Pauline M. Meadow

The relationship between the O-antigenic lipopolysaccharides and serological specificity in strains of Pseudomonas aeruginosa of different O-serotypes.

SUMMARY: The lipopolysaccharides from fifteen strains of Pseudomonas aeruginosa used for serotyping have been isolated and analysed. They all contained heptose, glucose, rhamnose, glucosamine, galactosamine, 2-keto-3-deoxyoctonic acid and alanine. Many of them contained additional sugars and amino compounds, some of which have been shown to be amino sugars. Their composition and structure suggested the existence of a common region of low molecular weight in all the lipopolysaccharides. They could be classified by their chemistry and that of their degradation products into twelve chemogroups, nine of which contained one serotype and three of which contained two serotypes. All the lipopolysaccharides contained the same fatty acids, 3-OH 10:0, 2-OH 12:0, 3-OH 12:0 and 12:0. The isolated lipopolysaccharides reacted with the homologous Habs type antisera. The serological specificity was limited to the high molecular weight regions thought to correspond to side chains.

The extractable lipids of Pseudomonas aeruginosa

Abstract 1. 1. The composition, distribution and turnover of the extractable lipids of Pseudomonas aeruginosa 8602 have been studied. 90% of these extractable lipids were phospholipids, phosphatidyl ethanolamine being the major component under all conditions of growth. There were smaller amounts of bis-phosphatidyl glycerol and phosphatidyl glycerol and a fourth unidentified phospholipid. The phospholipids contained myristic acid, A-hexadecanoic acid, N-octadecanoic acid, hexadec-9-enoic acid and octadec-11-enoic acids as well as the C17 cyclopropane acid, methenyl-hexadecanoic acid, and the C19 cyclopropane acid, methenyloctadecanoic acid. No differences were detected between the extractable lipids of various cell fractions. 2. 2. The major changes in phospholipid and fatty acid composition throughout the growth cycle and under various growth conditions were in the proportion of cyclopropane acids present. These acids increased during the stationary phase and when the cells were grown under conditions of O2 limitation and decreased in limiting Mg2+. 3. 3. Cyclopropane acid synthesis was measured by the incorporation of [ Me- 14 C ] methionine during the stationary phase. It occurred faster in phosphatidyl glycerol than in phosphatidyl ethanolamine. Bis-phosphatidyl glycerol apparently was not a substrate. The amount of octadecenoic acid converted into C19 cyclopropane acid was higher in the 1 than in the 2 position of the phospholipids.

The Isolation and Characterization of Lipopolysaccharide-defective Mutants of Pseudomonas aeruginosa PAC1

Mutants with defective lipopolysaccharides (LPSs) were isolated from Pseudomonas aeruginosa PACIR (Habs serogroup 3) by selection for resistance to aeruginocin from P. aeruginosa PI6 Carbenicillin-sensitive mutants were isolated from P. aeruginosa PACI but not all had defective LPSs. Rough colonial morphology and resistance to bacteriophage II9X appeared to be independent of LPS composition. The LPSs from five mutants were analysed and compared with that of the parent strain. Separation of partially-degraded polysaccharides from LPS from PACI on Sephadex G75 yielded two different high molecular weight fractions and a phosphorylated low molecular weight fraction (L). The mutant LPSs lacked most or all of the high molecular weight fractions but retained some low molecular weight material. That from PACI and two of the mutants was separated by elution from Biogel P6 into two fractions. One, L2, was the core polysaccharide while the other, LI, contained short antigenic side-chains attached to the core like the semi-rough (SR) LPSs of the Enterobacteriaceae. The two mutants which gave the LI fraction with Habs 3 and PACI antisera as did the parent strain. The other three mutants were unreactive and their LPSs contained core components only. One appeared to have a complete core while the other two lacked rhamnose and rhammose plus glucose respectively. Thus there may be four types of LPS in PACI: one contains unsubstituted core polysaccharide and yields L2 on acid hydrolysis, another has short antigenic side-chains of the SR type and yields the LI fraction, while the two high molecular weight fractions are derived from core polysaccharides with different side-chains.

