Murein (peptidoglycan) structure of Vibrio fetus comparison of a venereal and an intestinal strain

The murein of a venereal and an intestinal strain of Vibrio fetus was isolated by extraction with hot 4% sodium dodecyl sulfate, indicating the absence of covalently bound protein. Murein was composed of muramic acid, glucosamine, alanine, glutamic acid and diaminopimelic acid in molar ratios of 1:1:2:1:1. Approx. 30% of Dpm molecules were involved in peptide cross linkages and analyses of lysozyme split products indicated a structure similar to that of other Gram-negative genera. Evidence was obtained for the occurrence of chromatographically distinct fractions of the disaccharide tetrapeptide (GlcNAcMurNAclAladGlumesoDpmdAla). Digestion products also included variable concentrations of free murein peptides and glucosamine, whose origin is unexplained. In no instance were differences observed between mureins of intestinal and venereal strains of V. fetus.     

Characterization of the cell wall murein of Thiobacillus versutus

Amino acid analysis of pure murein isolated from cells of Thiobacillus versutus grown in complex medium revealed the typical constituents of most mureins from gram-negative cells, i.e. muramic acid, glucosamine, glutamic acid, alanine and diaminopimelic acid in molecular ratio of 0.58: 0.79: 1.0: 1.76:1.07, respectively. The presence of glycine and leucine was also demonstrated (0.20 and 0.08 compared to glutamic acid). Glycine was also present in the murein of cells grown in chemically defined synthetic medium. The crosslinkage of T. versutus murein was approximately 36% --much higher than for most other gram-negative species. High pressure liquid chromatography analysis of muropeptide composition following muramidase digestion of T. versutus murein revealed essentially the same pattern as for Escherichia coli under similar conditions of digestion and separation with, however, some differences in the minor peaks.     

Die Aminosäuresequenz des Mureins von Staphylococcus epidermidis (Winslow and Winslow) Evans,Stamm 66

Zusammenfassung Das Murein eines aus Milch isolierten Stammes von Staphylococcus epidermidis weist folgende Molverhältnisse auf (auf- bzw. abgerundete Zahlen): Mur:GlcNH₂:Ala:Glu:Lys:Gly=1:1:3:1:1:4. Das Verhältnis D-Ala:L-Ala ist 1:2,03. Die Glutaminsäure liegt in der D-Konfiguration und als Amid vor.Durch die Isolierung und Identifizierung der Peptide des Partialhydrolysats des Mureins konnte die Aminosäuresequenz erschlossen werden. Die Sequenz des an die Muraminsäure gebundenen Tetrapeptides (L-Ala-D-GluNH₂-L-Lys-D-Ala) stimmt mit dem der meisten anderen Bakterien überein. Die Quervernetzung wird durch das Peptid (Gly)_4–5-L-Ala hergestellt, das mit dem N-terminalen Glycin an die Carboxylgruppe des D-Alanins und mit dem C-terminalen L-Alanin an die -Aminogruppe des Lysins zweier benachbarter Tetrapeptide gebunden ist. Die Dinitrophenylierung des Mureins ergab, daß 2% des Lysins (-Aminogruppe), 3% des gesamten Alanins und 7% des gesamten Glycins N-terminal vorliegen. Demnach ist die Quervernetzung nur zu rund 60% realisiert. Neben unvernetzten mehr oder weinger vollständigen Interpeptidbrücken kommen auch unvollständige Peptide vor, bei denen nur L-Alanin an die -Aminogruppe des Lysins gebunden ist. In mindestens 2% der Fälle fehlt die Interpeptidkette völlig.

