Total Cell Wall Research Articles

Structural and biochemical alterations of Nocardia asteroides cell walls during its growth cycle.

Analysis of Nocardia asteroides 14759 cell walls were done to determine the chemical and structural composition during the growth cycle. It was found that the ultrastructural profiles of the cell wall become altered as the cultures aged. Chemical analysis revealed corresponding shifts in cell wall components as the culture went from lag to logarithmic to stationary phases of growth. The peptidoglycan from lag-phase cells (5 h) represented 15% of the total cell wall weight, and the percentage of peptidoglycan progressively increased so that, in 1-week stationary-phase cells, it represented approximately 40% of the total wall weight. In lag-phase cells it was found that 36% of the cell wall weight was lipid in nature, whereas stationary-phase cells had only 7% lipid in their wall. The overall sugar composition of the walls remained relatively constant at about 28 to 31% however, the arabinose to galactose ratio changed from approximately 1:1 in lag-phase to 2:1 in stationary-phase cells. Gas-liquid chromatography demonstrated that the fatty acids making up the cell wall lipids changed relative to one another as the cells aged. Based upon the removal of lipids by ethanol-ether, chloroform, and alkaline methanol extraction, it was shown that the classes of loosely and firmly associated lipids changed as the cells aged. Further, it was found that a carotenoid-like pigment associated to a C22 fatty acid increased in the cell wall as the culture stopped growing. Peptidolipid or lipoprotein was found to make up a significant part of the cell wall. This component increased in amount and varied in amino acid content as the culture aged. Analysis of the totally extracted basal layer of the cell wall (peptidoglycan plus arabinogalactan) showed that it too changed as the cells grew and fragmented. The data presented established that the cell wall of N. asteroides was structurally and chemically complex and that a progression of chemical and physical processes occurred within the wall as the cells developed through their growth cycle.

The relative importance of the total cell wall and quantity of digested cell wall in the regulation of the voluntary intake of grass hays by sheep

SUMMARYIn 18 digestibility experiments with sheep, 9 diets consisting of single-grass hays and 9 diets each consisting of equal quantities of a good and poor quality hay were fed. Correlations between the voluntary intake of organic matter, digested organic matter, total cell wall and digested cell wall in all possible combinations were investigated. There were no significant differences among the simple correlation coefficients for hays fed singly or as mixtures, even though they were eaten in very different amounts. The simple pooled correlationswere significant (P < 0·01) among voluntary intake, digested organic matter and cell wall. The cell wall was significantly correlated with digested cell wall, but there was no simple correlation between voluntary intake and digested cell wall. However, when considered together in a multiple linear model they explained 58% of the variation in voluntary intake compared with 43% explained by the total cell wall alone. The additional variation accounted for by the digested cell wall was significant (P < 0·05).The relative importance of the total cell wall and digested cell wall is discussed in relation to selection in grass breeding.

Cell-Wall Fractions and In Vitro Digestibility of Peruvian Feedstuffs1

Forty-four feedstuffs from the temperate, subtropical and tropical regions of Peru were analyzed for in vitro true dry matter digestibility (IVTDMD), crude protein, and cell-wall constituents. Included were cultivated and native grasses and legumes, crop residues, and other materials. Among the cultivated forage species there were noticeable differences in maturity effects on IVTDMD and concentration of cell-wall fractions; maturity and species effects were not necessarily related to “tropical” or “temperate” species type. The results were used to test species differences in the relationship between chemical fractions and IVTDMD by the use of linear regression analysis. The model which consistently gave highest R2 values when applied to various data subgroups and individual forage species is represented by the equation IVTDMD = 117.2 − .59 ± .05 HC − .76 ± .09 C − 1.93 ± .18 L where HC = hemicellulose, C = cellulose and L = permanganate lignin; this equation being derived from a fit of all observations (n = 354) and giving an R2 value of .64. The same model applied to the subgroupings of grasses, legumes and fibrous crop residues yielded R2 values of .54, .78 and .69, respectively. A prediction based only on the total cell-wall fractions gave results slightly inferior to the model which included HC, C and L separately.

