A New Class of Heptose‐Defective Mutant of Salmonella typhimurium

Abstract

Salmonella typhimurium rfa-657 strains, including both mutants and transductants, were shown to contain in their lipopolysaccharides both the (usual) L and the D-iSOmer of glycero-Dnzanno-heptose. By the application, in sequence, of' two different extraction procedures S and R form lipopolysaccharides could be isolated scparately from a representative rfa-657 strain. The R lipopolysaccharide was predominantly of the Re chemotype, only a few 2-keto-3-deoxyoctonate (KDO) residues being substituted by heptose (Rd, chemotype) or by longer core chains. The D-heptose was enriched in the R lipopolysaccharide, while the S lipopolysaccharide contained mainly the natural L-isomer. It was found that D-hcptose can replace L-heptoses I to 111 of the wild-type core. However, an elongation to completeness of the core stubs containing D-heptose seldom occurs.The heterogeneity in structure of the lipopolysaccharide produced by this class of (leaky) mutants is assumed to be due to an altered 6-epimerase which, in the wild form, catalyses the conversion of NDP-D-glycero-D-manno-heptose into NDP-L-glyyeero-D-manno-heptose and which is determincd by the gene rfa-657 situated between cysE and pyrE. The symbol rfaD is proposed for this gene. Abbreviations. KDO, 2-keto-3-deoxyoctonate. Note. All values of the retention time, t~, refer to 2,3,4,6-tetra-0-methyl-glucose. MATERIALS AND METHODS Bacterial &rains, Phages and LipopolysaccharidesStrain SL 1027 is a smooth, genetically marked derivative of S. typhimurium strain LT2, and SL 3600 is a "part-rough'' (i.e. phenotypically intermediate between S and R ) mutant, rfa-657, of SL 1027, isolated from a mutagen-treated culture by selection for resistance to Felix 0 phage [3] a.nd SL 3149, the two SL 3600 reisolates, respectively.The first lot of lipopolysaccharide examined was extracted a t Preiburg from a batch of SL 3600 cells grown on nutrient agar, washed and acetone-dricd a t Stanford. Later one crop each of strains SL 3147,

Vibrio cholerae Strains Possess Multiple Strategies for Abiotic and Biotic Surface Colonization

“…One transposon insertion disrupted the rfaD gene (VC0240), which has been shown in Salmonella to be involved in the biosynthesis of a conserved sugar in the LPS molecule (34). The second mutation was in a region of the TP chromosome not found in the N16961 genome.…”

Biochemistry Of Endotoxins

“…A chemically synthesized standard has not been reported. Mutants defective in the rfa D gene are thought to lack the epimerase that converts ADP-D-glycero-D-manno-heptose to ADP-L-glycero-D-manno-heptose (162)(163)(164), but the supporting enzymolo gy is inconclusive. Mutants in rfaD supposedly incorporate D-glycero-D manno-heptose into LPS (162)(163)(164) .…”

Cloning, characterization, and DNA sequence of the rfaLK region for lipopolysaccharide synthesis in Salmonella typhimurium LT2

“…Although glycosyl transferase activity for each step in outer core synthesis has been demonstrated in vitro (25,43,48), loss of transferase activity in particular mutants has been demonstrated only for rfaG (47), rfaB (72), and rfaI (26). The rfaD gene encodes the 6-epimerase required in heptose synthesis (32). Addition of the 0-somatic side chains to the completed oligosaccharide core requires at least two genes.…”