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The amino acid sequence of the murein of Staphylococcus epidermidis (winslow and winslow) evans, strain 66

Summary A strain of Staphylococcus epidermidis was isolated from raw milk. Its murein contained muramic acid, glucosamine, alanine, D-glutamic acid, L-lysine and glucine at a molar ratio of about 1:1:3:1:1:4. The ratio D-Ala: L-Ala is 1:2.03. D-glutamic acid is present as an amide.By partial acid hydrolysis of the cell wall and subsequent isolation and identification of the peptides the amino acid sequence of the murein was elucidated. The tetrapeptide, bound to muramic acid is identical with that of most bacteria: L-Ala-D-GluNH₂-L-Lys-D-Ala. The crosslinking of the murein is performed by the peptide (Gly)_4–5-L-Ala. L-Ala is attached to the -aminogroup of lysine, while the N-terminal glycine is bound to the C-terminal D-alanine of an adjacent tetrapeptide. About 2% of lysine, 3% of alanine and 7% of glycine of the murein are dinitrophenylizable, indicating that about 2% of the tetrapeptides are not substituted by an interpeptide chain, and that 40% of the interpeptide chains are more or less incomplete (10% consist of L-alanine only) and are not bound to a C-terminal D-alanine.

     

Die Aminosäuresequenz eines glycinhaltigen Mureins einiger Stämme von Lactobacillus bifidus

Zusammenfassung Das Murein (Peptidoglycan) von 6 Stämmen Lactobacillus bifidus, die aus der Faeces von Brustkindern oder aus dem Darminhalt von Bienen isoliert worden waren, wies folgendes Molverhältnis auf (auf- bzw. abgerundete Zahlen): Mur:GlcNH₂:Ala:Glu:Lys:Gly=1:1:2:1:1:1. Das Verhältnis l-Ala: d-Ala=1,1:1. Glutaminsäure liegt als Amid vor.Durch die Analyse der Peptide des Partialhydrolysats konnte folgende Aminosäuresequenz erschlossen werden: Das Tetrapeptid besitzt wie bei den meisten Bakterien die übliche Sequenz l-Ala-d-Glu-l-Lys-d-Ala. Glycin ist einerseits an die -Aminogruppe des Lysins, andererseits an die Carboxylgruppe des C-terminalen d-Alanins gebunden und stellt somit die Quervernetzung des Mureins her. Die Dinitrophenylierung der Zellwand ergab, daß rund 50% des Glycins und einige Prozent des Lysins eine freie Aminogruppe tragen. Die Quervernetzung ist demnach nur zu rund 50% durchgeführt.

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The amino acid sequence of the glycine containing murein of some strains of Lactobacillus bifidus

Summary The murein (peptidoglycan) of 6 strains of L. bifidus, isolated from faeces of breast fed infants or from the intestine of bees, respectively, contained muramic acid, glucosamine, alanine, glutamic acid, lysine and glycine at a molar ratio of 1:1:2:1:1:1. The ratio of l-alanine: d-alanine is 1,1: 1. The analysis of the peptides obtained by acid partial hydrolysis indicated that the amino acid sequence of the tetrapeptide is identical with that of most bacteria (l-Ala-d-Glu-l-Lys-d-Ala). Glutamic acid is present as an amide.Glycine is involved in the crosslinking of adjacent muropeptides by forming a bridge between the -aminogroup of lysine and the carboxyl group of a C-terminal d-alanine. About 50% of the glycine is N-terminal, indicating that only 50% of the possible cross linkages are realized.The murein of these strains of L. bifidus resembles the murein of staphylococci, but differs by the number of glycine molecules. While a pentameric glycylpeptide occurs in the murein of staphylococci, only one molecule of glycine is involved in the crosslinkage of the murein described here.

     

Die Aminosäuresequenz des Threonin und Serin enthaltenden Mureins von Bifidobacterium longum Reuter

