Identification of genes involved in the production of a novel antifungal agent (7, 10, 12-trihydroxy-8(E)-octadecenoic acid) in Pseudomonas aeruginosa
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The gram-negative bacterium Pseudomonas aeruginosa catalyzes the conversion of ricinoleic acid into a novel trihydroxy fatty acid, 7, 10, 12-trihydroxy-8(E)-octadecenoic acid (TOD), that has a potent antifungal activity against important crop pathogens, including Magnaporthe grisea the causative agent of rice blast disease. Natural crop-protecting agents such as TOD offer several advantages over synthetic agents, including improved ecological compatibility and environmental safety. Unfortunately, because many naturally occurring crop-protecting agents are produced only in trace amounts, it has been difficult to isolate large enough quantities of these antimicrobial agents to be economically feasible. Thus, a bacterium such as P. aeruginosa that is genetically amenable and produces an antifungal agent is ideal for genetic manipulation to achieve improved TOD production. The long-term goal of this research is to develop efficient processes for improving production of TOD from P. aeruginosa to mass-produce an economically competitive and environmentally friendly biological crop protection agent. As an initial step towards this goal, a genetic study was undertaken to identify the genes that are required for production of TOD in P. aeruginosa. In order to facilitate the genetic screen, a bioassay was developed to assess TOD’s antifungal activity against M. grisea in 96-well microtiter plates using either pure TOD or P. aeruginosa culture supernatants. In order to identify the genes involved in the bioconversion, a transposon mutagenesis was performed and a library of ~15,000 Tn5-B21 insertion mutants of the P. aeruginosa strain NRRL B-23260 (an environmental isolate) was constructed. From the genetic screen, 28 transposon insertion mutants that were defective for TOD production were identified. Molecular studies identified eleven mutants that appeared to have single transposon insertions. Genetic mapping of these eleven mutants was conducted to identify the genes that had been interrupted by transposon insertion. From this analysis, eight genes were identified including genes encoding alkylated DNA repair proteins, ferrous iron transport proteins, pseudouridylate synthase, a hypothetical protein/magnesium transport protein, a transport protein of ABC transport, and XcpP of the general secretory pathway. Three of the mutants have been successfully complemented with the wild-type genes to demonstrate their involvement in the microbial conversion of ricinoleic acid to TOD.
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