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 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.

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.

scholarly journals Isolation and partial characterization of apiogalacturonans from the cell wall of Lemna minor

1970 ◽  
Vol 116 (4) ◽  
pp. 569-579 ◽  
Author(s):  
David A. Hart ◽  
Paul K. Kindel
Keyword(s):  
Cell Wall ◽  
Sodium Chloride ◽  
Cell Walls ◽  
Lemna Minor ◽  
Extraction Procedure ◽  
Ammonium Oxalate ◽  
Periodate Oxidation ◽  
Dry Weight ◽  
Weight Basis ◽  
Sephadex Column

1. A mild, reproducible extraction procedure, using 0.5% ammonium oxalate, was developed for the isolation of polysaccharides containing d-apiose from the cell wall of Lemna minor. On a dry-weight basis the polysaccharide fractions extracted with ammonium oxalate made up 14% of the material designated cell walls and contained 20% of the d-apiose originally present in the cell walls. The cell walls, as isolated, contained 83% of the d-apiose present in L. minor. 2. After extraction with ammonium oxalate, purified polysaccharides were obtained by DEAE-Sephadex column chromatography and by fractional precipitation with sodium chloride. With these procedures the material extracted at 22°C could be separated into at least five polysaccharides. On a dry-weight basis two of these polysaccharides made up more than 50% of the material extracted at 22°C. There was a direct relationship between the d-apiose content of the polysaccharides and their solubility in sodium chloride solutions; those of highest d-apiose content were most soluble. 3. All the polysaccharides isolated appeared to be of one general type, namely galacturonans to which were attached side chains containing d-apiose. The d-apiose content of the apiogalacturonans varied from 7.9 to 38.1%. The content of esterified d-galacturonic acid residues in all apiogalacturonans was low, being in the range 1.0–3.5%. Hydrolysis of a representative apiogalacturonan with dilute acid resulted in the complete removal of the d-apiose with little or no degradation of the galacturonan portion. 4. Treatment of polysaccharide fractions with pectinase established that those of high d-apiose content and soluble in m-sodium chloride were not degraded, whereas those of low d-apiose content and insoluble in m-sodium chloride were extensively degraded. When the d-apiose was removed from a typical pectinase-resistant polysaccharide, the remainder of the polysaccharide was readily degraded by this enzyme. 5. Periodate oxidation of representative polysaccharide fractions and apiogalacturonans and determination of the formaldehyde released showed that about 50% of the d-apiose molecules were substituted at either the 3- or the 3′-position.

scholarly journals Sortase C-Mediated Anchoring of BasI to the Cell Wall Envelope of Bacillus anthracis

2007 ◽  
Vol 189 (17) ◽  
pp. 6425-6436 ◽  
Author(s):  
Luciano A. Marraffini ◽  
Olaf Schneewind
Keyword(s):  
Cell Wall ◽  
Bacillus Anthracis ◽  
Active Site ◽  
Diaminopimelic Acid ◽  
Side Chain ◽  
Carboxyl Group ◽  
Amino Group ◽  
Active Site Cysteine ◽  
New Hosts ◽  
Host Death

ABSTRACT Vegetative forms of Bacillus anthracis replicate in tissues of an infected host and precipitate lethal anthrax disease. Upon host death, bacilli form dormant spores that contaminate the environment, thereby gaining entry into new hosts where spores germinate and once again replicate as vegetative forms. We show here that sortase C, an enzyme that is required for the formation of infectious spores, anchors BasI polypeptide to the envelope of predivisional sporulating bacilli. BasI anchoring to the cell wall requires the active site cysteine of sortase C and an LPNTA motif sorting signal at the C-terminal end of the BasI precursor. The LPNTA motif of BasI is cleaved between the threonine (T) and the alanine (A) residue; the C-terminal carboxyl group of threonine is subsequently amide linked to the side chain amino group of diaminopimelic acid within the wall peptides of B. anthracis peptidoglycan.