Zusammenfassung Von 18 Stämmen von Bifidobacterium longum Reuter und einem Stamm von B. lactentis Reuter wurden die Zellwände in üblicher Weise hergestellt. Sie enthielten Mur, GlcNH₂, d-Glu, Ala, l-Orn (l-Lys), Thr, Ser in einem Molverhältnis von rund 1:1:1:4:1:1:1. Die Molverhältnisse änderten sich nicht, wenn die Zellwände mit Trichloressigsäure oder heißem Formamid extrahiert wurden. In einigen Stämmen trat mehr als 1 Mol Glutaminsäure pro Mol Diaminosäure auf. Die zusätzliche Glutaminsäure hatte die l-Konfiguration. Sie war kein Bestandteil des Mureins, sondern einer lysozymunempfindlichen, unbekannten Zellwandkomponente, vermutlich einer Polyglutaminsäure. l-Ornithin war in den meisten Stämmen die dominierende Diaminosäure, während l-Lysin nur mit einem Anteil von 10–20% vertreten war. In 2 Stämmen war l-Lysin dominierend (90%). Die Aminosäuresequenz wurde durch die Analyse der Oligopeptide aus Partialhydrolysaten bestimmt. Die an Mureinsäure gebundenen Peptiduntereinheiten hatten die auch von anderen Mureinen bekannte Sequenz: l-Ala-d-Glu-l-Orn (oder l-Lys)-d-Ala. Glutaminsäure ist wahrscheinlich amidiert, wie aus dem Auftreten von rund 1 Mol NH₃ im Hydrolysat der Zellwände zu schließen ist. Die Interpeptidbrücke besteht aus dem Peptid l-Ala-Thr-l-Ala-l-Ser. Sie ist mit dem C-terminalen Serin an die -Aminogruppe der Diaminosäure der Peptiduntereinheit gebunden. Die Quervernetzung erfolgt zwischen dem N-terminalen Alanin der Interpeptidbrücke zum C-terminalen d-Alanin einer Peptiduntereinheit. Da 4% des gesamten Alanins und 3% der -Aminogruppe des Ornithins dinitrophenylierbar sind, ist anzunehmen, daß die Quervernetzung nur zu etwa 80% verwirklicht ist.

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The amino acid sequence of the threonine and serine containing murein of Bifidobacterium longum reuter

Summary Cell walls of 18 strains of Bifidobacterium longum Reuter and one strain of B. lactentis Reuter were prepared in the usual way. They contained Mur, GlcNH₂, d-Glu, Ala, l-Orn (l-Lys), Thr, Ser in a molar ratio of about 1:1:1:4:1:1:1. The ratio was not changed when the cell walls were extracted by trichloroacetic acid or hot formamide. In some strains more than 1 mole glutamic acid per mole of diamino acid was present. The additional glutamic acid was of the l-rather than the d-form. It was not a constituent of the murein, but of an unknown lysozyme insensitive cell wall component, probably a polyglutamic acid. l-Ornithine was the dominating diamino acid in most strains but l-lysine was also present in a portion of 10 to 20%. In 2 strains l-lysine was dominating (90%).The amino acid sequence was determined by analysing the oligopeptides arising during partial acid hydrolysis. It was shown that the peptide subunits attached to the muramic acid are the same as those of other mureins: l-Ala-d-Glu-l-Orn (or l-Lys)-d-Ala. glutamic acid is probably amidated, since about 1 mole of NH₃ is released by acid hydrolysis of the cell walls. The interpeptide bridge consists of the peptide l-Ala-Thr-l-Ala-l-Ser which is bound by its C-terminal serine to the -amino group of the diamino acid of one peptide subunit and by its N-terminal l-alanine to the C-terminal d-alanine of another peptide subunit. About 4% of the total alanine and 3% of the -amino groups of ornithine of the cell wall can be dinitrophenylated. This indicates that about 20% of the peptide subunits are not crosslinked.

     

Discovery of new inhibitors of the bacterial peptidoglycan biosynthesis enzymes MurD and MurF by structure-based virtual screening

The ATP-dependent Mur ligases (MurC, MurD, MurE and MurF) successively add l-Ala, d-Glu, meso-A₂pm or l-Lys, and d-Ala-d-Ala to the nucleotide precursor UDP-MurNAc, and they represent promising targets for antibacterial drug discovery. We have used the molecular docking programme eHiTS for the virtual screening of 1990 compounds from the National Cancer Institute ‘Diversity Set’ on MurD and MurF. The 50 top-scoring compounds from screening on each enzyme were selected for experimental biochemical evaluation. Our approach of virtual screening and subsequent in vitro biochemical evaluation of the best ranked compounds has provided four novel MurD inhibitors (best IC₅₀ = 10 μM) and one novel MurF inhibitor (IC₅₀ = 63 μM).     

