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<p>Host-associated microbial communities play central roles in health and stability of agricultural and natural ecosystems. This project focuses on three models: (1) Salmonella-tomato interactions; (2) Medicago-Rhizobium symbiosis; and (3) coral-associated microbial communities. These three system are directly relevant to the state's agricultural production and natural ecosystems. Further characterization of interactions in these three models will also offer an opportunity to define evolutionarily conserved mechanisms by which diverse host-associated microbial communities are structured and maintained.All three of the models on which my research focuses have important impacts on the economy of Florida. The number of produce-associated outbreaks of salmonellosis is on the rise and is comparable to the outbreaks associated with the consumption of meats and poultry. Fourteen major foodborne pathogens cause damage to the US economy at a rate of $14.1 billion per year. Conservatively, produce-related outbreaks account for 18.1 and 16.6 % of all E. coli O157:H7 and Salmonella outbreaks respectively. The outbreaks of foodborne illness linked to the consumption of fruits and vegetables led to the ranking of produce as the 4th riskiest food, estimated to cause over 1 million illnesses annually. Based on USDA ERS data, tomato is the most consumed fresh vegetable: in 2009, Americans purchased 18.7 lbs of fresh tomatoes per capita. Florida ranks first (or second, depending on the metric) nationally in the acreage, production, and value of fresh market tomato. In the 2010-2011 growing season, 30,700 acres were under cultivation for the fresh tomato maret. Fresh market tomatoes comprise about 40% of Florida's fresh market vegetable cash receipts. About 33,000 workers are directly involved in tomato production and harvest each year. The issue of food safety represent one of the greatest threats to sustainability and profitability of Florida tomato industry. Based on USDA ERS data, following the June-August 2008 outbreak of salmonellosis caused by S. enterica sv. St. Paul (which was initially wrongly blamed on tomatoes from Florida) the price of tomatoes at the point of first sale dropped from 56.8 cents/lb in June to 25.6 cents/lb in August. This nearly obliterated Florida tomato industry. Promoting safety of produce from Florida will help avoid future attribution errors and is seen as one of the major components of increasing competitiveness of Florida tomatoes on international markets. The goal of this project is to contribute to the development of the comprehensive approach to improving microbiological safety of tomatoes through cultivar selection, minor changes in the production practices combined with education.Based on USDA ERS data, Medicago spp are important pasture legumes in temperate and arid climates. M. sativa (alalfa) alone accounts for 60% of all hay produced in the U.S., and is the fourth most important crop in the United States both in terms of acreage and production value. M. truncatula (barrel medic) and Melilotus spp (sweet clover) are gaining recognition as promising annual pasture legumes, especially in arid regions, or as a winter cover crop/green mulch in warmer climates or as emergency annual hay crops. The symbiosis with Sinorhizobium contributes direct, timely and self-regulating amounts of nitrogen needed for growth of Medicago spp in the field. Better understanding of the steps required to establish, fine-tune and maintain the symbiosis should contribute to the development of superior inoculum strains or to management practices that optimize symbiotic benefits.In Florida, coral reefs contribute at least $2.9 billion to local economies annually, and provide employment for approximately 39,000 people. Florida reefs are a major tourist attraction: visitors spend $460-1,087/person/trip. Florida coral reefs provide habitat for over 4,000 fish species and support commercial and recreational fishing industries which generate ~ $60 million. The capitalized reef user value in southeast Florida is $8.5 billion.OBJECTIVESThe goal of this project is to define mechanisms of interactions within host-associated microbial communities, focusing specifically on the interactions of co-evolved commensals/symbionts and opportunistic pathogens. Even though interest in microbiome research has exploded recently, little is still known about the mechanisms by which host-associated microbiota contribute to the health of any host. </p><p>Objective 1. The overall goal of this objective is to promote safety of vegetables by assessing how intrinsic crop properties and agronomic practices affect persistence of human pathogens in the production environment and also affect vulnerability of crops to accidental contamination post-harvest. A better understanding of these parameters and their interactions will promote microbiological safety of produce. </p><p>Objective 2. We hypothesize that bacterial small regulatory RNA and post-transcriptional changes in gene expression mediated by the RNA-binding protein Hfq play central roles in maintaining the productive symbiotic relationship. Therefore, the goal of this objective is to characterize contribution of Hfq to the symbiotic regulation of rhizobial small regulatory RNA genes. Levels of srRNA gene expression within nodules will be characterized by qRT-PCR and expression patterns determined with reporter fusions. In vitro experiments will focus on comparing stability of complexes formed by Hfq with target bacterial mRNA and bacterial srRNA, and will determine the kinetics of each molecule's binding. Eventually, we plan to characterize the entire Symbiotic Hfq Regulon by co-immunoprecipitation of the non-coding and mRNA bound to Hfq within bacteroids. </p><p>Objective 3. A recognition of the unique roles of the eukaryotes' microbial associates in evolution, development and responses to stressors helped define the term "meta-organism" or a "holobiont" as a complex multi-organismal symbiosis. Long-lived colonial invertebrates, such as corals, which lack adaptive immunity are thought to rely extensively on their associated microbiota for nutrient acquisition and defense, and the composition of coral-associated microbial communities correlates with the health status of the holobiont. We hypothesize that the co-evolved "primary" commensals of corals provide functions needed for the fitness of the entire holobiont and its responses to environmental stressors. We further hypothesize that, under some conditions, commensals either escape regulation by the host and themselves contribute to the appearance of the disease signs or are outcompeted by opportunists which invade the holobiont. This hypothesis is based on the observations that shifts in the composition of the coral-associated microbiome correlate with environmental stress and disease and that members of the coral microbiome produce substances that inhibit virulence behaviors in pathogens (e.g., surface colonization, attachment, enzymes involved in host mucus degradation). Therefore, the third objective of this proposal is to define the effect of environmental drivers on the physiology and biochemistry of the holobiont and to evaluate resilience of the host when functionality of the associated microbiome is affected by environmental stressors (e.g. increased temperature). </p>
Over the last decade, scientists have come to realize that interactions between higher organisms and their microbial associates are central to the health of plants and animals. Most of these discoveries were made in animal models of human diseases. Significantly less is known about the role of microbes in health and stability of aquatic ecosystems and in ensuring microbiological safety of foods. Therefore, with this project, we will focus on the characterization of food safety outcomes in Salmonella-tomato interactions; the roles of novel types of signal interactions in Medicago-Rhizobium symbiosis; and promoting health of coral-associated microbial communities. In Florida, addressing these three questions is important as they represent pressing needs of the state, in which marine ecosystems are critical for tourism and fisheries; vegetable production has been affected by outbreaks of foodborne illness and pasture-based beef production requires wise investment of resources into forages. We anticipate that upon completion of this work, safer crop production practices will be identified, opportunities to improve health of marine ecosystems will be defined and the ability of forage legumes (such as alfalfa and medics) to form more efficient symbioses will be described. In process, at least two cohorts of post-doctoral scientists will be better prepared for independent careers, graduate and undergraduate students will gain valuable research experience.