The extraction of cell walls of Pseudomonas aeruginosa with aqueous phenol. Material from the phenol layer

1. A method is described for the fractionation of cell walls of Pseudomonas aeruginosa by using aqueous phenol. 2. With this technique, cell walls are quantitatively separated into five fractions: two water-soluble fractions (AqI and AqII), a residual insoluble fraction (R), and two phenol-soluble fractions (PhMP and PhMS). 3. The compositions of fractions PhMP and PhMS are discussed. Fraction PhMP consists almost entirely of protein, which is soluble in aqueous sodium dodecyl sulphate. Analytical ultracentrifugation of the solution yields a single peak, but several components may be resolved by gel electrophoresis. 4. Fraction PhMS consists principally of phospholipid (approx. 44%) and fatty acids (approx. 27%), although smaller amounts (approx. 13%) of protein are present.     

Isolation and analysis of sacculi from Streptococcus sanguis.

Sacculi were prepared from Streptococcus sanguis 34 by exhaustive extraction of bacteria with hot 1% sodium dodecyl sulfate-0.5% 2-mercaptoethanol. Lyophilized residue was dissociated by brief sonication to single bodies closely resembling streptococci in phase-contrast microscopic density, staining properties, and morphology. Electron micrographs revealed bodies that contained variable amounts of cellular contents and were bounded by intact cell walls. Chemical analyses of sacculi demonstrated the presence of peptidoglycan, carbohydrate, protein, and phosphate. The hexose content of sacculi varied 10-fold depending upon the composition of the growth medium. When sacculi were subjected to treatment with 5 M LiCl, 8 M urea, 40% phenol (25 degrees C), or dimethyl sulfoxide most of the nitrogen and carbohydrate present was recovered in the insoluble fraction. These data suggest that sacculi contain the cell wall fraction of the extracted bacteria and that most of the carbohydrates and proteins of sacculi are firmly bound to the insoluble fraction, which contains the peptidoglycan matrix.     

Ultrastructure of the cell envelope and amino acid composition of the murein of Clostridium symbiosum

Abstract The cell envelope of the Gram-negative staining Clostridium symbiosum is 18 nm thick. It appears triple-layered and consists of an inner electrondense layer of about 5 nm, a lighter zone of 4 nm and an outer electron-dense layer of 9 nm. The inner layer corresponds to the murein sacculus, since the isolated peptidoglycan sacculi showed a thickness of 3–5 nm. Analysis showed that it belongs to the A₂pm-direct murein type. The outer layer could be removed by sodium dodecylsulfate. It contained mainly protein, small amounts of sugars and essentially no lipid, indicative of an S-layer rather than a typical Gram-negative type of outer membrane. Furthermore, l -alanine aminopeptidase activity characteristic of Gram-negative aerobic bacteria was absent in this organism and in other anaerobic Gram-negative bacteria tested. This demonstrates that such activity is an unreliable tool for the classification of anaerobic eubacteria. In spite of the thin murein layer, which is the likely reason for the Gram-negative reaction, the anaerobic growth, peritrichous flagellation and endospore formation indicate that this organism belongs to the genus Clostridium .     

In vitro synthesis of cross-linked murein and its attachment to sacculi by PBP1A from Escherichia coli

The penicillin-binding protein (PBP) 1A is a major murein (peptidoglycan) synthase in Escherichia coli. The murein synthesis activity of PBP1A was studied in vitro with radioactive lipid II substrate. PBP1A produced murein glycan strands by transglycosylation and formed peptide cross-links by transpeptidation. Time course experiments revealed that PBP1A, unlike PBP1B, required the presence of polymerized glycan strands carrying monomeric peptides for cross-linking activity. PBP1A was capable of attaching nascent murein synthesized from radioactive lipid II to nonlabeled murein sacculi. The attachment of the new material occurred by transpeptidation reactions in which monomeric triand tetrapeptides in the sacculi were the acceptors.     

Lipid A mutants of Salmonella typhimurium. Purification and characterization of a lipid A precursor produced by a mutant in 3-deoxy-D-mannooctulosonate-8-phosphate synthetase.