Peptidoglycan structure in cell walls of parental and filamentous Streptococcus cremoris HP

1974 ◽  
Vol 20 (7) ◽  
pp. 905-913 ◽  
Author(s):  
K. G. Johnson ◽  
I. J. McDonald
Keyword(s):  
Cell Wall ◽  
Glutamic Acid ◽  
Cell Walls ◽  
Free Amino ◽  
Muramic Acid ◽  
Chemical Analyses ◽  
Amino Groups ◽  
Streptococcus Cremoris ◽  
Peptidoglycan Structure ◽  
Filamentous Cell

Cell walls were prepared from parental and filamentous cells of Streptococcus cremoris HP. In addition to aspartic acid, glutamic acid, alanine, and lysine in a 1:2:3:1 ratio, such preparations contained hot formamide-extractable material composed of glucosamine, glucosa-mine-6-phosphate, glucose, galactose, and rhamnose. Parental and filamentous cell walls contained, respectively, 210 and 225 disaccharide units per milligram. The ratio of muramic acid: peptide subunits was about 1.3 for both preparations.Enzymic and chemical analyses revealed that glycan strands are incompletely substituted, peptide cross-bridging is not mediated by D-alanyl-L-alanyl linkages, peptide subunits are linked together to form large moieties, and no significant differences in peptidoglycan structure exist between parental and filamentous cell walls.Analysis by dinitrophenylation techniques disclosed the presence of significant quantities of glucosamine and muramic acid residues with free amino groups in the peptidoglycans of both cell wall preparations. Conversion of such groups by dinitrophenylation or N-acetylation greatly enhanced the response of cell walls to lysozyme digestion.

Inhibition of lysozyme by positively charged groups in the mucopeptide of the bacterial cell wall

1967 ◽  
Vol 167 (1009) ◽  
pp. 443-445 ◽  
Keyword(s):  
Cell Wall ◽  
Small Molecules ◽  
Bacterial Cell ◽  
Cell Walls ◽  
Bacterial Cell Wall ◽  
Amino Groups ◽  
Small Molecular Weight ◽  
Hydrolysis Of ◽  
Isolated Cell ◽  
Positively Charged

The elegant work that has been presented this afternoon has been concerned with the structure of lysozyme in relation to its action on model substrates of small molecular weight, or its inhibition by equally small molecules. The ‘natural’ substrate is presumably the mucopeptide of bacterial cell wall, which is a large, highly complex and insoluble molecule. A portion of a possible structure of a mucopeptide (Tipper & Strominger 1965) in this case from Staphylococcus aureus , is given in figure 51. Evidently in order to facilitate hydrolysis of the glycosidic links on C-1 of muramic acid (and, in the absence of O-acetyl groups, the enzyme is very good at this) lysozyme molecules must be able to approach closely to the relevant part of the polysaccharide backbone. One of the factors that appears to influence this approach of enzyme and substrate is the presence of positive charges on the mucopeptide. Isolated cell walls of Corynebacterium tritici were completely resistant to lysozyme, but could be made sensitive by the action of formamide at 150 °C for 15 min (Perkins 1965). It was found that this procedure did not remove more than 10% of the non-mucopeptide carbohydrate present, but it did formylate the free amino groups of diaminobutyric acid that occurred in the untreated wall. Acetylation by a mild procedure, followed by treatment with alkali to remove any O-acetyl groups, also caused the walls to become susceptible to dissolution by lysozyme.

scholarly journals Facile Synthesis and Metabolic Incorporation of m-DAP Bioisosteres Into Cell Walls of Live Bacteria

2020 ◽  
Author(s):  
Alexis J. Apostolos ◽  
Julia M. Nelson ◽  
Marcos M. Pires
Keyword(s):  
Cell Wall ◽  
Bacterial Cell ◽  
Cell Walls ◽  
Drug Targets ◽  
Solid Phase ◽  
Bacterial Species ◽  
Building Blocks ◽  
Structural Features ◽  
Diaminopimelic Acid ◽  
Bacterial Cells

