Specificity of cell wall labeling with tritiated diaminopimelic acid in Bacillus subtilis

1973 ◽  
Vol 19 (8) ◽  
pp. 1049-1051
Author(s):  
Siegfried Maier
Keyword(s):  
Cell Wall ◽  
Bacillus Subtilis ◽  
Glutamic Acid ◽  
Cell Walls ◽  
Diaminopimelic Acid ◽  
Chromatographic Purification ◽  
Membrane Chromatography

The suitability of tritiated 2,6-diaminopimelic acid (3H-DAP) as a label specific for cell walls was explored in Bacillus subtilis BC 102 grown in a medium enriched with 3H-DAP and an excess of L-lysine. Fractionation of labeled cells showed 57% of the activity in the cell wall and 28% in the membrane. Chromatography of labeled wall hydrolysates revealed two activity peaks: 62% in DAP and 29% in glutamic acid – alanine. Labeled membrane was devoid of activity in the DAP position. Chromatographic purification of the 3H-DAP improved specificity, giving 7% of the activity in the membrane and 85% in the wall. In such walls DAP accounted for 82% of the total wall activity. Therefore, only 69% of the total fixed purified 3H label remained with DAP in the wall.

scholarly journals The extraction of cell walls of Pseudomonas aeruginosa with aqueous phenol. The insoluble residue and material from the aqueous layers

1967 ◽  
Vol 105 (2) ◽  
pp. 759-765 ◽  
Author(s):  
K. Clarke ◽  
G. W. Gray ◽  
D. A. Reaveley
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Cell Wall ◽  
Cell Walls ◽  
Lipid A ◽  
Diaminopimelic Acid ◽  
Insoluble Residue ◽  
Muramic Acid ◽  
Gram Negative ◽  
Gram Negative Organisms ◽  
Residue Fraction

1. The insoluble residue and material present in the aqueous layers resulting from treatment of cell walls of Pseudomonas aeruginosa with aqueous phenol were examined. 2. The products (fractions AqI and AqII) isolated from the aqueous layers from the first and second extractions respectively account for approx. 25% and 12% of the cell wall and consist of both lipopolysaccharide and muropeptide. 3. The lipid part of the lipopolysaccharide is qualitatively similar to the corresponding material (lipid A) from other Gram-negative organisms, as is the polysaccharide part. 4. The insoluble residue (fraction R) contains sacculi, which also occur in fraction AqII. On hydrolysis, the sacculi yield glucosamine, muramic acid, alanine, glutamic acid and 2,6-diaminopimelic acid, together with small amounts of lysine, and they are therefore similar to the murein sacculi of other Gram-negative organisms. Fraction R also contains substantial amounts of protein, which differs from that obtained from the phenol layer. 5. The possible association or aggregation of lipopolysaccharide, murein and murein sacculi is discussed.

Controlled lysis of bacterial cells utilizing mutants with defective synthesis of D-alanine

1988 ◽  
Vol 34 (3) ◽  
pp. 256-261 ◽  
Author(s):  
Michael P. Heaton ◽  
Robert B. Johnston ◽  
Thomas L. Thompson
Keyword(s):  
Cell Wall ◽  
Bacillus Subtilis ◽  
Cell Walls ◽  
Alanine Racemase ◽  
Growth Media ◽  
Bacterial Cells ◽  
Cell Wall Formation ◽  
Fermentation Processes ◽  
Intracellular Enzymes ◽  
Dense Cell

An alanine racemase (EC 5.1.1.1) mutant (Dal−) of Bacillus subtilis required small amounts of D-alanine to synthesize an osmotically stable cell wall in certain growth media. Investigation of the conditions which caused lysis in hypotonic media revealed that in addition to complex media, such as nutrient broth and acid-hydrolyzed casein, glycine inhibited stable cell wall formation. D-Alanine prevented the glycine inhibition. Up to 99% lysis occurred in both dilute and dense cell suspensions (optical densities up to 110) within 2.5 h after adding 1% glycine to late log phase cultures. Intracellular enzymes recovered from the lysate were as active as those from lysozyme-disrupted cells. No amino acid tested other than glycine induced lysis. Dal− mutants can be used for controlled lysis of bacterial cells to facilitate the isolation of normal intracellular constituents and bioengineered products from fermentation processes. Cell walls of most bacteria contain D-alanine; thus, this strategy should be applicable to a wide variety of microorganisms.

