The Linkage of Pyrophosphorylethanolamine to Heptose in the Core of Salmonella minnesota Lipopolysaccharides

Abstract

Mild acid hydrolysis of the P+ lipopolysaccharide ol' Salmonella minnesota mRz, a UDPglucose synthetase-less mutant, leads to the formation of 5 degradation products whose structures were studied. Free 2-keto-3-deoxyoctonate (KDO), KDO-7-phosphorylethanolamine, and fraction P-(Hep-Hep-KDO) have been identified previously as degradation products of a Plipopolysaccharide (glycolipid). Fractions P+ (Hep-(Hep-4-phosphate)-KDO) and P P N (Hep-(Hep-4-pyrophosphorylethanolamine)-KDO) occur only i i i the P+ mutant. It was shown that fraction Pf is not an artifact which was formed from fraction PPN during partial hydrolysis. The simultaneous occurrence of fractions P+ and PPN reflects microheterogenicity of the lipopolysaccharide. Possible reasons for this are discussed.Salmonella R forms synthesize defective lipopolysaccharides consisting of lipid A to which are linked smaller or larger parts of the core polysaccharide [I]. Two classes of mutants, designated as P-and P f , have been identified [I, 21. Lipopolysaccharides of Pmutants, which were designated as glycolipids [Z], can be recognized by their relatively lower phosphate content. On acid hydrolysis free heptose and ethanolamine (and only small amounts of phosphorylethanolamine) are liberated. It has been shown that this ethanolamine derives a t least partly from the core where it is present as a phosphorylethanolamine group linked to the 7-position of a 2-keto-3-deoxyoctonate residue [3].I n contrast, lipopolysaccharides of P+ mutants contain relatively more phosphate, and on hydrolysis heptosephosphate and phosphorylethanolamine (besides ethanolamine) are liberated.The present paper describes investigations performed with lipopolysaccharides of Salmonella Pf mutants. The results show that the excess of phosphate is present as phosphate or pyrophosphorylethanolamine groups which are linked to the 4 position of heptose I units of the core. MATERIAL AND METHODS Bacteria and LipopolysaccharidesThe Salmonella minnesota R mutant mRz is a UDP-glucose synthetase-less mutant of the chemotype Rd,P+ [4]. The S. minnesota R mutant mR7 belongs to the chemotype RdL,P-due to defects in the transfer of glucose I and of phosphate groups to heptose units [2,4].Unusual Abbreviation. KDO, 2-keto-3-deoxyoctonate.P+ lipopolysaccharides and P-glycolipids were isolated by the phenol-chloroform-petroleum ether and purified by ultracentrifugation [5,6]. Analytical MethodsFor :full details of these methods see [3]. Heptose and maiinose were determined with H,SO,/cysteine [7], in s'ome cases mannose was also estimated with mannose isomerase according to Palleroni and Doudoroff [El]. Glucose was determined with glucose oxidase, 2-keto-3-deoxy-octonate (KDO) with periodate/tliiobarbituric acid [9] N NaOH [17]. After I0 min a t room temperature 100 pl of 0.1 N NaOH were added and the mixture allowed to stand at; room temperature for a further 30min. The solution was adjusted to pH 4 with acetic acid and extracted twice with chloroform. The water phase wi%s evaporated to dryness and salt re...

Role of the rfaG and rfaP genes in determining the lipopolysaccharide core structure and cell surface properties of Escherichia coli K-12

“…The evidence for this is that acid cleavage sufficient to cleave the pyrophosphoryl group internally would also be expected to attack the pyrophosphoryl-sugar linkage to release PPEA, but we did not observe a significant number of rfaP+ core oligosaccharide molecules in which heptose I was completely dephosphorylated. An identical conclusion was reached by Lehmann et al (28), who found that acid treatment of KDO-PPEA released only PPEA. Thus, the question is whether the heterogeneity in the substitution of heptose I is due to an inefficient attachment of PEA to P during synthesis or to enzymatic removal of PEA to generate P during maturation of the LPS.…”

“…The RcsC protein has a short periplasmic domain, spans the inner membrane, and has a sizable cytoplasmic domain (47) Fig. 8 (10,17,28,32) which showed that the most dramatic LPS structural defect in rfaP mutants was the loss of P or PPE from the heptose I residue. The additional loss of rfaG appears to alter the sensitivity of cells towards novobiocin, as seen in Fig.…”

“…There are negatively charged groups in the inner core-lipid A region of LPS which are not affected by deep rough mutations, including phosphoryl and/or pyrophosphoryl groups attached to lipid A (49), PEA attached to some of the branch KDO residues (21,28), and the carboxyl groups of KDO. However, these either do not participate in divalent cation-mediated cross-linking or do so at very low efficiency, since high levels of divalent cations do not rescue the detergent sensitivity of deep rough mutants.…”

“…The fractions from CS1959 were not characterized by MS/ MS. KDO substituted by phosphorylethanolamine (PEA) was not detected in this study, although it has been reported as a partial substituent in LPS from rfa+ E. coli K-12 (21) and in type RcP-LPS from Salmonella spp. (21,28). However, it should be noted that the fractions consisting primarily of free KDO were not characterized extensively and that the cultures from which the LPS was obtained were grown at 30°C.…”

A New Class of Heptose‐Defective Mutant of Salmonella typhimurium

“…of Heptose Phosphate It has been shown previously that in the more completed core structures of Ra through Rc mutants some of the heptose residues carry phosphate groups [21]. I n order to evaluate whether D-heptose in the lipopolysaccharide of mutant SL 3147 is likewise phosphorylated, the heptose phosphates were isolated according to the procedure of Slein and Schnell [Wj.…”