Die Aminosäuresequenz von 2,4-Diaminobuttersäure enthaltenden Mureinen bei verschiedenen coryneformen Bakterien und Agromyces ramosus

Zusammenfassung Bie 17 Stämmen von coryneformen Organismen wurde 2,4-Diaminobuttersäure als Bestandteil des Mureins gefunden. In 15 Fällen ergab die genauere Analyse die gleiche Aminosäuresequenz, wie sie schon früher von Perkins (1968) bei Corynebacterium insidiosum beschrieben wurde. In diesem Falle ist die L-2,4-Diaminobuttersäure ein Bestandteil der Peptiduntereinheit, während die D-2,4-Diaminobuttersäure die Quervernetzung zwischen dem Glutaminsäurerest und dem C-terminalen Alanin zweier benachbarter Peptiduntereinheiten herstellt. Das Murein gehört demnach zur Gruppe B nach Schleifer u. Kandler (1972). Die -Aminogruppe der L-2,4-Diaminobuttersäure ist in einigen Fällen acetyliert, in anderen Fällen ist sie frei.Das Murein der beiden anderen Stämme unterscheidet sich in seiner Primärstruktur dadurch, daß nur L-2,4-Diaminobuttersäure vorkommt. Im Falle von C. bovis ist wie bei einigen coryneformen pflanzenpathogenen Stämmen die Diaminosäure der Peptiduntereinheit durch Homoserin ersetzt und die Quervernetzung erfolgt durch das Dipeptid -Gly-L-Dab zwischen Glutaminsäure und D-Alanin. Dieses Murein gehört demnach ebenfalls zur Gruppe B. Dagegen ist das Murein von Arthrobacter sp. Ar 22 eine neue Variante der Gruppe A. Die L-2,4-Diaminobuttersäure ist hier ein Glied der Peptiduntereinheit und die Quervernetzung zwischen der -Aminogruppe der 2,4-Diaminobuttersäure und dem D-Alaninrest einer benachbarten Peptiduntereinheit wird durch das Pentapeptid -L-Asp-L-Ala-Gly-L-Ala-L-Ala gebildet. Außerdem ist die Position 1 der Peptiduntereinheit nicht mit L-Alanin, sondern mit Glycin besetzt. Letzteres ist bisher nur bei Mureinen der Gruppe B, aber nicht bei denen der Gruppe A gefunden worden. Ebenfalls neu ist das Vorkommen von L-Asparaginsäure anstelle der bisher gefundenen D-Form.

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The amino acid sequence of 2,4-diaminobutyric acid containing mureins of various coryneform bacteria and Agromyces ramosus

Summary In 17 strains of coryneform bacteria, 2,4-diaminobutyric acid was found to be a component of the murein (peptidoglycan). A detailed analysis showed that 15 strains contain a murein with the same amino acid sequence as that found in Corynebacterium insidiosum by Perkins (1968). In this case the L-2,4-diaminobutyric acid is a component of the peptite subunit while the D-2,4-diaminobutyric acid serves as interpetide bridge between D-glutamatic and the C-terminal D-alanine residue. Therefore this murein belongs to group B according to Schleifer and Kandler (1972). The -amino group of L-2,4-diaminobutyric acid is in some species acetylated, in others free.The murein of the remaining two strains differs by the lack of D-2,4-diaminobutyric acid. Only L-2,4-diaminobutyric acid is found. In the case of C. bovis, the diamino acid of the peptide subunit is replaced by L-homoserine as found in various plant pathogenic coryneform bacteria. The interpeptide bridge consists of the dipeptide -Gly-2,4-Dab. It connects the D-glutamic acid of one peptide subunit with the C-terminal D-alanine residue of an adjacent peptide subunit. Therefore this murein belongs also to group B.The murein of Arthrobacter sp. Ar 22 is a new varition of group A, however. Here the L-2,4-diaminobutyric acid is a component of the peptide subunit. The interpeptide bridge consists of the pentapeptide -L-Asp-L-Ala-Gly-L-Ala-L-Ala. It connects the -amino group of L-2,4-diaminobutyric acid and the C-terminal D-alanine residue of two peptide subunits. Position 1 of the peptide subunit is occupied by glycine instead of L-alanine as found in all the other mureins of group A so far. Another new feature of this murein is the occurrence of the L-form instead of the D-form of aspartic acid.

