K-061. Validation of Missing Gene Functions in the Rhamnose Metabolic Pathway of Bacillus, Streptomyces, and Salmonella

K. M. Stanton, A. A. Best;
Hope Coll., Holland, MI.

Genome sequencing projects yield vast amounts of information about microbes, yet each project reveals a large fraction of genes with no known function. This pool of genes represents an opportunity to enrich our understanding of metabolic diversity in microbes. Salmonella enterica serovar Typhimurium, Streptomyces coelicolor and Bacillus subtilis are known to utilize rhamnose, though not all of the genes involved have been identified. Advanced bioinformatics genome context techniques were used to identify candidate genes for these missing functions. Rhamnulose-1-phosphate aldolase (rhaD, rhaE) and lactaldehyde dehydrogenase (rhaZ, rhaW) perform the final two steps in the catabolism of rhamnose. In S. enterica, rhaD is of known function based on high sequence similarity with Escherichia coli rhaD, but the function of rhaZ requires verification. In contrast, B. subtilis and S. coelicolor are predicted to have a fused gene product responsible for both functions, RhaE/W. The candidate genes were cloned, sequenced, and overexpressed in E. coli. The recombinant proteins were purified using affinity chromatography and used in biochemical assays to test their predicted functions. The assays required the reconstruction of the first two steps in the rhamnose catabolic pathway, the activity of which was confirmed in a coupled assay with glycerol kinase. Assays using the experimental proteins RhaD/RhaZ or RhaE/W yielded inconclusive results; buffer conditions are being altered to address potential co-factor requirements. The substrates for both the aldolase and the dehydrogenase reactions are being synthesized to allow for direct validation of enzyme functions and for modification of substrate concentrations for determination of kinetic properties of the enzymes.

140/K. Metabolism and Enzymology - II

Tuesday, 10:30 am | Poster Hall