Receptor Sites for R-type Pyocins and Bacteriophage E79 in the Core Part of the Lipopolysaccharide of Pseudomonas aeruginosa PAC1

Pseudomoms aeruginosa strains and growth conditions. The parent strains PAC1 (NCIB 10848) and PAC~R and the LPS-defective mutants ~~~556, ~~~605 and ~~~610 have all been described previously (Koval & Meadow, 1977; Meadow et al., 1978). PAC~R was used as the parent strain for subsequent isolations because it is insensitive to several bacteriophages active against PAcl, but its LPS composition is indistinguishable from that of the latter (Chester & Meadow, 1975). ~~~611 and ~~~609 were isolated from PAC~R by selection for resistance to the pyocin produced by P. aeruginosa NIHD. Pyocin-producing strains were obtained as follows: strains ISE, sero6, ~21, ~~430,1s4, ISD, ~31, ISC and ISA

Evidence for the occurrence of Permeases for tricarboxylic acid cycle intermediates in Pseudomonas aeruginosa.

SUMMARY: Washed suspensions and cell-free extracts of Pseudomonas aeruginosa grown on Lemco agar, to which intermediates of the tricarboxylic acid cycle had been added, were tested for their ability to oxidize succinate, fumarate, malate, pyruvate, acetate, α-oxoglutarate and citrate. Whole organisms had lag periods of 2-3 hr. before citrate was oxidized rapidly, except when citrate or acetate had been added to the growth medium. There were lag periods of about 10 min. before rapid and linear oxidation of succinate by organisms grown on acetate; of fumarate by organisms grown on acetate or pyruvate; and of acetate by organisms grown on malate, pyruvate, α-oxoglutarate, fumarate or succinate. There were no lags for malate, pyruvate or α-oxoglutarate by organisms grown on any of the substrates tested. Organisms grown on malonate or Lemco agar to which no additions had been made had lags for all the substrates. Glucose-grown organisms had lags for all the intermediates except succinate. Only malonate-grown organisms oxidized malonate rapidly and linearly, and organisms grown without malonate had lag periods of 2-3 hr. before oxidizing this substrate. Cell-free extracts from organisms with lag periods before the oxidation of citrate, acetate, fumarate, malate, succinate and α-oxoglutarate were shown to oxidize these substrates without a lag period. Pyruvate and malonate were not oxidized by any of the extracts including those from organisms grown on these substrates. Chloramphenicol inhibited the adaptation to substrates by whole organisms but was without effect on the oxidation by cell-free extracts. The significance of these findings in relation to the hypothesis of specific permeases for the transport of organic molecules into the cell is discussed.

The Effect of Lipopolysaccharide Composition on the Ultrastructure ofPseudomonas aeruginosa

The surface structure of Pseudomonas aeruginosa PACl and PAClR and of lipopolysaccharide-defective mutants derived from them was studied by negative-staining and thin-section electron microscopy and compared with that of a rough mutant with wild-type lipopolysaccharide. The rough mutant and the parent strains had fairly smooth outer layers. Negatively stained preparations of all the mutants lacking polymerized O-antigenic sidechains, including a semi-rough mutant, showed numerous blebs on the surface. In thin sections of these mutants occasional extrusions from the surface were seen. They appeared to consist of material extruded from the outer membrane, but there was no evidence to suggest they were complete unit membranes. Polymerized O-antigenic side-chains in the lipopolysaccharide appear to be required to produce the wild-type appearance of the outer membrane in P. aeruginosa.

Synthesis of the Hydroxyacids in Lipid A of Pseudomonas aeruginosa

Summary: Washed suspensions and particulate fractions of Pseudomonas aeruginosa paci incorporated radioactivity from [i-14C]decanoic acid into bound 3-hydroxydecanoate, and from [i-14C]dodecanoate into bound 2- and 3-hydroxydodecanoate. Washed suspensions incorporated [i-14C]acetate into all three hydroxyacids, which are components of Lipid A in this organism. Degradation studies suggested that 2-hydroxydodecanoate was synthesized by direct hydroxylation of dodecanoate. Coenzyme A was required for synthesis of both 2- and 3-hydroxydodecanoates, synthesis of the former being stimulated by pyridine nucleotide coenzymes. Coenzyme A could not be replaced by acyl carrier protein which inhibited synthesis, but it was not required if [i-14C]dodecanoyl Coenzyme A was used as substrate. The hydroxyacids appeared to be incorporated into lipopolysaccharide since the radioactivity incorporated was extracted by hot 45% phenol and labelled hydroxyacids were then released only after acid or alkali hydrolysis.