scholarly journals The configuration of 2,6-diamino-3-hydroxypimelic acid in microbial cell walls

1969 ◽  
Vol 115 (4) ◽  
pp. 797-805 ◽  
Author(s):  
H R Perkins
Keyword(s):  
Cell Wall ◽  
Hydroxy Group ◽  
Cell Walls ◽  
Optical Rotation ◽  
Periodate Oxidation ◽  
Ring Closure ◽  
Diaminopimelic Acid ◽  
Amino Group ◽  
Amino Groups ◽  
Peptide Linkage

β-Hydroxydiaminopimelic acid, together with some diaminopimelic acid, occurs in the cell-wall mucopeptide of certain Actinomycetales. These components were converted into their di-DNP derivatives and separated by chromatography. Hence the relative proportions present in the cell walls of a number of species were measured. The problem of acid-induced inversion of configuration was studied. Of the diaminohydroxypimelic acids isomer B (see Scheme 2; amino groups meso, hydroxy group threo to its neighbouring amino group) always predominated but a small proportion of isomer D (amino groups l, hydroxy group erythro) also occurred. The configuration of the diaminohydroxypimelic acids was determined by periodate oxidation to glutamic γ-semialdehyde, which underwent spontaneous ring-closure. Reduction with sodium borohydride produced optically active proline, the configuration of which was determined by direct measurement of the optical rotation of DNP-proline. Un-cross-linked diaminohydroxypimelic acid in the cell wall was oxidized with periodate in the presence of ammonia. Since the remaining amino group was bound in peptide linkage, ring-closure was prevented and borohydride reduction of the aldehyde–ammonia presumed to be present resulted in the formation of ornithine. The quantity of ornithine was used as a measure of the degree of cross-linking.

scholarly journals STUDIES ON THE CHEMICAL STRUCTURE OF THE STREPTOCOCCAL CELL WALL

1962 ◽  
Vol 115 (1) ◽  
pp. 49-62 ◽  
Author(s):  
Richard M. Krause ◽  
Maclyn McCarty
Keyword(s):  
Cell Wall ◽  
Glutamic Acid ◽  
Cell Walls ◽  
Soluble Group ◽  
Chemical Structure ◽  
Streptococcal Cell Wall ◽  
Whole Cell ◽  
Cell Wall Lysis ◽  
C Cell ◽  
Serological Studies

The trypsinized cell walls of Group C streptococci contain two components, the group-specific carbohydrate and a mucopeptide polymer. Hot formamide extraction of Group C cell walls results in a soluble group-specific carbohydrate fraction and an insoluble mucopeptide residue. This mucopeptide, similar in composition to that of Groups A and A-variant streptococci, contains N-acetylglucosamine, N-acetylmuramic acid, alanine, glutamic acid, lysine, and glycine. It is dissolved by the muralytic enzymes, including lysozyme, which does not attack the whole cell wall. Lysis of the cell wall by phage-associated lysin results in the release of soluble fragments composed of the elements of mucopeptide. Group C carbohydrate extracted with formamide is composed primarily of N-acetylgalactosamine and rhamnose. Serological studies suggest that the specificity of Group C carbohydrate is determined by the N-acetylgalactosamine.

Die gemeinsame Wurzel der Lyse von Escherichia coli durch Penicillin oder durch Phagen

1957 ◽  
Vol 12 (7) ◽  
pp. 421-427 ◽  
Author(s):  
W. Weidel ◽  
J. Primosigh
Keyword(s):  
Cell Wall ◽  
Cell Walls ◽  
Building Blocks ◽  
Diaminopimelic Acid ◽  
Wall Structure ◽  
Gram Positive ◽  
E Coli ◽  
Growing Cell ◽  
Cell Wall Disruption ◽  
Entire Cell

One of the two layers of the E. coli B cell wall is shown to possess the chemical composition typical of a gram-positive microorganism. It is this layer which lends support and strength to the entire cell wall structure, its rigidity depending up on the incorporation of building blocks made up from alanine, glutamic acid, diaminopimelic acid, muramic acid and glucosamine.Phage enzyme is an agent capable of removing these stabilizing units from the „gram-positive “ layer, thereby causing it to collapse. Penicillin appears to prevent the biosynthetic incorporation of the same stabilizing units into growing cell walls, thus producing eventually the effect of cell wall disruption in a basically similar way.The rather manifold aspects of these findings are discussed at some length.

