Low-molecular-weight solutes released during mild acid hydrolysis of the lipopolysaccharide of <i>Pseudomonas aeruginosa</i>. Identification of ethanolamine triphosphate

Drewry, David T.; Gray, G. W.; Wilkinson, Stephen G.

doi:10.1042/bj1300289

Cited by 22 publications

(7 citation statements)

References 17 publications

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“…A much higher phosphate content, up to 10 or more phosphate groups, has been reported for LPS of P. aeruginosa, and most of them are considered to be located in the core region [12–14], although some phosphate may be present as triphosphate residues [13]. On mild acid hydrolysis, much of the phosphate can be released as P i , PP i and ethanolamine mono‐, di‐ and triphosphates [42,43]. No more phosphate groups could be retained in the core after alkaline degradation of the LPS of P. aeruginosa [10] as di‐ and triphosphates are unstable under alkaline conditions as well (P. M. Sánchez Carballo et al, unpublished data).…”

Section: Resultsmentioning

confidence: 99%

Elucidation of the structure of an alanine‐lacking core tetrasaccharide trisphosphate from the lipopolysaccharide of Pseudomonas aeruginosa mutant H4

Carballo

Rietschel

Kosma³

et al. 1999

European Journal of Biochemistry

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Lipopolysaccharide (LPS) of Pseudomonas aeruginosa rough mutant H4 was isolated by hot water/phenol extraction followed by a modified phenol/chloroform/petroleum ether procedure. Upon SDS/PAGE, the LPS showed a strong major band corresponding to the expected rough-type LPS. Additional faint high molecular-mass bands revealed that the O-chain was present, indicating that the H4 mutant is genetically unstable. Mild acid hydrolysis of the LPS removed lipid A and released a phosphorylated core oligosaccharide that was purified by gel-permeation chromatography and high-performance anion-exchange liquid chromatography. The oligosaccharide contained two residues of L-glycero-D-manno-heptose (Hep) and one residue each of 3-deoxy-D-manno-oct-2-ulosonic acid (Kdo) and GalNAc. Upon matrix-assisted laser desorption/ionization mass spectroscopy in the negative ion mode, the main fraction expressed a peak for the molecular ion [M-H]- at m/z 1106.41, which was compatible with a carbamoylated, trisphosphorylated tetrasaccharide. The structure was further investigated using one- and two-dimensional homonuclear and heteronuclear correlated NMR spectroscopy at pD 3 and, after borohydride reduction, at pD 9. The NMR data of the two phosphorylated tetrasaccharides recorded at different pD allowed determination of the positions of the three phosphate (P) groups and the carbamoyl group (Cm) thus establishing the following structure of the core oligosaccharide: [equation: see text] Two unusual structural features in the core oligosaccharide of P. aeruginosa were identified for the first time, i.e. the replacement of an amide-linked alanyl group in GalN with an acetyl group and the phosphorylation at position 6 of HepII.

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Section: Resultsmentioning

confidence: 99%

Elucidation of the structure of an alanine‐lacking core tetrasaccharide trisphosphate from the lipopolysaccharide of Pseudomonas aeruginosa mutant H4

Carballo

Rietschel

Kosma³

et al. 1999

European Journal of Biochemistry

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“…These three observations suggested the existence of an altered rough core in a portion of the LPS of the mutant strain. Since fraction III material represented degraded LPS (7,34), it was not studied in detail.…”

Section: Resultsmentioning

confidence: 99%

Chemical and chromatographic analysis of lipopolysaccharide from an antibiotic-supersusceptible mutant of Pseudomonas aeruginosa

Kropinski¹,

Kuzio²,

Angus³

et al. 1982

Antimicrob Agents Chemother

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Lipopolysaccharides extracted from Pseudomonas aeruginosa strain K799 and its antibiotic-supersusceptible derivative Z61 were analyzed chemically and chromatographically. The side-chain polysaccharides purified by gel exclusion chromatography were compositionally identical, being composed of fucosamine (2-amino-2,6-dideoxygalactose), quinovosamine (2-amino-2,6-dideoxyglucose), and an unidentified amino sugar. In addition, low amounts of the core-specific components (glucose, rhamnose, alanine, and galactosamine) were found associated with the side chains from both strains. In the preceding paper (1), we provided evidence that the basis of the generally enhanced susceptibility to antibiotics of mutant Z61, when compared with its progenitor strain K799, was a more permeable outer membrane. In particular, the outer membrane of strain Z61 was more permeable to the hydrophilic antibiotic nitrocefin (1) and to other P-lactams (41), despite an apparently unaltered porin protein F, an outer membrane protein shown to form water-filled channels in model membrane systems (2a, 14). Indirect evidence was provided that mutant Z61 had an unspecified lipopolysaccharide (LPS) alteration (1). To gain further information regarding the mutation present in strain Z61, we characterized the LPS alteration in greater depth. Due to the rather complex method used to isolate mutant Z61 (41), we prepared a series of spontaneous revertants (1). Full revertants were isolated which were indistinguishable from the wild-type strain K799 in a wide variety of properties (1), including some LPS properties (see below). This suggests that Z61 and K799 differ by a single major mutation which is responsible for the antibiotic susceptibility and permeability differences between the strains as well as the