     

Murein structure of Acetobacterium woodii

The isolated cell walls of Acetobacterium woodii contain a murein of the crosslinkage type B. d-Orinithinyl residues function as interpeptide bridges between the -carboxyl group of d-glutamic acid and the carboxyl group of the terminal d-analyl residue of an adjacent peptide subunit. The usual l-alanyl residue in position 1 of the peptide subunit is replaced by a l-seryl residue. As yet this murein type was only found in Eubacterium limosum, an organism which was supposed to be related to Acetobacterium because of some metabolic similarities.     

Analyses of MbtB, MbtE, and MbtF suggest revisions to the mycobactin biosynthesis pathway in Mycobacterium tuberculosis

The production of mycobactin (MBT) by Mycobacterium tuberculosis is essential for this bacterium to access iron when it is in an infected host. Due to this essential function, there is considerable interest in deciphering the mechanism of MBT assembly, with the goal of targeting select biosynthetic steps for antituberculosis drug development. The proposed scheme for MBT biosynthesis involves assembly of the MBT backbone by a hybrid nonribosomal peptide synthetase (NRPS)/polyketide synthase (PKS) megasynthase followed by the tailoring of this backbone by N(6) acylation of the central l-Lys residue and subsequent N(6)-hydroxylation of the central N(6)-acyl-l-Lys and the terminal caprolactam. A complete testing of this hypothesis has been hindered by the inability to heterologously produce soluble megasynthase components. Here we show that soluble forms of the NRPS components MbtB, MbtE, and MbtF are obtained when these enzymes are coproduced with MbtH. Using these soluble enzymes we determined the amino acid specificity of each adenylation (A) domain. These results suggest that the proposed tailoring enzymes are actually involved in precursor biosynthesis since the A domains of MbtE and MbtF are specific for N(6)-acyl-N(6)-hydroxy-l-Lys and N(6)-hydroxy-l-Lys, respectively. Furthermore, the preference of the A domain of MbtB for l-Thr over l-Ser suggests that the megasynthase produces MBT derivatives with β-methyl oxazoline rings. Since the most prominent form of MBT produced by M. tuberculosis lacks this β-methyl group, a mechanism for demethylation remains to be discovered. These results suggest revisions to the MBT biosynthesis pathway while also identifying new targets for antituberculosis drug development.     

Purification and biochemical characterization of Mur ligases from Staphylococcus aureus

The Mur ligases (MurC, MurD, MurE and MurF) catalyze the stepwise synthesis of the UDP-N-acetylmuramoyl-pentapeptide precursor of peptidoglycan. The murC, murD, murE and murF genes from Staphylococcus aureus, a major pathogen, were cloned and the corresponding proteins were overproduced in Escherichia coli and purified as His₆-tagged forms. Their biochemical properties were investigated and compared to those of the E. coli enzymes. Staphylococcal MurC accepted l-Ala, l-Ser and Gly as substrates, as the E. coli enzyme does, with a strong preference for l-Ala. S. aureus MurE was very specific for l-lysine and in particular did not accept meso-diaminopimelic acid as a substrate. This mirrors the E. coli MurE specificity, for which meso-diaminopimelic acid is the preferred substrate and l-lysine a very poor one. S. aureus MurF appeared less specific and accepted both forms (l-lysine and meso-diaminopimelic acid) of UDP-MurNAc-tripeptide, as the E. coli MurF does. The inverse and strict substrate specificities of the two MurE orthologues is thus responsible for the presence of exclusively meso-diaminopimelic acid and l-lysine at the third position of the peptide in the peptidoglycans of E. coli and S. aureus, respectively. The specific activities of the four Mur ligases were also determined in crude extracts of S. aureus and compared to cell requirements for peptidoglycan biosynthesis.     

The murein of Thiobacillus neapolitanus

Amino acid analysis of pure murein isolated from cells of T. neapolitanus revealed the typical constituents of most mureins form Gram-negative bacteria. i.e. glutamic acid, alanine and diaminopimelic acid, but the molecular ratio ot these was unusual, being approximately 1: 1: 1. The reduced amount of alanine was explained by the absence of monomers containing tetrapeptide side chains, as revealed by h. p. 1. c. analysis, [(3)H]glutamic acid, [(3)H]diaminopimelic acid and [(3)H]N-acetylglucosamine were incorporated into the murein and allowed to determine the degree of its crosslinkage (28%) and the occurrence of turnover.     

