Aldoheptose Biosynthesis. E. coli K-12 aldoheptose (i.e., L-glycero-D- mannoheptose) mutants carrying the cysE-pyrE linked mutation rfaD or rfa-2, were previously isolated and genetically characterized. These mutations result in L-glycero-D-mannoheptose deficient Lipopolysaccharide (LPS). Thus, the rfaD and rfa-2 mutants produce a truncated LPS that is primarily lipid A-2-keto-3-deoxyoctulosonic acid structure (i.e., chemotype Re). These mutants possess an outer membrane (OM) that is characteristically more permeable to a number of hydrophobic agents. The molecular genetics and biology of the rfaD gene has been completed and reported by this laboratory. An efficient rfaD gene expression system and a two-step purification protocol for the rfaD gene product (ADP-L-glycero-D- mannoheptose-6-epimerase) was developed. The enzyme thus purified was found to be greater than 95% pure, and it was highly active. Key kinetics and physical characterization studies of the epimerase are complete. Preliminary crystals of the epimerase have been obtained. Structural and functional homology of the Pseudomonas aeruginosa rfaD gene and its E. coli counterpart is indicated. The second mutation, designated rfa-2, has been exploited to define the rfa-2 gene and resolve its sequence and flanking nucleotides, as well as determine its physical location on the E. coli chromosome relative to the rfaD gene. A rfa-2-6xHISTAG gene fusion has been constructed to facilitate the purification of the rfa-2 gene product. Interspecific complimentation studies demonstrated that the E. coli K-12 rfa-2 gene complements the chemotype Re LPS mutant of S. typhimurium, designated rfaC. The role of the rfa-2 gene product as a heptosyl transferase is under investigation.
