Stephen G. Wilkinson

The Effect of Ethylenediaminetetra-acetic Acid on the Cell Walls of Some Gram-Negative Bacteria

SUMMARY: A comparative survey of amino acid, amino sugar, sugar and lipid components of the cell walls of strains of Pseudomonas aeruginosa, Alcaligenes faecalis, Escherichia coli and Proteus mirabilis was made. The cell walls of P. aeruginosa and A. faecalis, against which ethylenediaminetetra-acetic acid (EDTA) has a potent bactericidal action, differed from those of the other organisms principally in their sugar components and in their high content of phosphorus. EDTA at alkaline pH selectively solubilized a high proportion of the carbohydrate and phosphorus present, apparently as lipopolysaccharides, in the walls of sensitive organisms. It is suggested that metal cations and lipopolysaccharides in the cell walls of P. aeruginosa and A. faecalis may be essential to the structural integrity of these organisms.

Phenotypic variation of lipid composition in Burkholderia cepacia : a response to increased growth temperature is a greater content of 2-hydroxy acids in phosphatidylethanolamine and ornithine amide lipid

Burkholderia cepacia produces an unusual range of polar lipids, which includes two forms each of phosphatidylethanolamine (PE) and ornithine amide lipid (OL), differing in the presence or absence of 2-hydroxy fatty acids. By using chemostat cultures in chemically defined media, variations in the lipid content and the proportions of individual lipids have been studied as a function of (a) growth temperature, (b) growth rate and (c) growth-limiting nutrient (carbon, magnesium, phosphorus or oxygen). Total cellular lipid in carbon-limited cultures was lowest at high growth temperatures and low growth rates. Increases in growth temperature over the range 25-40 degrees C led to increases in the proportions of molecular species of PE and OL containing 2-hydroxy acids, without changing the PE:OL ratio. Growth temperature did not alter the balance between neutral and acidic lipids, but the contribution of phosphatidylglycerol to the latter increased with rising growth temperature and growth rate. Pigmentation of cells and the presence of flagella were also temperature-dependent. Change in growth rate also affected the PE:OL ratio and the extent to which monoenoic acids were replaced by their cyclopropane derivatives. Whereas similar lipid profiles were found for carbon-, magnesium- and oxygen-limited cultures, ornithine amides were the only polar lipids detected in phosphorus-limited cells.

Polar lipids and fatty acids of Pseudomonas cepacia

The polar lipids and fatty acids produced by the reference strains for seven different O serogroups of Pseudomonas cepacia have been identified. Similar results were obtained for all strains. Contrary to a previous report, the only significant phospholipids in this species are phosphatidylethanolamine and bis(phosphatidyl)glycerol, which contributed 57-83% and 17-43%, respectively, of the total lipid phosphorus. The former lipid was found as two chromatographically distinct fractions. In the less polar fraction and in bis(phosphatidyl)glycerol, the major fatty acids were hexadecanoic acid, cis-9,10-methylenehexadecanoic acid, cis-octadec-11-enoic acid, and cis-11,12-methyleneoctadecanoic acid. In the more polar fraction of phosphatidylethanolamine, the fatty acid in one position is a 2-hydroxy acid, mainly 2-hydroxyhexadecenoic acid, 2-hydroxyhexadecanoic acid, 2-hydroxyoctadecenoic acid, or 2-hydroxymethyleneoctadecanoic acid. Compared with other phospholipids, this fraction of phosphatidylethanolamine was depleted in cis-9,10-methylenehexadecanoic acid. Each strain also produced two ornithine amide lipids. In the major lipid, 3-hydroxyhexadecanoic acid was amide-bound to the alpha-amino group and was itself probably esterified by a 2-hydroxy acid, mainly 2-hydroxyoctadecenoic acid or the derived cyclopropane acid. In the minor ornithine amide lipid, the ester-bound acids were mainly methyleneoctadecanoic acid and hexadecanoic acid. The unusual lipid profiles of P. cepacia are of chemotaxonomic interest.