AbstractBacterial cell walls contain peptidoglycan (PG), a scaffold that provides proper rigidity to resist lysis from internal osmotic pressure and a barrier to protect cells against external stressors. It consists of repeating sugar units with a linkage to a stem peptide that becomes highly crosslinked by cell wall transpeptidases (TP). Because it is an essential component of the bacterial cell, the PG biosynthetic machinery is often the target of antibiotics. For this reason, cellular probes that advance our understanding of PG biosynthesis and its maintenance can be powerful tools to reveal novel drug targets. While synthetic PG fragments containing L-Lysine in the 3rd position on the stem peptide are easier to access, those with meso-diaminopimelic acid (m-DAP) pose a severe synthetic challenge. Herein, we describe a solid phase synthetic scheme based on the widely available Fmoc-protected L-Cysteine building block to assemble meso-cystine (m-CYT), which mimics key structural features of m-DAP. To demonstrate proper mimicry of m-DAP, cell wall probes were synthesized with m-CYT in place of m-DAP and evaluated for their metabolic processing in live bacterial cells. We found that m-CYT-based cell wall probes were properly processed by TPs in various bacterial species that endogenously contain m-DAP in their PG. We anticipate that this strategy, which is based on the use of inexpensive and commercially available building blocks, can be widely adopted to provide greater accessibility of PG mimics for m-DAP containing organisms.

scholarly journals Peptidoglycan biosynthesis by preparations from Bacillus licheniformis: cross-linking of newly synthesized chains to preformed cell wall

1974 ◽  
Vol 139 (3) ◽  
pp. 781-784 ◽  
Author(s):  
J. Barrie Ward ◽  
Harold R. Perkins
Keyword(s):  
Cell Wall ◽  
Bacillus Licheniformis ◽  
Free Amino ◽  
Membrane Preparation ◽  
Diaminopimelic Acid ◽  
Cross Linking ◽  
Amino Group ◽  
Peptidoglycan Biosynthesis ◽  
Acetyl Group ◽  
Degraded Product

A wall-plus-membrane preparation from a Bacillus licheniformis mutant incorporated radioactivity from a peptidoglycan precursor in which the free amino group of diaminopimelic acid was blocked by 14C-labelled acetyl group. This incorporation was penicillin-sensitive. The enzymically degraded product contained cross-linked dimers, showing that newly synthesized peptidoglycan chains had been cross-linked to the pre-existing cell wall.

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.

Glucosamine substitution and muramidase susceptibility in Bacillus anthracis

1984 ◽  
Vol 30 (5) ◽  
pp. 553-559 ◽  
Author(s):  
Gene F. Zipperle Jr. ◽  
John W. Ezzell Jr. ◽  
Ronald J. Doyle
Keyword(s):  
Acetic Anhydride ◽  
Bacillus Anthracis ◽  
Growth Rates ◽  
Cell Walls ◽  
Diaminopimelic Acid ◽  
Amino Sugars ◽  
Muramic Acid ◽  
Amino Groups ◽  
Lytic Action ◽  
Number Of Cells

Cell walls of Bacillus anthracis were found to be resistant to lysozyme, and partially resistant to mutanolysin, a muramidase from Streptomyces globisporus. Following treatment with acetic anhydride, it was observed that the walls were highly susceptible to hydrolysis by lysozyme or mutanolysin. Analyses of cell walls, prior to and following derivatization with fluorodinitrobenzene, revealed that approximately 88% of the glucosamine residues and 34% of the muramic acid residues of the peptidoglycan contained unsubstituted amino groups, thereby providing an explanation for the resistance of the walls to lysozyme. The walls of B. anthracis were approximately 19% cross-linked, based on the findings that 81% of the diaminopimelic acid residues could be modified by fluorodinitrobenzene. Walls of B. thuringiensis 4040 and B. cereus ATCC 19637 also contained high percentages of unsubstituted amino sugars, and unless acetylated, were also relatively resistant to lysozyme and mutanolysin. When B. anthracis, B. cereus, or B. thuringiensis were grown in the presence of 100 μg/mL lysozyme, there was a decrease in the average number of cells per chain, but there was no decrease in growth rates, suggesting that the enzyme was acting at septa. It is unlikely that lysozyme and autolysins act synergistically in Bacillus, because azide anion, which activates autolysins, did not enhance the lytic action of lysozyme in B. anthracis, B. cereus, or B. thuringiensis.

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