Research Project Summary Information
NYS Department of Education
This project is based on extensive past and ongoing work, both through NYSERDA and others. NYSERDA has an existing project to scale-up fermentation of a microbe that makes a toxin that selectively kills zebra mussels. The microbe is then killed and the whole cell is used as the control agent.
Researchers hoped to: mechanically or enzymatically lyse P. fluorescens to release the toxin from the cells, assess the toxin's sensitivity to proteolytic enzymes and heat, assess the toxin's stability over time, prepare a concentrated form of the bioactive toxin using a molecular exclusion membrane, separate the toxin from other molecules via chromatographic separation methods, such as reverse phase separation, ion exchange separation, or hydrophobic interaction chromatography; and identify the bioactive toxin by: mass spectrometry, NMR spectrometry, and/or protein sequencing.
Since researchers were unable to develop an effective method to deliver the solubilized toxin molecules to the zebra mussels on particles, they altered the biochemical experimental approach and decided to search the literature for documented products from P. fluorescens that matched characteristics of the biotoxin. A likely candidate appeared to be glycine dehydrogenase, and thus testing focused on determining whether or not this enzyme was the biotoxin produced by strain CL0145A.
Successful control of zebra mussels could improve operations at New York State (NYS) facilities, using cooling water from infested water bodies. Safe, non-chemical (typically chlorine bleach) alternatives are needed. Success could have accelerated commercialization of the mass fermentation process by allowing researchers to inexpensively and rapidly assess toxin quantities in fermentations, increase patent protection (since all strains producing the same toxin would be covered), and ultimately, lead to production of a pure toxin (as opposed to a whole-cell fermentation broth). A pure toxin could reduce effects on non-target species and possibly be produced more economically either synthetically or via gene amplification in a surrogate species.
Experiments in which CL0145A cells were treated with an irreversible flavoenzyme inhibitor, diphenyleneiodonium chloride, confirmed that glycine dehydrogenase activity was inhibited, but the cells were still highly toxic to zebra mussels. Thus, we concluded that glycine dehydrogenase is not the molecule that causes mussel death. Although the identity of the biotoxin was not elucidated in this project, as had been hoped, valuable information about its characteristics has been gained. Treatment of toxic cells with lysozyme or deoxycholate appeared to separate the toxin molecules from the cells, suggesting that the toxin is associated with the outer membrane of the cells. Protease treatments decreased toxicity, suggesting that the membrane-associated toxin is likely a protein.
NYS Department of Education
109 South Union St., 2nd Floor
Rochester, NY 14607
Indigenous/Renewable Energy Resources
NYSERDA Contact Information
R&D - Environment & Energy Res