Life is hard if you are a plant. You can’t run away from a hungry herbivore, and you can pick up nasty fungal infections from playing in the dirt all day. Fungal pathogens that infect agricultural crops are a major problem for farmers. To control these pests, several million pounds of fungicides are used every year causing significant health and environmental problems. One strategy to reduce fungicide use is to employ certain soil microbes that produce antifungal compounds. These antifungal compounds, in turn, protect the plant without excessive application of agrochemicals. This protective relationship between microbe and plant is referred to as biological control.(1)
Our ability to use biological control strains in agriculture is somewhat limited however, due to a variety of factors(2,3) affecting antibiotic production. Sometimes the problem is that the biocontrol strain is unable to sufficiently colonize the root surfaces or to express biosynthetic genes necessary for antifungal production. Several biochemical signals have been identified that control these traits and include N-acyl homoserine lactones (a quorum-sensing metabolite), global regulatory proteins, and sigma factors. Other factors, however, depend on environmental conditions such as soil characteristics, plant root exudates, or the surrounding microbial population. While much of the research has focused on those factors intrinsic to the biocontrol strain, much less effort has been placed on understanding the exogenous factors controlling antibiotic production.
My long-term research interest is to identify external chemical signals that influence antibiotic biosynthesis in biological control organisms. In order to investigate these signals, it is first necessary to detect and isolate small organic metabolites from bacterial or fungal cultures. Therefore, students in my lab will initially focus on developing the analytical methods and bioassays for detecting, isolating, and structurally characterizing secondary metabolites in liquid cultures.
The fluorescent Pseudomonads are a group of gram-negative bacteria that are commonly isolated from the rhizosphere. These bacteria have been widely studied for their biological control activity, which in part, results from the production of a variety of natural products.(4) My short-term research objective is to develop the analytical capability to isolate and characterize secondary natural products from liquid bacterial cultures. The ability to reliably produce and detect compounds is critical for studying factors that control their synthesis. The two projects described below provide a focus to establish expertise in this area.1. Isolation and characterization of flaviorin from Pseudomonas aurantiaca BL915.
An antifungal metabolite, tentatively named flaviorin, has been detected in the culture supernatant of P. aurantiaca BL915. Sequence analysis of the gene cluster responsible for flaviorin production suggests that it originates from a pathway closely related to known fatty acid or polyketide pathways.(5) Several of the predicted enzyme functions, however, are unusual and have not been found previously within gene clusters encoding fatty acid or polyketide pathways. Therefore, it is likely that flaviorin biosynthesis may represent a novel polyketide pathway. Nevertheless, the chemical structure of flaviorin has yet to be determined, although there is spectroscopic evidence suggesting that it possess a polyene carbon skeleton.(6) The product can be extracted using organic solvents and has been detected photometrically via HPLC. Development of this project will focus on the chromatographic isolation and structural characterization of flaviorin.2. Isolation of unknown antibacterial metabolite of Pseudomonas fluorescens A506.
Fire blight is a plant disease that affects orchards and is caused by the bacterial pathogen Erwinia caratovora. Recent work (7) has identified an antibacterial activity produced by the biocontrol strain P. fluorescens A506 that is effective at inhibiting the pathogen in vitro. The antibacterial activity of A506 can be concentrated using ion exchange chromatography, yet we have not been able to purify the active components in sufficient quantities to determine either how many compounds a present or their structures. One of the challenges of isolating these compounds has been their extreme water solubility, which precludes the use of traditional organic extraction and separation methods. Aside from the possibility of identifying new biologically active compounds, developing separation and isolation methods for very water-soluble, organic compounds will provide tools to explore areas of natural product chemistry that have generally been avoided due to the difficulty of working with water soluble compounds.