The primary goal of this project is characterize the expression and annotate the function of two currently"hypothetical" gene sequences of the well known marine microbial genome of Roseobacter denitrificans Och114. These hypothetical genes are found in almost every completed bacterial genome to date, but often more DNAsequence data is generated without proper verification of function. This effort will involve genetic engineering to manipulate the genome of Roseobacter denitrificans Och114 to elucidate the functions of four selected genes, two which are known (controls) and involved in structure, development, motility, and chemotaxis, and also two which are unknown or hypothetical, both possibly functioning in sugar metabolism. The molecular biology experimental approach will provide a better understanding of the mechanisms of gene regulation and metabolic networks in microbial systems. Genetic engineering through standard molecular biology practices such as plasmid transformation will assist in shuttling the target hypothetical genes of interest between Escherichia coli and R. denitrificans using carefully constructed plasmid vectors, derivates of pBBR1-MCS, so that site-directed mutagenesis can be performed. This will modify the genes to decrease their gene expression and gene products which will be phenotypically noticeable in culture and under microscopy. Computer based bioinformatics tools will also provide ways to analyze and select and compare target genes. These techniques can be applied to many cultivable microbes, both terrestrial and marine, to provide insight into the annotation and characterization of novel genes of interest, most importantly, those involved in secondary metabolism of pharmaceuticals, antimicrobial agents, and anti-tumor agents used incancer treatment and prevention. This project will assist in training at least one graduate and one undergraduate student in sophisticated molecular biology methods such as quantitative PCR, site directed mutagenesis and plasmid cloning.