STUDIES OF THE BIOSYNTHESIS OF 3,6-DIDEOXYHEXOSES - MOLECULAR-CLONINGAND CHARACTERIZATION OF THE ASC (ASCARYLOSE) REGION FROM YERSINIA-PSEUDOTUBERCULOSIS SEROGROUP VA

The 3,6-dideoxyhexoses are found in the lipopolysaccharides of gram-ne gative bacteria, where they have been shown to be the dominant antigen ic determinants. Of the five 3,6-dideoxyhexoses known to occur natural ly four have been found in various Strains of Salmonella enterica (abe quose, tyvelose, paratose, and colitose) and all five, including ascar ylose, are present among the serotypes of Yersinia pseudotuberculosis. Although there exists one report of the cloning of the rfb region;har boring the abequose biosynthetic genes from Y. pseudotuberculosis sero group IIA, the detailed genetic principles underlying a 3,6-dideoxyhex ose polymorphism in Y. pseudotuberculosis have not been addressed. To extend the available information on the genes responsible for 3,6-dide oxyhexose formation in Yersinia spp. and facilitate a comparison with the established rfb (0 antigen) cluster of Salmonella spp;, we report the production of three overlapping clones containing the entire gene cluster required for CDP-ascarylose biosynthesis. On the basis of a de tailed sequence analysis, the implications regarding 3,6-dideoxyhexose polymorphism among Salmonella and Yersinia spp. are discussed. In add ition, the functional cloning of this region has allowed the expressio n of E(p) (alpha-D-glucose cytidylyltransferase), E(od) (CDP-D-glucose 4,6-dehydratase), E(1) -6-deoxy-L-threo-D-glycero-4-hexulose-3-dehydr ase) E(3) (CDP-6-deoxy-Delta(3,4)-glucoseen reductase), E(ep) deoxy-D- glycero-D-glycero-4-hexulose-5-epimerase), and E(red) deoxy-L-glycero- D-glycero-4-hexulose-4-reductase), facilitating future mechanistic stu dies of this intriguing biosynthetic pathway.