Turfgrass Wear Tolerance Mechanisms: II. Effects of Cell Wall Constituents on Turfgrass Wear Tolerance1

AbstractThis investigation was conducted to assess the relationship of cell wall constituents of seven cool‐season turfgrass species to wear tolerance. The turfgrass literature contains no data of this nature. The percent total cell wall (TCW), lignocellulose (ADF), cellulose, hemicellulose, and lignin were determined on a gram dry weight and mg/dm2 basis. Species differed significantly in cell wall constituents for both methods of determination. The relative ranking of the species based on the content of various cell wall constituents expressed on a gram dry weight basis was as follows: Cascade chewings fescue (Festuca rubra var. commutata Gaud) > Pennlawn red fescue (F. rubra L.) and Kentucky 31 tall fescue (F. arundinacea Schreb.) > Manhattan perennial ryegrass (Lolium perenne L.) > Merion Kentucky bluegrass (Poa pratensis L.) > Italian ryegrass (L. multiflorum Lam.) > rough bluegrass (P. trivialis L.). However, the species ranked as follows when the cell wall components were expressed on mg/dm2 basis: Kentucky 31 > Manhattan and Merion > Pennlawn and Italian ryegrass > Cascade > rought bluegrass. Cell wall constituents reported on a g/dry wt. basis were not correlated individually to wear tolerance. However, the combined effects of TCW, ADF, cellulose, and lignin accounted for 96% of the observed wear tolerance variation among the seven turfgrass species studied. TCW, ADF, cellulose, and hemicellulose contents expressed as mg/dm2 were significantly correlated individually to wear tolerance. Their combined effects accounted for 97% of the variation in inter‐species wear tolerance. Total cell wall content expressed on a mg/dm2 basis accounted for 98% of the variation in wear tolerance among the seven turfgrass species.Cell wall constituents were found to increase significantly with plant maturity, with the exception of hemicellulose. TCW increased significantly during the period from July to September, but declined in all species during October. The TCW, ADF, cellulose, hemicellulose, and lignin contents of leaf blades were significantly less than for the leaf sheath for all species.

Turfgrass Wear Tolerance Mechanisms: I. Wear Tolerance of Seven Turfgrass Species and Quantitative Methods for Determining Turfgrass Wear Injury1

AbstractThe relative wear tolerance of seven cool‐season turfgrass species was determined for both sled (foot‐like) and wheel (vehicular) wear injury. Four methods of evaluating wear tolerance differentials were used: 1) visual rating of wear injury, 2) percent total cell wall content (TCW), 3) percent verdure, and 4) percent chlorophyll/unit area remaining after wear treatment. Manhattan perennial ryegrass (Lolium perenne L.) was most tolerant to wheel wear; Kentucky 31 tall fescue (Festuca arundinacea Schreb.) and Merion Kentucky bluegrass (Poa pratensis L.) ranked second; Pennlawn red fescue (F. rubra L.) and Italian ryegrass (L. multiflorum Lam.) were intermediate; while Cascade chewings fescue (F. rubra var. commutata Gaud.) and rough bluegrass (P. trivialis L.) ranked lowest. The relative ranking for sled wear was slightly different from that for the wheel. Visual ratings indicated that Manhattan, Kentucky 31, and Merion were equally tolerant to sled wear. However, Merion was the most wear tolerant to sled injury, according to ratings based on the percent verdure remaining after treatment. Manhattan and Kentucky 31 ranked second and third, respectively; while, Cascade chewings fescue and rough bluegrass were almost destroyed by the crushing, tearing action of the sled.Wear tolerance differentiation among species for the four methods tested was in high agreement. However, percent verdure remaining was the preferred method for quantitatively evaluating wear tolerance differentials. It eliminated arbitrary decisions that were inherent in the visual rating system, and involved fewer procedural steps than either the percent TCW or percent chlorophyll content determinations.