The Sensitivity of Pseudomonads to Ethylene-diaminetetra-acetic Acid

Surmmary Forty-one strains representing 21 nomen species of the genus Pseudomonas and 2 of the genus Alcaligenes were screened for sensitivity to ethylene-diaminetetra-acetic acid (EDTA). Sensitivity of organisms was assessed by determining the release of intracellular solutes and the loss of viability under the action of EDTA. The latter criterion was found to be the more useful for the differentiation of sensitive and resistant organisms. Strains of Pseudomonas diminuta, P. geniculata, P. iodinum, P. maltophilia, P. pavonacea and possibly P. rubescens were considered as resistant to EDTA. Strains of P. alcaligenes were clearly differentiated from strains of Alcaligenes faecalis, which were highly resistant to EDTA. The practical value and possible taxonomic significance of the results are discussed.

Lipopolysaccharides from Pseudomonas maltophilia: Composition of the lipopolysaccharide and structure of the side-chain polysaccharide from strain N.C.I.B. 9204

Lipopolysaccharide was extracted from defatted cell-walls of Pseudomonas maltophilia N.C.I.B. 9204. The major fatty acid components were 9-methyldecanoic acid, 2-hydroxy-9-methyldecanoic acid, 3-hydroxy-9-methyldecanoic acid, 3-hydroxy-dodecanoic acid, and 3-hydroxy-11-methyldodecanoic acid. Monosaccharide components of the phosphorylated core-oligosaccharide were D-glucose, D-mannose, D-galacturonic acid, 2-amino-2-deoxyglucose, and a 3-deoxyoctulosonic acid. The putative O-specific polysaccharide was composed mainly of 2-amino-2-deoxy-D-glucose, D-arabinose, and 6-deoxy-L-talose, but also contained an O-acetyl group and small proportions of rhamnose and 6-deoxy-3-O-methyltalose. Degradative and n.m.r. (1H and 13C) studies showed that the polymer had a branched trisaccharide repeating-unit with the following structure; the O-acetyl group was tentatively assigned to C-2 of the 6-deoxytalopyranosyl residue. (Formula: see text).

Lipopolysaccharide from the O14 type strain of Serratia marcescens: Structural studies of a polymeric fraction

Abstract The lipopolysaccharide from Serratia marcescens C.D.C. 4444-60, the reference strain for O-serogroup 14, has been analysed. All of the phosphorus of the lipopolysaccharide appeared to be present in the lipid A region as orthophosphoric mono-and di-ester residues. Mild hydrolysis of the lipopolysaccharide with acid released orthophosphate, a 3-deoxy-2-octulosonic acid, and an unidentified related compound. The major components of the core oligosaccharide were probably d -glucose and l - glycero - d - manno -heptose; lesser components included the 3-deoxy-2-octulosonic acid, d - glycero - d - manno -heptose, and 2-amino-2-deoxyglucose. Products of high molecular weight included a polysaccharide having the disaccharide repeating-unit →4)-α- d -Gal p NAc-(1 → 2)-β- d -Rib f -(1 →. The same polysaccharide was isolated from the lipopolysaccharide of a second O14 strain of S. marcescens , but was absent from a third. All three lipopolysaccharides contained polymeric material rich in glucose and mannose.

Lipid Composition and Chemotaxonomy of Pseudomonas putrefaciens (Alteromonas putrefaciens)