We describe here the isolation, purification, and structural characterization of a lipid A precursor synthesized under nonpermissive conditions by a mutant of Salmonella typhimurium conditionally defective in the synthesis of the 3-deoxy-D-mannoctulosonate (2-keto-3-deoxyoctonate, KDO) region of the lipopolysaccharide. The precursor was isolated free from lipopolysaccharide, murein, and phospholipids by extraction of delipidated cells with 90% phenol/CHCL3/petroleum ether. The molecule was recovered from the phenol phase after precipitation of lipopolysaccharide with H2O and subsequently purified by DEAE-cellulose chromatography. Structural analyses showed that the lipid A precursor is a phosphorylated glucosamine disaccharide containing one ester and two amide-linked residues of beta-hydroxymyristate. In contrast to lipid A, the precursor disaccharide lacks ester-linked 12:0 and 14:0 fatty acids as well as KDO. The molecule contains 2 phosphate residues both of which were identified as phosphomonoesters by 31P NMR spectroscopy. One of the phosphomonoesters is located in position 1 of the reducing terminal glucosamine residue; the location of the other phosphomonoester was not determined. The structure of the precursor provides strong support for the conclusion that KDO incorporation occurs at an early stage in lipid A biosynthesis prior to the incorporation of ester-linked saturated fatty acids.     

Evidence for multisite growth of Escherichia coli murein involving concomitant endopeptidase and transpeptidase activities.

During diaminopimelic acid starvation of Escherichia coli W7, a large fraction of the preexisting murein cross-links are opened by murein endopeptidase and the resulting uncross-linked material is degraded. This is reflected morphologically in a general loss of rigidity of the murein sacculus long before lysis occurs. In growing cells, a dynamic situation is demonstrable. When cells whose murein sacculi are uniformly labeled with [14C]diaminopimelic acid were chased with unlabeled DAP, a significant, rapid shift of [14C]diaminopimelic acid from the donor to the acceptor half of dimers was observed. The shift can be explained by the presence of about 100 separate sites where new murein strands were being inserted between old radioactive strands of murein. Thus, the gradual loss of rigidity of the murein sacculus as endopeptidase continues to function during starvation of E. coli W7 suggests an even distribution of the active endopeptidases. This is consistent with the kinetic data which suggest that endopeptidase, along with murein synthetase and transpeptidase, acts at about 100 distinct sites to elongate the murein sacculus.     

Carbohydrate composition of the phenol-soluble lipopolysaccharides of Citrobacter freundii

Phenol-soluble lipopolysaccharides were obtained from the interphase and phenol phase fractions of 44% aqueous phenol-extracted Citrobacter species. Upon detailed investigation of C. freundii 8090, the two lipopolysaccharide fractions were found to contain different amounts of lipid A, although qualitative composition was similar. Both contained lipid A, 2-keto-deoxyoctonic acid, heptose, phosphate, d-glucose, galactose, rhamnose, 2-acetamido-2 deoxy-d-glucose, 3-acetamido-3,6-dideoxy-d-glucose, O-acetyl, and trace amino acids. Partially purified phenol-phase lipopolysaccharide partitioned into the phenol-soluble phase when refractionated with 44% aqueous phenol, and was further found to be soluble in 88% phenol, 95% ethyl alcohol, and chloroform-methanol (2:1).     

Novel type of murein transglycosylase in Escherichia coli.

The purification and properties of a novel type of murein transglycosylase from Escherichia coli are described. The purified enzyme appears as a single band on sodium dodecyl sulfate-polyacrylamide gels and has an apparent molecular weight of approximately 65,000 as estimated by gel filtration and gel electrophoresis. It degrades pure murein sacculi from E. coli almost completely into low-molecular-weight products. The two prominent muropeptide fragments in the digest are the disaccharide-tripeptide N-acetylglucosamine-N-acetylmuramic acid-L-alanine-D-iso-glutamic acid-meso-diaminopimelic acid and the corresponding disaccharide-tetrapeptide N-acetylglucosamine-N-acetylmuramic acid-L-alanine-D-iso-glutamic acid-meso-diaminopimelic acid-D-alanine. The unique feature of these compounds is that the disaccharide has no reducing end group and that the muramic acid residue possesses an internal 1 leads to 6 anhydro linkage. The new lytic enzyme is designated as a murein: murein transglycosylase. Its possible role in the rearrangement of murein during cell growth and division is discussed.     

The structure of the heptose-3-deoxy-D-mannooctulosonic-acid region in a mutant form of Aeromonas salmonicida lipopolysaccharide

Lipopolysaccharide was isolated from a phage-selected mutant of a wild strain of Aeromonas salmonicida by the aqueous phenol method. The lipopolysaccharide consisted of the R form, containing per mole, three moles of L-glycero-D-manno-heptopyranose, one mole of 3-deoxy-D-manno-2-octulosonic acid (dOclA) and lipid A. The dOclA was not fully assayable by the thiobarbituric acid methods usually used, but its degradation product was detected, after Smith degradation of the lipopolysaccharide, either as free 3-deoxy-2-heptulosonic acid (after hydrolysis) or substituted by a mannopyranosyl residue derived from heptose. Mass spectrometry indicated that the dOclA existed in the furanose form and was substituted by the heptose trisaccharide through position six. Methylation analysis, chemical degradation, chromium trioxide oxidation and nuclear magnetic resonance spectroscopy were used to identify the structure of the core oligosaccharide as: L alpha DHepp(1----2)L alpha DHepp(1----3)L alpha DHepp(1----6)dOclAf(2----.     