Characterization of the Carbohydrate Fragments Obtained from Saccharomyces cerevisiae Mannan by Alkaline Degradation

Abstract Mild alkaline degradation of Saccharomyces cerevisiae X2180 wild type mannan, under conditions that effect β elimination of the substituted serine and threonine residues, releases mannose, mannobiose, mannotriose, and mannotetraose. Methylation analysis demonstrated that these fragments have the same structures as those that are obtained by acetolysis of the polysaccharide component of the yeast mannan. Similar treatment of a mutant mannan that lacks the mannotetraose unit in the polysaccharide component produced only mannose, mannobiose, and mannotriose. Thus, the single mutation alters both the tetrasaccharide units in the polysaccharide component of the mannan and those that are attached to serine and threonine. Because these studies were carried out on the total cell wall mannan, rather than a single molecular species, we cannot say that both the polysaccharide chains and the shorter oligosaccharides linked to serine and threonine are present in the same molecule. Strong alkaline degradation in the presence of sodium borotritide of the mannan residue recovered from the β elimination reaction yielded a protein-free polysaccharide that gave radioactive N-acetylglucosaminitol on acetolysis. Thus, the mannose-containing polysaccharide of the mannan is attached to glucosamine, and it is probable that the linkage to protein is through a di-N-acetylchitobiose to asparagine as in many other glycoproteins. The mannan isolated from the wild type and mutant strains of S. cerevisiae by procedures that avoid exposure to alkali had a molecular weight of 133,000. On treatment with 0.1 n sodium hydroxide at room temperature, the molecular weight was reduced to about 40,000. These conditions could saponify acyl ester bonds but are unlikely to break phosphodiester linkages or any other linkages except those of the oligosaccharides attached to serine and threonine.

The constancy of the digested cell wall in grass

SUMMARYThe quantity of in vitro digested cell wall was determined in the separated leaf and stem of Digitaria decumbens (pangola grass) fertilized at three different levels of nitrogen and cut after two different periods of regrowth. The treatment means ranged from 39·6 to 41·5 g digested cell wall per 100 g forage organic matter in leaf, and from 37·5 to 41·6 g in stem. For both leaf and stem the lowest mean amounts of digested cell wall were associated with the highest levels of fertilizer and the longer periods of regrowth. The results for nitrogen fertilizer were consistent with those from previous in vivo digestibility experiments, but when the effect of the total cell wall on digested cell wall was taken into account there were inconsistencies. The previous in vivo digestibility data were used in conjunction with the present results in support of a view that interpretation of digestibility data for grasses is best made by reference to a constant digested cell wall of 39·5 g/100 g forage organic matter.

Detachment and Chemical Characterization of the Regularly Arranged Subunits from the Surface of an Acinetobacter

Acinetobacter sp. strain MJT/F5/199A carries an array of tetragonally arranged subunits on its outer surface. The subunits can be detached from isolated cell walls by incubation with 1 M urea or by washing with water after treatment with 10 mM ethylenediaminetetraacetic acid or ethyleneglycol-bis(beta-aminoethylether)N,N'-tetraacetic acid. After removal of the urea, they reaggregate into the same ordered array at air-water interfaces in the presence of MgCl(2). The detached subunits were characterized as an acidic protein of molecular weight 65,000. They represent one-fifth of the total cell wall protein.

The characterization of a chitin-associated D-glucan from the cell walls of Aspergillus niger

Exhaustive extraction of the cell walls of Aspergillus niger with 10% NaOH solution leaves an alkali-resistant residue containing chitin and glucan as the major components. The glucan in this residue comprises 58.7% of the total cell wall glucan and was characterized by permethylation, and identification of the resulting O- methyl- D-glucoses obtained after hydrolysis by gas-liquid chromagtography and mass spectrometry of the derived partially acetylated, partially methylated, [1- 2H]alditols. The glucan was separated from the chitin by acetylation of the alkali-resistance material, a procedure which separates a large portion of the total glucan as a chloroformsoluble acetate, abd by treatment of the alkali-insoluble residue with nitrous acid, a procedure which was found to render the complex soluble in dimethylsulfoxide and amenable, therefore, to permethylation. The data collected suggests that the preparation is an essentially linear glucan containing 85–95% 1 → 3 linkages and 10–15% 1 → 4 linkages. An analysis of the glycosidic linkages using NMR spectroscopy indicate that both α and β linkages are present in the ratio of 4:1. An identical glucan appears to be present in the cell walls of Penicillium chrysogenum as well as the spore cell walls of both organisms, as evidenced by methylation studies.