An enzymatic release of antibiotic-containing peptidoglycan fragments from streptomycin-producing Streptomyces griseus

1980 ◽  
Vol 26 (2) ◽  
pp. 141-145 ◽  
Author(s):  
Gy. Barabás ◽  
I. Szabó ◽  
A. Ottenberger ◽  
V. Zs.-Nagy ◽  
G. Szabó
Keyword(s):  
Amino Acids ◽  
Cell Wall ◽  
Glutamic Acid ◽  
Streptomyces Griseus ◽  
Activity Analysis ◽  
Antibiotic Activity ◽  
Diaminopimelic Acid ◽  
Autolytic Enzyme ◽  
A Cell

A cell wall bound autolytic enzyme of a streptomycin-producing strain of Streptomyces griseus was investigated. The peptidoglycan fragments released by the enzyme showed antibiotic activity. Analysis of these fragments proved that streptomycin is bound to a cell wall peptide. The peptide contained the four amino acids characteristic of Streptomyces cell wall: alanine, glycine, diaminopimelic acid, and glutamine or glutamic acid.

scholarly journals Autolysis of isolated cell walls of Bacillus licheniformis N.C.T.C. 6346 and Bacillus subtilis Marburg strain 168. Separation of the products and characterization of the mucopeptide fragments

1970 ◽  
Vol 119 (5) ◽  
pp. 849-860 ◽  
Author(s):  
R. C. Hughes
Keyword(s):  
Bacillus Subtilis ◽  
Bacillus Licheniformis ◽  
Cell Walls ◽  
Deae Cellulose ◽  
Casein Hydrolysate ◽  
Diaminopimelic Acid ◽  
Average Chain Length ◽  
Amide Bonds ◽  
Chain Lengths

1. Cell walls were isolated from Bacillus licheniformis N.C.T.C. 6346 and Bacillus subtilis Marburg strain 168 trp grown on casein hydrolysate into exponential phase. Autolysis was carried out and the soluble products, separated by chromatography on DEAE-cellulose, from the two wall preparations are broadly similar in composition and are in agreement with autolysis proceeding with hydrolysis of amide bonds between l-alanine and N-acetylmuramic acid residues in the mucopeptide components. 2. Peptides originating from the mucopeptide components were isolated and shown to be a monomer peptide, l-alanyl-d-glutamyl-meso-diaminopimelic acid and a dimer peptide containing two monomer peptides linked through a residue of d-alanine. Approximately one amide group is present for each equivalent tripeptide unit and is probably substituted on diaminopimelic acid residues. 3. Oligosaccharides originating from the mucopeptide components were isolated and after hydrolysis contained almost equimolar amounts of glucosamine and muramic acid and only very small amounts of amino acids. The number-average chain length, estimated by the release of non-reducing end groups of N-acetylglucosamine with exo-β-N-acetylglucosaminidase, is approximately ten hexosamine residues for oligosaccharides isolated from either organism. The oligosaccharides are polydisperse. 4. N-Acetylglucosamine residues are the only reducing terminals detectable in the oligosaccharides isolated from B. subtilis or B. licheniformis cell-wall autolysates. The number-average chain lengths of the oligosaccharides were determined by estimation of the content of these residues and are higher than those found by enzymic assay. Possible reasons for the discrepancy are discussed.

scholarly journals Autolysis of cell walls of Bacillus stearothermophilus B65 and the chemical structure of the peptidoglycan