The chemical composition of the lipopolysaccharide of Pseudomonas aeruginosa

1. Lipopolysaccharide was isolated from both cell walls and acetone-dried whole cells of Pseudomonas aeruginosa (N.C.T.C. 1999). 2. Closely similar products are obtained, although that from whole cells cannot be completely freed from small amounts (2-7%) of residual nucleic acids. 3. The lipid moiety (23-33%) has a similar amino sugar backbone to that of lipids of enterobacterial lipopolysaccharides, but contains different hydroxy acids (2- and 3-hydroxydodecanoic acid and 3-hydroxydecanoic acid). 3-Hydroxytetradecanoic acid is absent, and 3-hydroxydodecanoic acid is the main N-acylating acid. No clear evidence permitting a distinction between the possibilities that phosphodiester or glycosidic linkages exist between the glucosamine residues was obtained. 4. Identifiable sugars (glucose, rhamnose, 3-deoxy-2-octulonic acid and heptose) account for less than 20% of the lipopolysaccharide, and alanine, galactosamine and fucosamine are apparently components of the polysaccharide moiety. 5. The polysaccharide moiety is unusual in that it is not readily obtained from the lipopolysaccharide by treatment with dilute acetic acid, which does, however, solubilize much of the phosphorus of the lipopolysaccharide. 6. The ;polysaccharide' fraction (approx. 21%) obtained by treatment with dilute acetic acid contains only a small proportion of the total polysaccharide components, and in one case only 45% of the fraction was accountable for in terms of identifiable components. 7. Evidence suggests that unidentified nitrogenous components are concentrated in the residual material after removal of both the lipid and the ;polysaccharide' fraction from the lipopolysaccharide.     

Murein and lipopolysaccharide biosynthesis in synchronized cells of Escherichia coli K 12 and the effect of penicillin G,mecillinam and nalidixic acid

The incorporation of radioactive N-acetylglucosamine into murein and lipopolysaccharide of synchronized cells of Escherichia coli K 12 was followed over 100 min in the presence of antibiotics. At 20 min intervals cell walls were prepared. Lipopolysaccharide and murein sacculi were isolated and the radioactivity was quantified in both polymers. Labelled, newly synthesized murein was characterized according to murein subunits linked to lipoprotein, and the degree of crosslinkage. Furthermore, murein subunits containing anhydromuramic acid were determined, permitting the calculation of the average glycan chain length. The results indicated that penicillin G at 30 g/ml stimulated the incorporation of new murein subunits into sacculi followed by a sudden increase in lipopolysaccharide incorporation into the outer membrane. The degree of crosslinkage in murein synthesized in the presence of 30 g/ml penicillin G was higher than in the control, and almost twice as high as in murein synthesized in the presence of 20 g/ml nalidixic acid. Both antibiotics inhibited cell division at the concentrations indicated. Murein synthesized in the presence of 2 g/ml mecillinam also showed higher crosslinkage. However, about twice as much anhydromuramic acid-containing subunits were observed as in the control. At the same time lipopolysaccharide incorporation into the outer membrane was stimulated two- to three-fold.Abbreviation GlcNAc N-acetylglucosamine     

Isolation of ribonucleic acid from animal tissue by use of chaotropic agents

A method is presented for solubilizing and isolating pulse-labeled RNA from isolated nuclei, whole cells, and the insoluble material which collects at the interface between the phenol and aqueous layers during phenol extraction procedures. For this we have used solutions of the chaotropic salt, lithium trichloroacetate. It appears that the method may be useful for extracting the more stable RNA species as well. The RNA extracted from whole cells and phenol interface precipitate is undegraded, as determined by acrylamide gel electrophoresis after exposure to dimethyl sulfoxide. Pulse-labeled RNA extracted from isolated nuclei shows a marked tendency to aggregate and this fraction is being investigated further.     