Digestibility of Roughage Materials Ensiled with Sodium Chlorite

Feasibility of increasing the digestibility of roughage materials was investigated by both in vitro and in vivo techniques. Various substrates were ensiled at 18% dry matter with 0 to 7% NaClO2 (wet basis). Cell wall content, generally hemicellulose and lignin, decreased with increasing rates of NaClO2 treatment. Rice hulls and peanut hulls contain cutin, which was hydrolyzed upon treatment with sodium chlorite. Wheat straw lignin was decreased most efficiently with sodium chlorite (2.81. g/g of NaClO2), but peanut hull lignin was not decreased. The in vitro disappearance of total cell wall increased greatly by this approach. In vitro digestible cell wall consistently increased with increasing rate of NaClO2 treatment.Ground barley straw with water added (about 80% moisture) was ensiled for 60 days in 1.2 × 2.4-m wooden silos. Silage treatments were control, sodium chloride (10% or air-dry straw), and sodinm chlorite (15% of the air-dry straw). Dried silages and the original barley straw were ground and pelleted into four nutritionally balanced diets and fed to eight yearling wethers to determine ad libitum intake and digestibility in a Latin square design. Ad libitum digestible organic-matter intakes were similar for all diets, 1.53% of body weight, except for control silage (1.34%). Nitrogen retentions and urine outputs were higher for diets containing additional sodium. Diet digestibilities for organic matter and cell walls were ground straw, 55 and 28%; control silage, 52 and 22%; sodium chlorid silage, 55 and 28%; and sodium chlorite silage, 62 and 44%. Relative diet differences in organic matter and cell wall in vitro digestibility measurements agreed closely with in vivo values.

In vitro digestibility and carbohydrate composition of Digitaria decumbens and Setaria anceps grown at different levels of nitrogenous fertilizer

Two tropical pasture grasses, Digitaria decumbens cv. Pangola and Setaria anceps cv. Nandi, were compared on the basis of in vitro digestibility and carbohydrate composition. These species were grown at three different levels of applied nitrogen (280, 476, and 673 kg/ha) and continuously grazed at one stocking rate of 6.2 beasts per ha. The in vitro digestibility of both species apparently increased with applied nitrogen, Pangola being slightly superior at the higher nitrogen levels. Lignin did not appear to be a major factor influencing digestibility. Total extractable carbohydrates (ethanol + water-soluble) varied little with nitrogen level, although Pangola was consistently higher (c. 9.0 %) than Setaria (c. 7.2 %). Pangola had a higher total cell wall polysaccharide content than Setaria at all nitrogen levels. Numerical analysis showed a reduction in cell wall polysaccharides in both species with increasing nitrogen, this effect being more marked in Setaria. The concomitant liveweight gains showed an overall superiority in Pangola which accounted for c. 80 % of the variation in the system; a further 16% was attributable to the increased responsiveness of Setaria to applied nitrogen. Since the total cell wall polysaccharides were higher in Pangola than in Setaria, it appears that Pangola polysaccharides are more digestible than those of Setaria.

Nutrient Digestibility of Lower-fiber Rations in the Ruminant Animal1,2

Digestibility trials with growing steers related chemical composition as defined by detergent analyses to true and apparent digestible components in 14 lower-fiber rations. Linear relationships were significant when the digested fraction of a constituent was regressed on the total constituent consumed in the rations for cell-wall constituents, cellulose, hemicellulose, neutral detergent solubles, and nitrogen fractions, with less nutritive uniformity in the fibrous fractions. Total ration cellulose explained 77% (coefficient of determination) of the variation in digestible cellulose while lignification constituted only 20%. Total hemicellulose explained 59% of the variation in digestible hemicellulose with a small contribution (6%) from lignification. Decreased apparent digestibility of detergent solubles or increased fecal loss of nonfibrous energy were related to increased cell-wall fiber in the ration. Correlation (r = .97, P<.01) between digestible and total N indicated nutritive uniformity among N sources which included urea. Fecal N loss in ruminant animals was controlled by amount of ration N and composition of ration dry matter. A larger metabolic fecal protein excretion was associated with cell-wall N than with detergent soluble N (2.2 and 1.7% of ration DM). Apparent digestible dry matter was related to fecal endogenous loss of detergent solubles and N and to total cell-wall fiber, whether constituted mostly by cellulose or hemicellulose, in the ration. Cellulose, acid-detergent lignin, and cell wall fractions were inversely correlated (P<.01) to digestible dry matter; however, hemicellulose was not. Total N was not related statistically to digestible dry matter, but detergent soluble N (positive, P<.05) and cell wall N (negative, P<.01) were.