1970 ◽  
Vol 118 (5) ◽  
pp. 859-868 ◽  
Author(s):  
W. D. Grant ◽  
A. J. Wicken
Keyword(s):  
Glutamic Acid ◽  
Cell Walls ◽  
Bacillus Stearothermophilus ◽  
Teichoic Acid ◽  
Paper Electrophoresis ◽  
Diaminopimelic Acid ◽  
Peptide Fragments ◽  
Acid Complex ◽  
Acid Hydrolysate ◽  
Peptide Mixture

1. The cell walls of Bacillus stearothermophilus B65 contain glucosamine, muramic acid, alanine, α∈-diaminopimelic acid (Dap), glutamic acid, aspartic acid, glycine, and serine in the molecular proportions 0.60:0.64:2.30:0.85:1.00:0.11:0.13:0.31. 2. Both d- and l-alanine are present, but glutamic acid and diaminopimelic acid are present only as the d- and meso-isomers respectively. 3. The peptide fragments Ala-Dap, Dap-Ala, and Dap-Ala-Dap have been isolated from a partial acid hydrolysate of the cell walls. 4. The major products of autolysis of the cell wall were d-alanine, a peptide mixture, peptidoglycan material and a peptidoglycan–teichoic acid complex. 5. Separation of the peptide mixture into ten major peptides was achieved by DEAE-Sephadex and paper chromatography, and paper electrophoresis. 6. The structures of these peptides have been determined and they fall into four groups, the individual members of each group differing only in number or position of carboxamide substituents. 7. The structures are I, a tripeptide l-Ala–d-Glu-meso-Dap; II, a pentapeptide made up by the tripeptide (I) linked through the ∈-amino group of its diaminopimelic acid residue to the carboxyterminal of the dipeptide meso-Dap-d-Ala; III, a heptapeptide made up by a similar linkage between the tripeptide (I) and the tetrapeptide l-Ala-d-Glu-meso-Dap-d-Ala; IV, a possible undecapeptide made up by a further tetrapeptide similarly linked to the heptapeptide (III) structure. 8. The structure of the peptidoglycan and the actions of the autolytic enzymes are discussed in terms of these peptide structures.

scholarly journals The effect of magnesium ion deprivation on the synthesis of mucopeptide and its precursors in Bacillus subtilis

1969 ◽  
Vol 115 (3) ◽  
pp. 419-430 ◽  
Author(s):  
A J Garrett
Keyword(s):  
Bacillus Subtilis ◽  
Glutamic Acid ◽  
Trichloroacetic Acid ◽  
Amino Sugar ◽  
Diaminopimelic Acid ◽  
Magnesium Ion ◽  
Subtilis Strain ◽  
Amino Sugars ◽  
Intracellular Concentrations

1. Mg2+ or Mn2+ starvation causes suspensions of Bacillus subtilis strain W 23 to accumulate bound amino sugars that are soluble in trichloroacetic acid. 2. The presence of chloramphenicol or puromycin produces higher intracellular concentrations of amino sugars during Mg2+ starvation, but neither compound can stimulate the accumulation when Mg2+ is present. 3. The major component of the amino sugar fraction extracted from cells deprived of Mg2+ is a nucleotide containing uridine, phosphorus, N-acetylmuramic acid, alanine, glutamic acid and α∈-diaminopimelic acid in the molar proportions of 1:2:1:3:1:1. This compound represents at least 80% of the bound N-acetylhexosamine extracted by trichloroacetic acid. 4. Studies of the binding of this nucleotide with vancomycin support the proposal that it is the mucopeptide precursor UDP-N-acetylmuramyl-l-alanyl-d-glutaminyl- α∈-diaminopimelyl-d-alanyl-d-alanine. 5. A method is described for the isolation of this material labelled with [3H]α∈-diaminopimelic acid. 6. When Mg2+ is supplied to cells previously starved of Mg2+, the accumulated pool of amino sugars rapidly decreases. 7. The biosynthesis of mucopeptide is inhibited by 35–50% under conditions of Mg2+ starvation. The presence of EDTA increases this inhibition to 70%. The amount of N-acetylhexosamine that accumulates is balanced exactly by the associated fall in mucopeptide synthesis. 8. ‘Chase’ experiments show that the accumulated N-acetylhexosamine compound is utilized in mucopeptide synthesis.

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