State of the rigid-layer in cell walls of some gram-negative bacteria

Summary Rigid-layers in cell walls of Enterobacteriaceae and Pseudomonadales contain murein sacculi of identical chemotype. However, lipoprotein in covalent linkage to the murein participates to a different extent in rigid layer construction in the two taxonomic groups. In Proteus mirabilis and Escherichia coli lipoprotein particles of similar size and spatial arrangement are visible in the electron microscope as major rigid layer components. In the cell walls of Pseudomonadales covalent (lipo-)protein plays a far less prominent role. It is present as a minor constituent in rigid layers of Pseudomonas aeruginosa. Rigid layers of Spirillum serpens are naked murein sacculi devoid of any covalently attached protein.     

Murein biosynthesis in synchronized cells of Proteus mirabilis. Quantitative analysis of O-acetylated murein subunits and of chain terminators incorporated into the sacculus during the cell cycle

Cells of Proteus mirabilis, synchronized by sucrose density gradient centrifugation, were grown in complex medium containing radioactive N-acetylglucosamine. At various times, labelled murein sacculi were isolated and digested with endo-N,O-acetylmuramidase from Chalaropsis. The murein fragments thus obtained were separated into disaccharide peptides as the monomeric subunits and into peptide-cross-linked subunits by gel filtration. The subunits were further differentiated into O-acetylated and non-O-acetylated species, and into subunits containing anhydro-N-acetylmuramic acid which were glycan chain terminators in the native sacculi. Quantification of the subunit species gave the following results. At specific times during the cell cycle, murein subunits were lost from the polymer and a transient decrease in cross-linkage was observed. The overall degree of cross-linkage in mature murein, i.e. the ratio of peptide-cross-linked subunits versus uncross-linked subunits, was 1.15 as determined by regression analysis. Anhydro-N-acetylmuramic-acid-containing murein subunits representing glycan chain terminators were found either peptide-cross-linked or uncross-linked as monomers. Since these two subunit species were recovered in a defined ratio of 1.6, mature murein consisted of at least two different types of glycan chains. On average, each chain contained 15.4 murein subunits. About 60% of the murein subunits in mature murein were O-acetylated and showed a higher degree of cross-linkage than the non-O-acetylated portion. Finally, following the composition of the sacculus during the cell cycle revealed a complex precursor-product relationship between non-O-acetylated and O-acetylated subunits during murein maturation. The data allowed us to deduce several features of the assembly process of murein sacculi.     

Purification and properties of a membrane-bound lytic transglycosylase from Escherichia coli.

A membrane-bound lytic transglycosylase (Mlt) has been solubilized in the presence of 2% Triton X-100 containing 0.5 M NaCl from membranes of an Escherichia coli mutant that carries a deletion in the slt gene coding for a 70-kDa soluble lytic transglycosylase (Slt70). The enzyme was purified by a four-step procedure including anion-exchange (HiLoad SP-Sepharose and MonoS), heparin-Sepharose, and poly(U)-Sepharose 4B column chromatography. The purified protein that migrated during denaturing sodium dodecyl sulfate-polyacrylamide gel electrophoresis as a single band corresponding to an apparent molecular mass of about 38 kDa is referred to as Mlt38. Optimal activity was found in buffers with a pH between 4.0 and 4.5. The enzyme is stimulated by a factor of 2.5 in the presence of Mg2+ at a concentration of 10 mM and loses its activity rapidly at temperatures above 30 degrees C. Besides insoluble murein sacculi, the enzyme was able to degrade glycan strands isolated from murein by amidase treatment. The enzymatic reaction occurred with a maximal velocity of about 2.2 mg/liter/min with murein sacculi as a substrate. The amino acid sequences of four proteolytic peptides showed no identity with known sequences in the data bank. With Mlt38, the number of proteins in E. coli showing lytic transglycosylase activity rises to three.     

Subcellular distribution of the soluble lytic transglycosylase in Escherichia coli.

The localization of the major autolytic enzyme, the soluble lytic transglycosylase, in the different cell compartments of Escherichia coli was investigated by immunoelectron microscopy. Ultrathin sections were labeled with a specific antiserum against purified soluble lytic transglycosylase, and the antibody-enzyme complexes were visualized with colloidal protein A-gold. A preferential localization of the lytic transglycosylase in the envelope was observed, with only 20 to 30% of the enzyme left in the cytoplasm. Most of the enzyme associated with the cell wall was tightly bound to the murein sacculus. Sacculi prepared by boiling of cells in 4% sodium dodecyl sulfate could be immunolabeled with the specific antiserum, indicating a surprisingly strong interaction of the lytic transglycosylase with murein. The enzyme-substrate complex could be reconstituted in vitro by incubating pronase-treated, protein-free murein sacculi with purified lytic transglycosylase at 0 degrees C. Titration of sacculi with increasing amounts of enzyme indicated a limiting number of binding sites for about 1,000 molecules of enzyme per sacculus. Ruptured murein sacculi obtained after penicillin treatment revealed that the enzyme is exclusively bound to the outer surface of the sacculus. This finding is discussed in the light of recent evidence suggesting that the murein of E. coli might be a structure of more than one layer expanding by inside-to-outside growth of patches of murein.     