The major polar lipids in cells of Pseudomonas putrefaciens NCIB 10472 grown on nutrient agar were phosphatidylethanolamine, phoisphatidylglycerol, a glucosyldiacylglycerol, a glucuronosyldiacylglycerol and an ornithine amide lipid. An additional phospholipid, tentatively identified as acyl phosphatidylglycerol or bis-phosphatidic acid, was a trace component of the wall lipids from broth cultures, which lacked the glycolipids and the ornithine amide lipid. The wall lipids from broth cultures of three further strains of P. putrefaciens (NCIB 10471, NCIB 11156 and NCTC 10737) contained all of the above lipids, and in two cases (strains NCIB 10471 and NCIB 11156) had an unusually high content of free fatty acid. Fatty acid compositions of the extractable lipids were qualitatively similar for all four strains: the major components were iso-pentadecanoic acid, pentadecanoic acid, a cis-heptadecenoic acid and a cis-hexadecenoic acid. Anteiso fatty acids were minor components in strain NCIB 10472. Lipid mixtures in which the ornithine amide lipid was present also contained small amounts of beta-hydroxy fatty acids: in strain NCIB 10472 the major ones were the straight-chain and iso-branched C16 acids. Lipopolysaccharides from all four strains had similar, complex fatty acid compositions. The major non-hydroxy acids were the straight-chain and iso-branched C13 acids. beta-Hydroxy acids common to all strains included the straight-chain C11, C12, C13, C14 and C15 acids, together with branched-chain C13 and C15 acids probably belonging to the iso series. The lipopolysaccharide from strains NCIB 10472 also contained C12 and C14 hydroxy acids of the same series, and small amounts of C13 and C15 beta-hydroxy acids probably belonging to the anteiso series. The close resemblance in both polar lipid and fatty acid compositions between strains of P. putrefaciens and Pseudomonas rubescens is further evidence that these species are synonymous. Significant differences between the lipids and fatty acids of P. putrefaciens and those reported for a strain of Alteromonas haloplanktis do not harmonize with a proposal to transfer the former organism to the genus Alteromonas.

Structures of neutral glycans isolated from the lipopolysaccharides of reference strains for serratia marcescens serogroups o16 and o20

Neutral polymers have been isolated from the lipopolysaccharides of the reference strains for Serratia marcescens O16 and O20, serogroups which exhibit significant cross-reactivity. Both organisms produce a galactan with the disaccharide repeating-unit shown, and which apparently accounts for the serological observations. The same galactan has also been reported as the O4-specific polysaccharide of Pasteurella haemolytica. In S. marcescens O16, the galactan is apparently accompanied by a polymer of 2-substituted beta-D-ribofuranosyl residues.

Lipid composition and taxonomy of (Pseudomonas) echinoides: transfer to the genus Sphingomonas

Lipid components of [Pseudomonas] echinoides NCIMB 9420 have been studied as an aid to taxonomic relocation of the organism. Non-polar lipids include the carotenoid nostoxanthin and the ubiquinone Q-10. The major fatty acids are cis-vaccenic acid [18:1(11c)], hexadecanoic acid (16:0) and 2-hydroxy-tetradecanoic acid (2-OH-14:0), but 11-methyloctadec-11-enoic acid[11-Me-18:1(11)] is a significant minor component. The preponderant phospholipids are phosphatidylethanolamine and phosphatidylglycerol; minor lipids include bis(phosphatidyl)glycerol and an unidentified aminophospholipid. Several glycolipids are present, the major one being a glucuronosylceramide derived from sphinganine with amide-bound 2-OH-14:0. The lipid profile supports a proposal to reclassify the organism as Sphingomonas echinoides.

Polar lipids and fatty acids of Pseudomonas caryophylli, Pseudomonas gladioli and Pseudomonas pickettii

Summary: The polar lipids and their fatty acid components in Pseudomonas caryophylli, Pseudomonas gladioli and Pseudomonas pickettii have been identified. In addition to diphosphatidylglycerol and phosphatidylglycerol (a trace only for P. pickettii), all three species contained two forms of phosphatidylethanolamine differing in the presence or absence of α-hydroxy fatty acids. This seems to be a distinctive feature of species in Pseudomonas RNA homology group II. Also, P. caryophylli and P. gladioli (but not P. pickettii) produced two forms of ornithine amide lipid, differing in the nature (hydroxy or non-hydroxy) of the ester-linked fatty acid. In all three species, the major non-hydroxy acids were hexadecanoic acid, a hexadecenoic acid, an octadecenoic acid, and cyclopropane derivatives of the monoenoic acids. The α-hydroxy acids were the derivatives of the same components, while the amide-linked acid of the ornithine amide lipids was mainly or entirely 3-hydroxyhexadecanoic acid. The possible taxonomic implications of the data are discussed.