Nutritive Value Estimates in Apomictic Lines of Buffelgrass (Cenchrus ciliaris L.)1

AbstractThe cell wall content of a forage and the digestibility of the cell wall determine to a large extent the digestibility of the forage. A study was conducted with buffelgrass (Cenchrus ciliaris L.) to determine whether cell wall digestibility differences among lines are related to components of the cell wall or the total cell wall content. Seven lines of buffelgrass were evaluated in an in vivo digestibility study. Leaf, stem, and whole plant samples of 15 lines were evaluated at two harvests in an in vitro digestion study. Significant differences in dry matter digestibility were detected among lines in both the in vivo and in vitro studies. About 66% of the variation in digestibility was associated with the digesibility of cell wall. Correlations of digestibility with various cell wall components or component ratios were not sufficiently high to be of significance for prediction purposes.There were no differences among lines in in vitro dry matter digestibility of leaves, but there were for stems and whole plants. Differences in digestibility of cell wall were obtained for leaves, stems, and whole plants. Experimental lines differed in agronomic characteristics such as rhizome development, density, seed production, and color, but no significant correlations of these variables with digestibility could be shown. The most useful procedure for screening buffelgrass plants for quality seems to be in vitro dry matter digestibility with an associated neutral detergent fiber determination, whereby both cell wall content and cell wall digestibiity could be estimated.

An assessment of the quality of forage from its cell-wall content and amount of cell wall digested

SUMMARYGrasses and legumes comprising poor to good quality temperate and tropical species were fed to either cattle or sheep in 36 digestibility experiments. Cell wall in these forages was the ash-free and protein-free residue after sequential extraction with acidpepsin, organic solvents and either water for grasses or ammonium oxalate for legumes. The average amount of cell wall digested per 100 g forage OM was 40·0±0·59 g in grasses and 19·8±1·85 g in legumes. It was considered that within grasses and within legumes the physiology of ruminant digestion, rather than forage quality, was the main determinant of the average amount of cell wall digested and the difference between grasses and legumes was due to interaction of the ruminant digestion process with the physical structure of the cell wall. Of forage factors governing variation about the physiological average, the total cell wall had some effect on the amount of cell wall digested, but the lignin concentration in the cell wall had no effect.Among grasses and legumes the average, apparently undigested, protein-free non-cell-wall component was 6·2±0·13 g per 100 g forage OM. This component and digestible protein relative to total protein varied among different sets of data. It was concluded that only the component of digestible organic matter which was governed by the relative proportions of cell walls and cellular contents was predictable from chemical composition. It was considered that selection in plant breeding should be based on both digestible cell wall and cell-wall content instead of digestible organic matter.

Method for Determining Solutes in the Cell Walls of Leaves

A perfusion method is described whereby large discs of amphistomatous leaves are vacuum-perfused with water so that either successive fractions of perfusate may be analyzed for solutes or the infused water may be displaced and collected after equilibration with the leaf cells. With castor bean leaves, estimates of electrolyte concentration in cell wall water by the two methods were similar. Total electrolytes in leaf cell wall water of castor beans (Ricinus communis), sunflower (Helianthus annuus), and cabbage (Brassica oleracea capitata) from nonsaline cultures were about 2, 2, and 10 milliequivalents per liter, respectively, increasing to 4, 10, and 30 milliequivalents per liter under saline conditions. Electrolytes recovered in successive fractions were similar in composition, and continuous perfusion resulted in a steady release of solutes, the concentration in the perfusate varying inversely with the perfusion rate. Diffusional release of solutes from cells was less than expected at low perfusion rates, suggesting that solute reabsorption may increase as solute concentration in the perfusate increases with decreased perfusion rates. Perfusate concentration and composition were essentially unaffected by temperature (2 and 23 C) or by perfusing with 0.5 mm CaSO(4) rather than with water. Electrolytes in perfusates on an equivalent basis were Ca(2+), 30%; Mg(2+), 10%; and Na(+) + K(+), 60%, the proportions of sodium increasing from 10 to 50% in leaves (cabbage) that accumulated sodium under saline conditions. Salinity (added NaCl) of the root culture medium caused a 3- to 5-fold increase in total cell wall electrolyte concentration, but this amounted to an increase from less than 1 or a few per cent to no more than 7% (in cabbage) of the cell sap electrolyte concentrations. Solutes in the cell wall appear to be in dynamic equilibrium with intracellular solutes.

