Eine neue Gruppe von Salmonella R‐Mutanten
Authors: Risse H. J. , Dröge W. , Ruschmann E. , Lüderitz O. , Westphal O. , Schlosshardt J. 
Published: March 2005

From the wild types (S forms) of Salmonella minnesota and Salmonella ruiru R mutants (R forms) were isolated which produce hexose‐less cell wall lipopolysaccharides of chemotype Rd. These lipopolysaccharides are composed of lipid A (glucosamine, long‐chain fatty acids, phosphoric acid), 2‐keto‐3‐deoxy‐octonate (KDO), and l‐glycero‐d‐manno‐heptose (heptose), while those of the parent S forms contain additional galactosamine, glucosamine, galactose and glucose. R mutants with lipopolysaccharides of chemotype Rd could be differentiated into two groups, Rd1 and Rd2.

Group Rd1 comprises the S. minnesota mutants mR7 and mRz. Their lipopolysaccharides contain about 14% heptose. Partial hydrolysis of the mR7 lipopolysaccharide resulted in the formation of three main split products: lipid A, mR7A and mR7B. mR7A was identified as free 2‐keto‐3‐deoxy‐octonate, while mR7B was composed of heptose and 2‐keto‐3‐deoxy‐octonate. Depending on the method applied for the estimation of 2‐keto‐3‐deoxy‐octonate, the molar ratio of heptose/2‐keto‐3‐deoxy‐octonate was found to be 2:0.3 (on the basis of the thiobarbituric acid reaction) or 2:1 (according to the semicarbazide reaction). When permethylated mR7B was methanolyzed and the products analyzed by gas chromatography, two peaks (7.4 and 15.0 min) were observed. The 7.4 min peak was identical with one of the two peaks (7.4 and 9.6 min) obtained with permethylated authentic d‐glycero‐l‐manno‐heptose, the optical antipode of the bacterial heptose. The second peak at 15.0 min, which comprised about the same area as the 7.4 min peak, was derived from a partially methylated heptose. It is concluded that mR7B represents a heptose disaeeharide linked to 2‐keto‐3‐deoxy‐octonate and that such units are present in Rd1 lipopolysaccharides as terminal, nonreducing residues of the structure: heptosyl → heptosyl → 2‐keto‐3‐deoxy‐octonate–.

Group Rd2 comprises the S. minnesota mutants mR3 and mR4 and the S. ruiru mutant rR3. Their lipopolysaccharides contain about 7% neptose. By partial hydrolysis of the mR3 lipopolysaccharide four main split products were obtained: lipid A, mR3A, mR3B and mR3C. Fraction mR3A was identified with free 2‐keto‐3‐deoxy‐octonate. mR3B contained heptose, phosphate and ethanolamine in a molar ratio of 1:1:1, and, in addition, 1 mole (by thiobarbituric acid reaction) or 3 moles (by semicarbacide reaction) of 2‐keto‐3‐deoxy‐octonate. mR3C contained heptose and 2‐keto‐3‐deoxy‐octonate in a ratio of 1:1 (thiobarbituric acid reaction) or 1:2 (semicarbacide reaction), but no phosphate or ethanolamine. mR3B and mR3C were methylated, the products methanolyzed and analyzed by gas chromatography, From both oligosaccharides only one heptose peak (7.4 min) was obtained which was identical with the faster peak observed with mR7B and authentic heptose. It is concluded that in the mR3 lipopolysaccharide, and generally in Rd2 lipopolysaccharides, heptose is exclusively linked as a terminal, non‐reducing monosaccharide, presumably to 2‐keto‐3‐deoxy‐octonate: heptosyl → 2‐keto‐3‐deoxy‐octonate–.

The results indicate that Rd2 lipopolysaccharides act as precursors of Rd1 lipopolysaccharides in the biosynthesis of the wild type lipopolysaccharide and that the Rd1 structure (Hep → Hep → KDO –) is formed by transfer of one heptose unit to Rd2 lipopolysaccharides (with terminal Hep‐KDO).

Biosynthetic studies demonstrated that Rd1 lipopolysaccharides, but not Rd2 lipopolysaccharides, function as acceptor for the enzymatic incorporation of glucose from UDP‐glucose. Although the Rd1 mutants, mR7 and mRz, synthesize identical lipopolysaccharides, they are distinct regarding their enzymatic block involving the glucose anabolism. mR7 lacks glucosyl‐ I‐transferase activity, while mRz is defective in the enzyme UDP‐glucose‐synthetase. ‐ Rd2 mutants, with the incomplete heptosyl‐KDO core, are deficient with respect to the transfer of the second heptosyl residue to the first heptose; but they do contain‐as expected‐glucosyl‐I‐transferase which catalyzes the incorporation of glucose (glucose I) into the complete heptosyl‐ heptosyl‐KDO core of Rd1 lipopolysaccharides to form the glucosyl‐heptosyl‐heptosyl‐KDO structure of Salmonella Rc lipopolysaccharides.

Serologically, heptose proved to be an inhibitor of precipitation in both systems, mR7lanti mR7 (Rd1 system) and mR3/anti mR3 (Rd2 system), which is in agreement with the concept of terminal, non‐reducing heptose units occurring in both Rd1 and Rd2 lipopolysaccharides. However, in hemagglutination inhibition tests, the lipopolysaccharides of group Rd2 do not show serological cross‐reaction with Rd2 lipopolysaccharides, and vice versa.