Diaminopimelic Acid and Lysine Auxotrophs of Pseudomonas aeruginosa 8602

SUMMARY: Lysine auxotrophs have been isolated from Pseudomonas aeruginosa 8602. Three of the mutants were deficient in diaminopimelate decarboxylase and accumulated meso- and ll-diaminopimelic acid (DAP) but otherwise were indistinguishable from the parent strain. The fourth mutant required lysine for optimal growth, grew slowly on meso- but not ll-DAP, and the DAP which accumulated in large amounts was solely the ll-isomer. This mutant was deficient in diaminopimelate epimerase. No significant differences were detected between its wall composition and that of the parent strain but it was particularly sensitive to carbenicillin.

Characterization of Polyagglutinating and Surface Antigens in Pseudomonas aeruginosa

Mutants of Pseudomonas aeruginosa PAC1R (serotype O:3) which were resistant to bacteriophage D were isolated and shown to react with O:5d, O:9 and O:13 antisera as well as O:3. Antisera to the parent strain and to the three polyagglutinating (PA) mutants also showed cross-reactions. The mutants differed from the parent strain in their lipopolysaccharide (LPS) composition. The LPS from two of the three mutants yielded high molecular weight polysaccharide fractions. Although the high molecular weight fraction from one of the mutants contained the amino sugars and other components characteristic of the O:3 serotype strains, its mobility on Sephadex G75 was different from that of the parent strain. The high molecular weight material from the second mutant lacked the O-antigenic determinants but these were present in a semi-rough LPS fraction. The third mutant appeared rough and completely lacked the O-antigenic components. These three mutants were compared with the parent strain and with a non-agglutinating LPS-defective mutant which lacked both O-antigenic side chains and all neutral sugars in the outer core. Agglutination with absorbed sera and haemagglutination using purified LPS and ELISA detection suggested that wall components other than LPS were responsible for some of the cross-reactions observed. The components responsible were detected after SDS-PAGE of crude outer membrane fractions by a combination of Coomassie blue and silver-staining and antigenic components were detected by immunoelectrophoresis and ELISA-linked immunoblotting of the gels. The main antigenic determinants detected by antiserum to the parent strain were in the high molecular weight O-polysaccharide fractions and in the semirough fractions of the LPS, with some activity due to the H protein of the outer membrane. O:5d antisera reacted with unidentified high molecular weight polysaccharide fractions. Cross-reactions with the O:9 antiserum appeared to be due mainly to the F porin and, to a lesser extent, to the G and E proteins of the outer membrane. O:13 antiserum reacted with high molecular weight polysaccharide fractions but also with the rough core and F and H protein. Cross-reactivity of the other three mutant antisera could largely be interpreted in terms of the outer membrane components exposed in each strain. One reacted strongly with the F porin and the rough core, while the others reacted with a number of protein and LPS-derived fractions.(ABSTRACT TRUNCATED AT 400 WORDS)

The Accumulation of Nucleotides by Escherichia coli Strain 26–26

SUMMARY: Escherichia coli strain 26-26 (a mutant requiring lysine for growth) releases into the medium diaminopimelic acid, lipomucoprotein and nucleotides, including flavins, when grown with suboptimal concentrations of lysine. Cytidine diphosphate glycerol, cytidine diphosphate ribitol and a uridine-linked mucopeptide containing n-acetylmuramic acid, glutamic acid, mesodiaminopimelic acid and alanine were identified among the nucleotides extracted from the medium. Similar uridine diphosphate-linked mucopeptides were isolated from extracts made from bacteria at various stages of growth. In addition, uridine diphosphate-linked mucopeptides were isolated from bacterial extracts which were found to contain muramic acid and lysine but no diaminopimelic acid. The possible role of these compounds as precursors of cell wall structures is discussed.