2005 Annual Report 1.What major problem or issue is being resolved and how are you resolving it (summarize project aims and objectives)? How serious is the problem? What does it matter? The catfish aquaculture industry is the fastest growing sector of the U.S. agricultural economy, producing well over 500 million pounds live weight annually. However, prices for catfish have remained relatively flat while production costs have risen, resulting in very tight profit margins for producers and processors. In response, farmers are using increasingly intensive production practices to maximize yields and returns on production investments. The large amounts of fish feed and dense fish populations required for intensive fish production, contribute to dense bacterial and algal growths in production ponds. Although many types of bacteria and algae are beneficial, certain species of cyanobacteria and blue green algae produce the off-flavor compounds 2 methyl isoborneol (MIB) and geosmin. At very low concentrations, these compounds impart an unacceptable earthy/muddy flavor to fish tissues, rendering the crop undesirable to consumers. Catfish farmers have demonstrated their strong support for research to find safe, cost effective compounds, that can be added to ponds to mitigate off flavors; therefore, multiple approaches are underway. Molecular biological methods are being developed to allow forecasting of problematic periods and implementation of best management practices. Furthermore, it is currently unclear why certain species produce off-flavor metabolites at certain times of the year. To gain a better understanding of the regulation off-flavor metabolite expression, genes encoding the biosynthetic enzymes responsible for off-flavor metabolites will be cloned and studied. With a full understanding of this regulation, rationally-designed solutions to this problem can be developed. Furthermore, viruses that specifically kill problematic cyanobacteria, as well as bacteria that degrade off-flavor compounds, are being sought. At times 50 to 80% of available fish stocks can be off-flavor. Off-flavors can occur throughout the year, typically reaching greatest frequency in late summer. The occurrence of MIB and geosmin (earthy/muddy flavor) in ponds and fish is sporadic and unpredictable, and results in production/processing interruptions estimated to cost catfish producers alone, over 30 million dollars each year. Off-flavors also inhibit the adoption of more efficient production practices and processing strategies. Market restrictions caused by off-flavors depress farm gate sales, and require farmers to hold fish for extended periods, thus, increasing farmers' exposure to risk in the form of large scale fish kills. 2.List the milestones (indicators of progress) from your Project Plan. The goals and objectives for FY 2005 were to:. 1)isolate and characterize cyanophage specific for off-flavor-producing cyanobacteria;. 2)isolate and characterize bacterial strains that can utilize geosmin or MIB as a sole carbon source for use in bioremediation of off-flavor ponds;. 3)isolate and characterize the genes involved in geosmin and MIB biosynthesis. As was written in the project plan, if any one of these showed significant promise it would become the focus. The milestones are as follows: Objective 1 "Cyanophage." FY 2005: Establish assays for lysis of off-flavor-producing cyanobacteria by phage. Collect environmental samples. Begin testing samples. FY 2006: Continue collecting environmental samples and testing for lysis. Isolate phage stocks. FY 2007: 3: Determine MOI for stocks. FY 2008: Begin large scale testing for killing using individual phage stocks and combinations. FY 2009: Optimize large scale testing for killing using individual phage stocks and combinations. Objective 2 ABioremediation. FY 2005: Isolate geosmin and 2-methylisoborneol degrading bacteria. Isolate transposon mutants. FY 2006: Characterize transposon mutants. Identify strains. Carry out kinetic studies. Clone genes encoding the metabolic pathways. FY 2007: Complete cloning genes and characterize gene products (enzymes). FY 2008: Study regulation of metabolic pathways leading to isolation of constitutive mutants. FY 2009: Complete the sequencing of genes encoding the metabolic pathways. Objective 3: "Biosynthetic genes" FY 2005: Use cloned Streptomyces genes in blot hybridization and PCR cloning of homolog in producing species. Develop conjugation system in producing species. FY 2006: Clone remaining biosynthetic pathway genes. Use clones from year 1 to walk on chromosome for related genes. Knock out homologous genes. Develop transposon mutagenesis system. FY 2007: Knock out other genes potentially involved in biosynthetic pathway. Screen transposon mutants with ELISA. Clone transposon mutant genes. FY 2008: Identify regulatory genes. FY 2009: Determine interactions of regulators with regulated genes. 