Final Report Summary - BACTOFUNGICIDE (Genetics and quorum sensing regulation of antifungal and antioomycete biocontrol in plant-associated enterobacteria) Crop losses due to emerging and re-emerging plant pathogens are currently estimated in around 20% of the major crops worldwide and continue to challenge our ability to safeguard plant growth and health worldwide. Therefore, investigation addressed to the development of an environmentally sound and sustainable crop production is crucial – mainly when the use of chemical pesticides is highly restricted. With this in mind, it is not surprising that the use of beneficial microorganisms (biopesticides) is one of the most promising strategies for the (bio)control of plant diseases. In fact, the overall growth in the sales of biopesticides is increasing at an annual rate of 10% worldwide. The main goals in this Research Project, “Bactofungicide”, with Dr. Miguel A. Matilla as main researcher, were to investigate the genetics, genomics and functional genomics of several plant-associated enterobacteria in order to identify their mechanism(s) of bioactivity that are important for the biocontrol of fungal/oomycete diseases. The dissection of the regulation of these biocontrol properties was also one of the objectives to be investigated in this Research Project. Additionally, Dr. Matilla also investigated the anti-nematode properties observed in several biocontrol plant-associated bacterial strains. Special attention has been given to the investigation of the biosynthesis of a halogenated molecule, the haterumalide oocydin A, capable of killing plant pathogenic fungi and oomycetes, but also possessing strong anticancer properties. Although the chemical structure of oocydin A was known, the genes and the anabolic pathways responsible for its biosynthesis were unknown. Using genome sequencing, comparative genomics, random/site-directed mutagenesis approaches and chemical analyses, we identified and characterized a large polyketide synthase (PKS) gene cluster responsible for the biosynthesis of oocydin A (ooc). Furthermore, we showed that the ooc gene cluster is highly widespread – being present in several plant-associated bacteria belonging to Serratia and Dickeya genera. Using in silico analyses, and in collaboration with Dr. F. J. Leeper (Department of Chemistry, University of Cambridge) we proposed a model for the biosynthesis of oocydin A, and by extension, for other members of the haterumalide family of bioactive compounds. Although the biosynthesis of oocydin A has been accomplished by several research groups, the overall yield is still very low. Thus, the identification of the ooc gene cluster and the proposed biosynthetic model will provide the perfect background for the development of strategies orientated to increase the productivity of oocydin A, but also for the use the synthetic biology approaches in order to generate new chemical analogs of haterumalides showing enhanced biological properties – including antifungal, anti-oomycete and anticancer properties. The regulation of the biosynthesis of oocydin A was also investigated and the implication of different regulators in the expression of the ooc gene cluster demonstrated.During the phenotypic characterization of our bacterial strains, we observed that some of them showed strong nematicide properties against the model nematode, Caenorhabditis elegans. Plant diseases caused by pathogenic nematodes are economically very important. For example, the potato cyst nematodes of the Globodera genus cause losses estimated in more than 60 million euros in the United Kingdom each year. Annually, damages derived from plant parasitic nematodes are calculated at over 100 billion US Dollars worldwide. In this Research Project, we investigated the nematicide properties of several of our plant-associated strains and the regulation of their bioactivities. Research performed in this Project was facilitated due to the isolation of a new bacteriophage, phiMAM1. This phage is a very efficient generalized transducer, capable of moving chromosomal markers and plasmids between clinical and environmental isolates of Serratia and Kluyvera genera. Genome sequencing, phylogenetic analyses and morphological characterization of phiMAM1 allowed its classification within the new suggested genus “Viunalikevirus”. The high DNA homology, gene synteny and similar size observed between phiMAM1 and other viunalikeviruses suggested that all these phages may be efficient transducers. Indeed, we showed that previously isolated viunalikeviruses are excellent agents for functional genomics, being able to transduce chromosomal markers and plasmids at high efficiency. Furthermore, as a proof of principle of our hypothesis, we isolated new viunalikeviruses from the environment and demonstrated that all of them were very efficient generalized transducers. Importantly, phage therapy is an excellent alternative to the use of antibiotics (in medicine) and chemical pesticides (in agriculture) and several viunalikeviruses have been successfully used in phage therapy trials. However, our results suggest that the use of viunalikeviruses may be inappropriate in phage therapy strategies since they could be a source of dispersion of virulence factors and multidrug resistance genes, between others. In summary, we have used a multidisciplinary approach to investigate the regulation and biosynthesis secondary metabolites in several plant-associated bacteria. This Project has successfully addressed an important agricultural problem, the biological control of plant diseases and enhancement of crop productivity. The results obtained in this Project will have long term implications for agrochemical discovery and for crop/food security, particularly when the use of agrochemicals is being progressively diminished.