Two Cycles of Directional Selection for Improved Nutritive Value of Alfalfa1

A 6‐hour in vitro dry matter disappearance (6H‐DMD) procedure was used to evaluate individual progeny of ‘Vernal’ (Medicago sativa L. and Medicago falcata L.) and ‘DuPuits’ (Medicago sativa L.) clones selected for high or low 6H‐DMD values. Progeny derived from clones with high 6H‐DMD values had consistently higher 6H‐DMD values than progeny from low clones. Additional clones of ‘Culver’ MSA‐C4, MSB‐C4, Medicago glutinosa M.B., and Vernal were screened for high or low 6H‐DMD values over two harvests. Selected clones from Vernal and DuPuits progeny and from the five unselected populations were recombined for a second cycle of directional selection.Correlations between yield and 6H‐DMD were negative or near zero. Cell wall analysis of clones selected for a second cycle of improvement indicated a significant positive relationship between total cell wall constituents (NDF) and 6H‐DMD values of the forage.Second cycle clones had significant general and specific combining abilities for yield as well as leaf and NDF content. Progeny from clones with high 6H‐DMD values were higher in leaf content but lower in NDF content than progeny from clones with low 6H‐DMD values. Comparisons of selected two‐clone combinations in the first and second cycles indicated second cycle selection was effective in separating progeny means.

Chemical characterization, spatial distribution and function of a lipoprotein (murein-lipoprotein) of the E. coli cell wall. The specific effect of trypsin on the membrane structure.

A decrease of the absorbance at 578 nm of a cell wall suspension of mid log phase E. coli occurs when the suspension is incubated with trypsin. The reaction is so rapid that 55% of the total decrease is obtained within the first 2 min [ratio of enzyme to total cell wall protein = 1: 50 (w/w), room temperature]. The rate of the reaction is specific for trypsin when compared with other proteases, different lipases, lysozyme and other glycosidases. A peptide bond especially sensitive to trypsin could be localized within the complex cell wall by the demonstration that the decrease of the absorbance is paralleled by the splitting of the protein from the murein. This protein could be shown to be a lipoprotein with a part of the lipid probably covalently bound to the protein. It is called murein-lipoprotein. The link between the lipoprotein and the murein is -lysine. After trypsin digestion lysine is the only additional amino acid remaining at the murein. The ratio of the amount of lysine to the known constituents of the murein demonsstrates that on the average one lipoprotein molecule is covalently bound to every tenth repeating unit of the murein (N-acetylglucosamine–N-acetylmuramic acid–l-alanine-d-glutamic acid–meso-diaminopimelic acid–d-alanine). After 3 min incubation with trypsin, the isolated lipoprotein molecules have a lysine to arginine ratio of 4:4 as compared with 5:4 in the native molecule. The lipoprotein has an unusual amino acid comosition since it contains about 65% polar amino acids and no glycine, cysteine, proline, phenylalanine, and histidine could be found. On a weight basis the lipoprotein accounts for more than 40% of the rigid layer. Since the murein is held together exclusively by covalent bonds one can get a fairly accurate idea of the distribution of the lipoprotein molecules in the cell wall. About 105 lipoprotein molecules per cell should be distributed 103 A apart along the glycosidic chains of the murein. The lipoprotein has an important function in stabilizing the total structure of the cell wall. It seems that cleavage of only one peptide bond adjacent to the lysine link between the lipoprotein and the murein causes the rapid decrease of the absorbance and as shown by electron microscopic exmination of ultrathin sections of trypsin treated cell walls, two separated membrane structures appear which otherwise are closely adjacent to one another.