4a.What was the single most significant accomplishment this past year? The most significant accomplishment of FY 2005 was cloning the sesquiterpene cyclase gene from O. splendida. This accomplishment addresses Goal #2. Using degenerate PCR a DNA fragment containing part of the cyclase gene was isolated. Using inverse PCR the entire gene was isolated. This gene will allow us to begin to study the regulation of geosmin biosynthesis in this off-flavor-producing cyanobacteria. Short title: Isolation of sesquiterpene cyclase gene from O. splendida. 4b.List other significant accomplishments, if any. We have made significant progress in analyzing samples of a field study that began under the previous research project period. This work, when complete, will allow us to correlate cyanobacteria with weather, off-flavors, pond age, and begin to understand the interrelationships among cyanobacteria. Short title: DGGE analysis of cyanobacteria in Limco, Inc. catfish farm ponds. 4c.List any significant activities that support special target populations. None. 4d.Progress report. Objective 1: No work was attempted because all efforts were spent on Objective 3 and finishing a field study initiated in 2003. Objective 2: Several different bacterial strains were shown to convert MIB to distinct products (although no bacterium yet studied can completely degrade and grow with MIB). The chemical formed from MIB by Rhodococcus sp. strain T1, was isolated and identified by GC-MS and 13C- and 1H-NMR, as trans-2,3-dihydroxy-2-methylbornane. Two other products of the bacterial transformation of MIB have been partially identified. Objective 3: A DNA fragment was generated with PCR that likely is part of the gene for the first step in geosmin biosynthesis, a sesquiterpene cyclase, from O. splendida. Degenerate PCR primers were designed for this task using the S. coelicolor gene and two other homologous genes S. avermitilis and M. xanthus. The DNA fragment was only about 500 bp, however, using inverse PCR, we were able to extend this to nearly 4 Kb, and now have a clone with the entire gene and considerable flanking DNA. In the flank upstream is another gene encoding a geranyl geranyl pyrophosphatase that may also be involved in geosmin biosynthesis. To confirm that the cyclase is in the pathway we are proceeding on two paths: . 1)we are generating a construct for knocking out the gene following transformation;. 2)we have cloned the gene into an E. coli expression plasmid for over-expression towards purification and enzyme assays. Towards developing methods for introducing DNA into cultured field isolates, we have attempted using a Gene Gun and have had a low rate of success. This is thought to result from having no O. splendida DNA in the suicide plasmid we've been using so far. Towards developing forecasting of off-flavor episodes (a goal of the previous research project), denaturing gradient gel electrophoresis is being used to analyze the samples. 5.Describe the major accomplishments over the life of the project, including their predicted or actual impact. 1) A molecular assay was developed to determine which cyanobacteria, algal, or protozoan species are present in the catfish ponds. The assay uses polymerase chain reaction (PCR), followed by denaturing gradient gel electrophoresis (DGGE)to separate DNA from the species. The assay will allow producers to determine succession of organisms in ponds which will give them more lead time to harvest, or treat, if predictor species always precede problematic species. . 2) Many bacteria use signaling molecules to communicate between cells of the same species or even with other species. These molecules are important for dramatic changes in physiology in many organisms, and therefore, may be important in the regulation of geosmin and MIB expression. We have collected the spent media from cyanobacterial cultures and shown, using gas chromatography-mass spectrometry, that they contain putative signaling molecules (acyl homoserine lactones). These signaling molecules, or analogs of them, may possibly be used to inhibit off-flavor production. . 3)Viruses that infect cyanobacteria are likely involved in the ecology of the ponds. Water samples from the field study samples were tested using polymerase chain reaction. To date, we have determined that there are at least seven different kinds of these viruses present in the samples. . 4) The sesquiterpene cyclase gene from O. splendida was isolated. 6.What science and/or technologies have been transferred and to whom? When is the science and/or technology likely to become available to the end-user (industry, farmer, other scientists)? What are the constraints, if known, to the adoption and durability of the technology products? None to date. 7.List your most important publications in the popular press and presentations to organizations and articles written about your work. (NOTE: List your peer reviewed publications below). Mitigation of off-flavors in catfish aquaculture. Paper presented at the 49th Wind River Conference on Prokaryotic Biology, June 2005.