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“…Much of the phosphorus in the lipopolysaccharides of P. aeruginosa and P. alcaligenes is acid-labile [26,27], and probably occurs in condensed phosphates attached to the polysaccharides [28,65]. Such phosphates should possess and utilise metal-binding properties, and may serve to link the lipopolysaccharides to other wall components via metal-ion bridges.…”

Section: Discussionmentioning

confidence: 99%

Cell Walls, Lipids, and Lipopolysaccharides of Pseudomonas Species

Wilkinson¹,

Galbraith²,

Lightfoot³

1973

European Journal of Biochemistry

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Cell walls from two EDTA-sensitive and three EDTA-resistant pseudomonads grown in nutrient broth have been analysed. Each wall had a composition broadly characteristic of gramnegative bacteria, although the walls of Pseudomonas pavonacea and P. syncyanea were relatively rich in glycosaminopeptide and loosely-bound lipid, respectively. Phosphatidylglycerol was the only phospholipid common to all species, although phosphatidylethanolamine was absent only from P. diminuta. The latter species contained the distinctive range of glycolipids and a phosphoglucolipid identified previously in cells grown on nutrient agar. However, the glycolipids and an ornithine-containing lipid present in some other species (notably P. rubescens) when grown on nutrient agar were not detected. Except in P. rubescens, the major fatty acids in loosely-bound lipids were CIS, C16:l. and c18:1 acids: in P. rubescens, the major components were CIS, iso-Cl5, and C17:1 acids. Cyclopropane acids occurred in the lipids from P. stutzeri.Components of the lipopolysaccharides were identified and determined. The lipid A fractions contained fatty acids, phosphorus, and (except for P. diminuta) glucosamine. The fatty acids included both hydroxy and non-hydroxy acids. Apart from 2-hydroxydodecanoic acid (present in P. syncyanea), all the hydroxy acids were b-hydroxy acids, and 3-hydroxydodecanoic acid predominated in all species except P. rubescens. Odd-numbered hydroxy acids were minor components in P. diminuta and P. pavonacea and major components (CIS acids) in P. rubescens.The non-hydroxy acids consisted of saturated acids (C12 to C14) and orb-unsaturated acids (dehydration products from the b-hydroxy acids).Unusual features of the polysaccharide fractions from the lipopolysaccharides included the occurrence of non-phosphorylated heptose residues (P. diminuta), the presence of phosphorylated glucose residues and of aspartic acid ( P . pavonacea), the probable absence of 2-keto-3-deoxyoctonic acid and the presence of acid-labile galactose residues (P. rubescens), the virtual absence of glucose ( P . stutzeri), and the presence of alanine ( P . syncyanea). Amino sugars (glucosamine, galactosamine, and quinovosamine) occurred in the polysaccharides from P. stutzeri and P. syncyanea only. Following chromatography of the partly degraded polysaccharides on Sephadex, the distribution of components between possible side chain and core regions of the polysaccharides could be determined. The possible sigdcance of the results for the sensitivity of the organisms to EDTA and for their taxonomy is discussed.Although the cell walls and lipopolysaccharides of gram-negative bacteria have been studied extensively (see [1,2] for recent reviews), relatively little work has been done with pseudomonads other than Pseudomonas aeruginosa. Partly because of its outstanding resistance to many antibacterial agents, this organism has become a pathogen of some importance. It is perhaps surprising, therefore, that the

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Low-molecular-weight solutes released during mild acid hydrolysis of the lipopolysaccharide of Pseudomonas aeruginosa. Identification of ethanolamine triphosphate

Cited by 22 publications

References 17 publications

Elucidation of the structure of an alanine‐lacking core tetrasaccharide trisphosphate from the lipopolysaccharide of Pseudomonas aeruginosa mutant H4

Elucidation of the structure of an alanine‐lacking core tetrasaccharide trisphosphate from the lipopolysaccharide of Pseudomonas aeruginosa mutant H4

Chemical and chromatographic analysis of lipopolysaccharide from an antibiotic-supersusceptible mutant of Pseudomonas aeruginosa

Cell Walls, Lipids, and Lipopolysaccharides of Pseudomonas Species

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