2007 Annual Report 1a.Objectives (from AD-416) 1) Identify and characterize viruses that kill specific off-flavor producing cyanobacteria in aquaculture systems;. 2)Identify, characterize, and develop novel bacterial strains that metabolize (biodegrade) geosmin and 2- methylisoborneol, and develop them for use in eliminating those chemicals from aquaculture systems; and. 3)Identify and characterize genes and enzymes involved in the biosynthesis of off-flavor metabolites in aquaculture systems. Build on an understanding of the mechanisms used in regulating gene expression and in catalysis of individual reactions, and develop methods to control the production of geosmin and MIB. 1b.Approach (from AD-416) The cyanophage community in catfish ponds will be screened and its members and their cyanobacterial host strains will be identified. Virulent cyanophage that kill specific cyanobacteria (lytic) will be identified and isolated from the environment, and the kinetics of phage replication and cyanobacterial population reduction will be determined. Bacteria will be isolated and characterized regarding ability to degrade geosmin or MIB at relevant concentrations, metabolic pathways employed and metabolites produced, mechanisms for controlling geosmin and MIB degradative enzyme biosynthesis, and taxonomy. Towards development of methods for controlling off-flavor production, the genes and enzymes of the biosynthetic pathway of MIB and geosmin will be characterized. 3.Progress Report 1) Isolation and characterization of bacteria that are able to degrade off-flavor compounds geosmin and 2-methylisoborneol (MIB) have continued. Strains were isolated for their ability to grow on various terpenes (geosmin and MIB are terpenes). While many of these resemble previously isolated strains in their ability to transform MIB and geosmin, there have been some novel and important recent isolates. Two examples: (a) several different strains that dehydrate MIB yielding primarily the taste- and odor-less chemical 2-methylenebornane have been found; (b) another strain that is able to rapidly and completely remove both geosmin and MIB from culture media has been discovered. . 2)A 1.8 kb polymerase chain reaction (PCR) product was isolated in FY 2005 that contains a sesquiterpene cyclase gene from Oscillatoria splendida, which makes geosmin. This enzyme is related to cyclases from other organisms (S. coelicolor, S. avermitilis and M. xanthus). A mutation of the S. coelicolor cyclase gene eliminated geosmin biosynthesis. A larger DNA fragment was then isolated that flanks the cyclase gene. This fragment contains the gene for geranyl pyrophosphate synthase, the product of which is also a precursor of geosmin. The cyclase gene was then cloned into an E. coli expression plasmid and high level expression was observed. The enzyme has been purified to near homogeneity. Enzyme assays are being developed to test the role of this enzyme in geosmin biosynthesis. Antibodies to the purified enzyme are also being generated, with which homologous enzymes in other geosmin-producing species will be sought. 4.Accomplishments a) ELIMINATING OFF-FLAVOR COMPOUNDS IN CATFISH PONDS. -- Bioremediation is a process in which microorganisms (such as bacteria) are employed to treat and degrade pollutant chemicals. In-situ bioremediation has long been viewed as a potentially valuable technology for the removal of unwanted chemicals from the environment. This approach is being taken to remove the off-flavor compounds geosmin and MIB from catfish ponds as soon as they are produced by problematic algae. Bacteria that biodegrade geosmin and 2-methylisoborneol have been isolated, identified, and partially characterized. Following some additional characterization they should be ready to develop for use. If fully successful, these new bacteria will eliminate the geosmin and MIB off-flavor problem from catfish farming. This research aligns with NP 106 Component, "Quality, Safety, and Variety of Aquaculture Products for Consumers"; and addresses problem area D, "Off-flavor Delayed Harvesting", and problem area E, "Off-flavor Methodology." b) ISOLATION AND CHARACTERIZATION OF GENES INVOLVED IN GEOSMIN BIOSYNTHESIS. -- Whereas, not proven yet, the first genes involved in geosmin synthesis in cyanobacteria have been isolated. One of those genes, encoding a sesquiterpene cyclase, has been subcloned and the recombinant cyclase expressed in E. coli. The enzyme was not soluble initially, but following denaturation and renaturation it was. By characterizing these genes and their enzyme products, the regulation of geosmin biosynthesis can now begin to be understood at a molecular level. By understanding the regulation of geosmin biosynthesis methods, to control that regulation can be developed that should allow greater harvest yields of on-flavor catfish. This research aligns with NP 106 Component, "Quality, Safety, and Variety of Aquaculture Products for Consumers; and addresses problem area D, "Off-flavor Delayed Harvesting", and problem area E, "Off-flavor Methodology." 5.Significant Activities that Support Special Target Populations None. 6.Technology Transfer Number of non-peer reviewed presentations and proceedings 2