Die Aminosäuresequenz des DAP-haltigen Mureins von Lactobacillus plantarum und Lactobacillus inulinus

Zusammenfassung Von L. plantarum und L. inulinus wurden die Zellwände isoliert und durch Inkubation mit Trypsin gereinigt. Durch Extraktion mit TES und Formamid konnte das Murein (Peptidoglycan) bis zu rund 85% der Trockenmasse angereichert werden. Die Zellwände von L. plantarum enthielten rund 30% Teichonsäure des Ribit-Typs, die von L. inulinus waren frei von Teichonsäure.Im Hydrolysat der teichonsäurefreien Zellwände ergaben sich folgende aufbzw. abgerundete Molverhältnisse Mur: GlNH₂:Glu:DAPl-Alad-Ala=1:1:1:1:1:0,5. Außerdem waren 2 Mole Ammoniak enthalten, was das Vorliegen von Glu und DAP als Amide anzeigt. Die durch Hemmung mit d-Cycloserin angereicherte unvollständige Mureinvorstufe hatte ein Molverhältnis von UDP:Murl-Ala:Glu:DAP=1:1:1:1:1.Nach Dinitrophenylierung der Zellwand ließen sich rund 50% der gesamten DAP als mono-DNP-DAP nachweisen. Die Hydrazinolyse der Zellwand zum Nachweis C-terminaler Aminosäuren ergab 4% freies DAP und 0,8% freies Alanin.Durch die Analyse der in Partialhydrolysaten der Zellwand auftretenden Peptide konnte die folgende Aminosäuresequenz des an die Muraminsäure gebundenen Tetrapeptides bestimmt werden: l-Ala-d-Glu-l-Lys-d-Ala. Im Murein ist vermutlich nur etwa die Hälfte der Muraminsäure mit einem Tetrapeptid, die andere Hälfte mit einem Tripeptid, dessen d-Alanin fehlt, substituiert.Die Quervernetzung erfolgt zwischen der 2. Aminogruppe der DAP und der Carboxylgruppe des d-Alanins eines benachbarten Tetrapeptids.

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The amino acid sequence of the DAP-containing murein of Lactobacillus plantarum and Lactobacillus inulinus

Summary Cell walls of L. plantarum and L. inulinus were isolated and purified by incubation with trypsin. After extraction with TCA and formamide, 85% of the dry weight consists of murein (peptidoglycan).The cell walls of L. plantarum contained about 30% teichoic acid (ribit-type), whereas no teichoic acid was present in the cell walls of L. inulinus.The quantitative determination of amino sugars and amino acids in the hydrolysate of the cell walls showed the following molar ratios: Mur: Gl-NH₂:Glu:DAP l-Alad-Ala=1:1:1:1:1:0.5. In addition, 2 mols of NH₃ were found per mol of glutamic acid, indicating, that DAP as well as glutamic acid are present as amides.The UDP-activated cell wall precursor which was accumulated by inhibiting the cells by d-cycloserine showed the following molar ratios: UDP:Murl-Ala: Glu:DAP=1:1:1:1:1.After dinitrophenylation and hydrolysation of the cell wall 50% of the DAP were present as mono-DNP-DAP. Hydrozinolysis of the cell wall yielded 4% free DAP and 0.8% free alanine. This shows that only a very small amount of these amino acids are C-terminal in the whole murein.The analysis of various peptides from acid partial hydrolysates of the cell wall indicates the following amino acid sequence of the tetrapeptides attached to muramic acid: l-Ala-d-Glu-meso-DAP-d-Ala. Only half of the muramic acid molecules are substituted by tetrapeptides, while the other half carries a tripeptide in which the terminal d-alanine is missing.The cross-linking of the muropeptides is achieved by a peptide-bond between the second amino group of DAP and the carboxylgroup of the d-alanine of an adjacent muropeptide.