Mode of action of viomycin on Rhizobium meliloti

The biochemical mechanism of action of viomycin on R. meliloti involved an inhibition of protein synthesis. Adenine and amino acid incorporation into total nucleic acid and cell wall respectively was relatively unaffected. The antibiotic increased the level of 14C in the intracellular pool and "nucleic acids" fraction of cells fed 14C-amino acids but caused no cellular lysis. Mono- and di-valent cations protected cells from growth inhibition by viomycin.A viomycin-resistant mutant was similar to its sensitive parent in rate of uptake of glucose and glutamic acid by both growing and resting cells and in the rate of leakage when preloaded cells were suspended in buffered saline. Viomycin resistance did not appear to be episome-mediated or involve an extracellular degradative enzyme. The ability of TES buffer to eliminate viomycin resistance, coupled with the sensitivity to viomycin of cell-free protein-synthesizing systems derived from both sensitive and resistant cells indicates that resistance is probably the result of a selective reduction in antibiotic permeability.

Action of ethylenediaminetetraacetic acid, tris(hydroxymethyl)-aminomethane, and lysozyme on cell walls of Pseudomonas aeruginosa.

Incubation of isolated cell walls of Pseudomonas aeruginosa with tris(hydroxymethyl)aminomethane (Tris), ethylenediaminetetraacetic acid (EDTA), and lysozyme, either singly or in combinations, results in partial solubilization of the cell walls.When cell walls were incubated with a combination of Tris and EDTA, approximately 30% of the total cell wall and 32% of the lipopolysaccharide fraction were solubilized. The following percentages of the individual cell wall constituents were solubilized: protein, 19%; carbohydrate, 35%; lipid, 5%; ash, 54%; and, phosphorus 23%. When lysozyme was included with Tris and EDTA, approximately 36% of the total cell wall and 86% of the lipopolysaccharide fraction were solubilized. Solubilization of the individual cell wall constituents was similarly increased.Phospholipids, which make up 7–8% of the intact cell wall of P. aeruginosa, were not released by incubation of the cell walls with these agents. Fatty acids in addition to those found in the lipopolysaccharide, however, were detected in the solubilized cell wall materials.Incubation systems composed of Tris, of EDTA, and of Tris and lysozyme solubilized cell walls to a lesser extent than incubation systems of Tris and EDTA, and of Tris, EDTA, and lysozyme. Incubation of cell walls in water, furthermore, was effective in solubilizing approximately 8% of the cell walls. Muramic acid containing materials were solubilized only in the presence of lysozyme.Materials liberated from cell walls which were incubated in systems containing EDTA exhibited single symmetrical Schlieren peaks characterized by uncorrected sedimentation constants of approximately 7 S, suggesting the release of homogenous subunits from the cell walls.Intra- and inter-molecular cross-linking by divalent cations via phosphate groups contained in lipoprotein and lipopolysaccharide components of the cell wall of P. aeruginosa is proposed.

The glycerol teichoic acid from the cell wall of Bacillus stearothermophilus B65.

1. A glycerol teichoic acid has been extracted from cell walls of Bacillus stearothermophilus B65 and its structure examined. 2. Trichloroacetic acid-extractable teichoic acid accounted for 68% of the total cell-wall phosphorus and residual material could be hydrolysed to a mixture of products including those characteristic of glycerol teichoic acids. 3. The extracted polymer is composed of glycerol, phosphoric acid, d-glucose and d-alanine. 4. Hydrolysis of the polymer with alkali gave glycerol, 1-O-alpha-d-glucopyranosylglycerol and its monophosphates, glycerol mono- and di-phosphate, as well as traces of a glucosyldiglycerol triphosphate and a glucosylglycerol diphosphate. 5. The teichoic acid is a polymer of 18 or 19 glycerol phosphate units having alpha-d-glucopyranosyl residues attached to position 1 of 14 or 15 of the glycerol residues. 6. The glycerol residues are joined by phosphodiester linkages involving positions 2 and 3 in each glycerol. 7. d-Alanine is in ester linkage to the hydroxyl group at position 6 of approximately half of the glucose residues. 8. One in every 13 or 12 polymer molecules bears a phosphomonoester group on position 3 of a glucose residue, the possible significance of which in linkage of the polymer to other wall constituents is discussed.