Changes in murein composition during the cell cycle of Caulobacter crescentus

The amino acid and muropeptide compositions of murein (peptidoglycan) isolated from populations of Caulobacter crescentus predominantly composed of swarmer or stalked cells were determined and compared with the structure of murein sacculi obtained from a population of unsegregated cells. It appears that in spite of vast morphological alterations in the course of the cell cycle, the murein composition of the various cell types is not markedly different.     

Cell wall composition and incorporation of radio-labelled compounds by Veillonella alcalescens.

The cell wall of Veillonella alcalescens was shown to have a typically Gram-negative appearance and composition. The wall contains 24% lipid, 0.8% phosphorus, and 6.8% hexosamine. It is estimated to contain about 5% murein, unlike the 24% reported by other for Veillonella parvula. The amounts of 19 amino acids, including diaminopimelic acid, were determined. Though Veillonella sp. cannot metabolize sugars for energy, V. alcalescens incorporates ribose and fructose by separate, specific mechanisms and uses most of the incorporated sugar in nucleic acid synthesis. Large excesses of either sugar in the medium do not repress gluconeogenesis from the pyruvate level. We have been unable to detect phosphoglyceromutase (EC 2.7.5.3) by several assay methods but have no indication of a gluconeogenic pathway other than reverse glycolysis.     

Isolation and characterization of the unusual lipopolysaccharide component, 2-amino-2-deoxy-2-N-(27-hydroxyoctacosanoyl)-3-O-(3-hydroxy- tetradecanoyl)-gluco-hexuronic acid, and its de-O-acylation product from the free lipid A of Rhizobium trifolii ANU843

Two new, unusual lipid A components have been isolated and characterized from the free lipid A of Rhizobium trifolii ANU843. 2-Amino-2-deoxy-2-N-(27-hydroxyoctacosanoyl)-3-O-(3-hydroxy- tetradecanoyl)-gluco-hexuronic acid and its de-O-acylation product were purified from the chloroform/methanol extract of a mild acid hydrolysate of the lipopolysaccharide by chromatography on C18 reverse-phase columns and layers. The compositions of the two compounds were determined by releasing the acyl components by exhaustive acid-catalyzed methanolysis and identifying them as their methyl esters by gas chromatography and gas chromatography/mass spectrometry. The sugar component was identified by converting it to the alditol acetate derivative of glucosamine in a two-step reduction and identifying it as such by gas chromatography/mass spectrometry. The linkages of the fatty acyl components to the sugar residue and the configuration of the sugar component was confirmed by 1H and 13C NMR spectroscopy. The complete structures of the two compounds were further confirmed by fast atom bombardment mass spectrometry. It is still unsure whether the de-O-acylated derivative was formed from the di-acyl compound by de-O-acylation during acid hydrolysis. These structures represent the first report of 2-amino-2-deoxy-gluco-hexuronic acid in the free lipid A of a Gram-negative bacterium and confirms our earlier contention (Hollingsworth, R.I., and Carlson, R. W. (1989) J. Biol. Chem. 264, 9000-9303) of the involvement of 27-hydroxyoctacosanoic acid in the structure of the lipopolysaccharide of Rhizobium trifolii ANU843.     

Distribution of lipopolysaccharide and the detection of a new subfraction in the cell envelope of a marine pseudomonad.

The three outer layers of the cell envelope of marine pseudomonad B-16, the loosely bound outer layer, the outer membrane, and the periplasmic space layer, are the only ones containing appreciable amounts of both lipid and carbohydrate. These layers and a fraction released into the medium during growth of the cells were examined for the presence of common antigens by double immunodiffusion using anti-whole serum. Each of the layers, the medium fraction, and lipopolysaccharide (LPS) isolated from the organism were shown to contain two or more diffusible components showing reactions of identity. Thus LPS is found in each of the three outer layers of the cell envelope of this gram-negative bacterium. The periplasmic space layer was found to contain a fraction accounting for 20% of the dry weight of the layer, which was sedimentable at 30,000 x g and contained lipid, protein, and carbohydrate. Double-immunodiffusion tests indicated that the fraction contained at least one of the two antigens present in isolated LPS. A particulate material was released by the cells during growth which gave a positive test for 2-keto-3-deoxyoctulosonic acid and cross-reacted serologically with LPS.