     

Zur chemischen Zusammensetzung der Zellwand der Streptokokken

Zusammenfassung Das Murein (Peptidoglycan) eines aus Faeces isolierten Streptococcus, der in den wichtigsten Merkmalen mit Peptostreptococcus evolutus (Prevot) Smith übereinstimmt, weist folgende Molverhältnisse auf (aufgerundete bzw. abgerundete Zahlen): Mur:GlcNH₂:Ala:Glu:Lys:Gly=1:1:3:1:1:1. Das Verhältnis l-Alanin:d-Alanin=2,15:1. Die Glutaminsäure liegt in der d-Konfiguration und als Amid vor.Durch die Partialhydrolyse der Zellwände und die anschließende Isolierung und Identifizierung der Peptide konnte die Aminosäuresequenz des Mureins geklärt werden. Das Tetrapeptid stimmt mit der üblichen Sequenz l-Ala-d-Glu-NH₂-l-Lys-d-Ala der meisten übrigen Bakterien überein. Die Quervernetzung des Mureins wird durch das Peptid Glycyl-l-Alanin hergestellt, wobei l-Alanin an die -Aminogruppe des Lysins gebunden ist. Die Dinitrophenylierung der Zellwand ergab, daß 35% des Glycins und 6% des Lysins eine freie Aminogruppe aufweisen. Die Quervernetzung ist demnach nur zu höchstens 60% durchgeführt.

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The chemical composition of the cell walls of Streptococci III. The amino acid sequence of a glycine containing murein from Peptostreptococcus evolutus (Prevot) Smith

Summary Peptostreptococcus evolutus was isolated from feces. Its murein containes muramic acid, glucosamine, alanine, d-glutamic acid, lysine and glycine at a molar ratio of about 1:1:3:1:1:1. The ratio of l-alanine: d-alanine is 2,15:1. Glutamic acid is present as an amide.By acid partial hydrolysis of the cell walls and subsequent isolation and identification of the peptides the amino acid sequence of the murein was elucidated. The tetrapeptide is identical with that of most bacteria (l-Ala-d-Glu-NH₂-l-Lys-d-Ala). The crosslinking of the murein is performed by the peptide glycyl-l-alanine. l-alanine is attached to the -amino group of lysine while the amino group of glycine is bound to the carboxyl group of the c-terminal d-alanine of an adjacent tetrapeptide. About 35% glycine and 6% lysine of the murein are dinitrophenylisable indicating that maximally 60% of the possible cross-linkages are realized.

     

Identification of Mur, an atypical peptidoglycan hydrolase derived from Leuconostoc citreum

A gene encoding a protein homologous to known bacterial N-acetyl-muramidases has been cloned from Leuconostoc citreum by a PCR-based approach. The encoded protein, Mur, consists of 209 amino acid residues with a calculated molecular mass of 23,821 Da including a 31-amino-acid putative signal peptide. In contrast to most of the other known peptidoglycan hydrolases, L. citreum Mur protein does not contain amino acid repeats involved in cell wall binding. The purified L. citreum Mur protein was shown to exhibit peptidoglycan-hydrolyzing activity by renaturing sodium dodecyl sulfate-polyacrylamide gel electrophoresis. An active chimeric protein was constructed by fusion of L. citreum Mur to the C-terminal repeat-containing domain (cA) of AcmA, the major autolysin of Lactococcus lactis. Expression of the Mur-cA fusion protein was able to complement an acmA mutation in L. lactis; normal cell separation after cell division was restored by Mur-cA expression.     

Amino acid sequence of the murein of Planococcus and other Micrococcaceae

The amino acid composition and amino acid sequence of the murein (peptidoglycan) of 10 strains of planococci were studied. It is shown that the peptide subunit consists of muramyl-l-alanyl-gamma-d-glutamyl-l-lysyl-d -alanine. The cross-linking of two adjacent peptide subunits is mediated by d-glutamic acid which is bound to the epsilon-amino group of lysine by its gamma-carboxyl group and to the carboxyl group of d-alanine of an adjacent peptide subunit by its amino group. About 20 to 25% of the peptide subunits are not cross-linked. The murein structure of the different species and strains of Micrococcus, Staphylococcus, and Sarcina are compared. It is evident that the murein structure is a very good criterion for grouping the micrococci. In addition, some of these groups are fairly well defined by physiological properties as well as by their guanine + cytosine content of the deoxyribonucleic acid e.g., Micrococcus, Staphylococcus, Planococcus, Sarcina ureae. Other groups, represented by a single or a few strains only, such as M. varians NTCC 7281, M. radiodurans, M. freudenreichii ATCC 407, and M. luteus ATCC 398, need further investigation.     

