Lipopolysaccharide and Aldoheptose Biosynthesis in Transketolase Mutants of Salmonella typhimurium

Abstract

Genetic and biochemical evidence that sedoheptulose-7-phosphate is an obligatory precursor of the L-glycerc-D-mannoheptose residues of the lipopolysaccharide of Salmonella was obtained by isolation and characterization of transketolase-negative mutants of Salmonella typhimurium. These mutants, which are defective in synthesis of sedoheptulose-7-phosphate, were found to produce an incomplete heptose-deficient lipopolysaccharide, and were also sensitive to bile salts, a characteristic property of heptose-deficient mutants. Phenotypic repair of the defect in lipopolysaccharide synthesis was obtained by addition of exogenous sedoheptulose-7-phosphate to growing cultures of the mutant strains. Characterization of revertants isolated either as transketolase-positive or heptose-positive provided further evidence that the heptose deficiency resulted from mutation at the transketolase locus. On the basis of these findings a possible pathway for conversion of sedoheptulose-7-phosphate to L-glycero-Dxmannoheptose is proposed.L-Glycero-D-mannoheptose is a characteristic component of the lipopolysaccharides of SalmoneUa and related Gramnegative bacteria, and is a specific and major constituent of the innermost backbone region of rthe polysaccharide (Fig. 1). The pathway of biosynthesis of this sugar is unknown, although it has been assumed to be derived from sedoheptulose-7-phosphate (1, 2). To test this hypothesis we needed a mutant incapable of making sedoheptulose-7-P.While several reactions leading to formation of sedoheptulose-7-P are known, all depend on the presence of transketolase (TK): TK ribose-5-P + xylulose-5-P it sedoheptulose-7-P + glyceraldehyde-3-P [1] TK ribose-5-P + fructose-6-P 2::± sedoheptulose-7-P + erythrose-4-P TK fructose-6-P + glyceraldehyde-3-P >::xylulose-5-P + erythrose-4-P TA erythrose-4-P + fructose-6-P T± sedoheptulose-7-P + glyceraldehyde-3-P Ald erythrose4-P + dihydroxyacetone-P 2± sedoheptulose-1,7-diP [5]SDPase or sedoheptulose-1,7-diPsedoheptulose-7-P + Pi [6] FDPase In addition to reactions 1 and 2, catalyzed by transketolase, sedoheptulose-7-P can be derived from erythrose-4-P by reactions involving either transaldolase (reaction 4) or aldolase (reactions 5 plus 6); however, the only known sources of erythrose-4-P for these alternate pathways are reactions 2 and 3, which are themselves catalyzed by transketolase.Transketolase-negative mutants should therefore be deficient in synthesis of sedoheptulose-7-P, and, if this sugar is an obligatory precursor of L-glycero-D-mannoheptose, should produce an incomplete, heptose-deficient lipopolysaccharide. We now report that this is indeed the case, and that the defect in lipopolysaccharide synthesis can be corrected in vivo by addition of exogenous sedoheptulose-7-P to the culture medium.Isolation of transketolase-negative mutants was first described by Josephson and Fraenkel (3) in Escherichia coli. These workers developed a method of selection based on the fact that transketolase is essential both for catabolism of pentoses and for sy...

Biosynthesis of Inner Core Lipopolysaccharide in Enteric Bacteria Identification and Characterization of a Conserved Phosphoheptose Isomerase

“…Eidels and Osborn (28,44) (47) in Salmonella typhimurium suggests that rfaE encodes the ADPheptose synthase since addition of ADP-glyceromannoheptose to cell extracts of rfaE mutants restores the synthesis of a complete core LPS, but a similar gene in E. coli has not been identified.…”

The rfaE Gene from Escherichia coli Encodes a Bifunctional Protein Involved in Biosynthesis of the Lipopolysaccharide Core Precursor ADP- l - glycero - d - manno -Heptose

“…A transposon mutagenesis strategy using mini-Tn10 was utilized to isolate genes involved in the biosyn- (15,16). gmhA and gmhD are the only genes whose functions have been established biochemically (7,10).…”

“…From studies using Salmonella enterica, Eidels and Osborn (15,16) proposed that LDHep is synthesized from sedoheptulose 7-phosphate via four steps: (i) conversion of sedoheptulose 7-phosphate into D-glycero-D-manno-heptose 7-phosphate by a phosphoheptose isomerase, (ii) formation of D-glycero-D-manno-heptose 1-phosphate by a mutase reaction, (iii) transfer of a nucleotide via a phosphodiester linkage, and (iv) epimerization of D-glycero-D-manno-heptose 1-phosphate residue of the sugar nucleotide to LDHep (Fig. 1).…”

Isolation of a Mutant of Salmonella typhimurium Dependent on D-Arabinose-5-phosphate for Growth and Synthesis of 3-Deoxy-D-mannoctulosonate (Ketodeoxyoctonate)

“…Mutants blocked at various steps in biosynthesis of the polysaccharide, including incorporation of the first heptose residue (Fig. 1A), are readily obtained by phage-selection procedures (4). Such mutants are viable, but produce incomplete lipopolysaccharides lacking all parts of the polymer distal to the site of the biosynthetic lesion.…”