the ability of the pathogen to grow on coral mucus. These results provide insight into potential mechanisms of commensal interference with early colonization and infection behaviors in opportunistic pathogens and highlight an important function for the native microbiota in coral health.

In many pathogenic bacteria, quorum sensing (QS) controls expression of genes that are involved in virulence, production and resistance to antibiotics, formation and maintenance of microbial multicellular consortia on biotic and abiotic surfaces of medical and industrial importance. N-acyl homoserine lactones (AHL) are the best characterized quorum sensing signals in Gram-negative bacteria. Interference with AHL-mediated QS, therefore, is considered an attractive strategy for controlling virulence in pathogens. The search for AHL signals and their mimics has been facilitated by the development of sensitive bioassays, in which QS reporters luminesce in response to AHL signals. These bioassays have already led to the identification of dozens of compounds with QS modulating activities. The characterization of the mode of action of QS signals and their mimics requires follow-up biochemical studies. Here, we describe a set of luminescent reporters, which could be used in high, medium or low throughput format, for the discovery and validation of agonists or antagonists of the Las QS system of Pseudomonas aeruginosa. These nearly isogenic reporters contain truncations or point mutations in the AHL binding domain of the AHL receptor LasR, as well as mutations in the promoter for the gene encoding LasI AHL synthase. We also developed reporters for documenting the regulation of lasI and lasR promoters. The use of these reporters significantly streamlines identification and characterization of the Las QS signal agonists and antagonists prior to biochemical experiments. To test the usefulness of these reporters, we carried out bioassays with patulin, a known inhibitor of Las QS.

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Coral reefs are under increasing stress caused by global and local environmental changes, which are thought to increase the susceptibility of corals to opportunistic pathogens. In the absence of an easily culturable model animal, the understanding of the mechanisms of disease progression in corals remains fairly limited. In the present study, we tested the susceptibility of the tropical sea anemone Aiptasia pallida to an opportunistic coral pathogen (Serratia marcescens). A. pallida was susceptible to S. marcescens PDL100 and responded to this opportunistic coral pathogen with darkening of the tissues and retraction of tentacles, followed by complete disintegration of polyp tissues. Histological observations revealed loss of zooxanthellae and structural changes in eosinophilic granular cells in response to pathogen infection. A screen of S. marcescens mutants identified a motility and tetrathionate reductase mutants as defective in virulence in the A. pallida infection model. In co-infections with the wild-type strain, the tetrathionate reductase mutant was less fit within the surface mucopolysaccharide layer of the host coral Acropora palmata.