Analysis of the peptidoglycan structure of Bacillus subtilis endospores.

Peptidoglycan was prepared from purified Bacillus subtilis spores of wild-type and several mutant strains. Digestion with muramidase resulted in cleavage of the glycosidic bonds adjacent to muramic acid replaced by peptide or alanine side chains but not the bonds adjacent to muramic lactam. Reduction of the resulting muropeptides allowed their separation by reversed-phase high-pressure liquid chromatography. The structures of 20 muropeptides were determined by amino acid and amino sugar analysis and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. In wild-type spores, 50% of the muramic acid had been converted to the lactam and 75% of these lactam residues were spaced regularly at every second muramic acid position in the glycan chains. Single L-alanine side chains were found on 25% of the muramic acid residues. The remaining 25% of the muramic acid had tetrapeptide or tripeptide side chains, and 11% of the diaminopimelic acid in these side chains was involved in peptide cross-links. Analysis of spore peptidoglycan produced by a number of mutants lacking proteins involved in cell wall metabolism revealed structural changes. The most significant changes were in the spores of a dacB mutant which lacks the sporulation-specific penicillin-binding protein 5*. In these spores, only 46% of the muramic acid was in the lactam form, 12% had L-alanine side chains, and 42% had peptide side chains containing diaminopimelic acid, 29% of which was involved in cross-links.     

Chemical structure of the peptidoglycan of Vibrio parahaemolyticus A55 with special reference to the extent of interpeptide cross-linking.

The chemical structure of the cell wall peptidoglycan of Vibrio parahaemolyticus A55 was studied. Estimation of cross linkages between peptide subunits in the peptidoglycan by dinitrophenylation showed that about 30% of the total 2,6-diaminopimelic acid (A2pm) residues were involved in cross linkages. The presence of interpeptide bridges was also demonstrated by isolating bisdisaccharide peptide subunit dimers from Chalaropsis muramidase digests of the cell wall peptidoglycan by gel filtration followed by ion-exchange column chromatography, although most of the building blocks obtained were uncross-linked disaccharide peptide monomers. The chain length of a glycan moiety of the peptidoglycan obtained by treatment with the L-11 enzyme and gel filtration of the digest was also studied. The chain length varied from 7 to 44, but 30% of the glycan fragments had muramic acid at the reducing end and a chain length of 28 to 44. In conformity with the above structural study it was demonstrated that a particulate enzyme fraction obtained by differential centrifugation of a sonicated preparation of V. parahaemolyticus catalyzed a penicillin-sensitive transpeptidation reaction, using UDP-MurNAc-14C-pentapeptide and UDP-GlcNAc as substrates.     

Murein type and polysaccharide composition of cell walls from Renibacterium salmoninarum

Abstract The primary structure of the murein of Renibacterium salmoninarum ATCC33209 was determined. It contained lysine in the third position of the peptide subunit, a glycyl-alanine interpeptide bridge between lysine and the d -alanine of adjacent peptide subunits and a d -alanine-amide substitution at the α-carboxyl group of d -glutamic acid in position 2 of the peptide side chain.
Cell walls contained a considerable amount of polysaccharide with galactose as major sugar component. In addition N -acetyl-glucosamine, rhamnose and N -acetyl-fucosamine were detected.     

A novel type of meso-diaminopimelic acid-based peptidoglycan and novel poly (erythritol phosphate) teichoic acids in cell walls of two coryneform isolates from the surface flora of French cooked cheeses

The primary structure of the peptidoglycan and the teichoic acids of two coryneform isolates from the surface flora of French cooked cheeses, CNRZ 925 and CNRZ 926, have been determined. In the peptidoglycan, meso-diaminopimelic acid was localized in position three of the peptide subunit. It contained an d-glutamyl-d-aspartyl interpeptide bridge, connecting meso-diaminopimelic acid and d-alanine residues of adjacent peptide subunits. The -carboxyl group of d-glutamic acid in position two of peptide subunits was substituted with glycine amide. The teichoic acid pattern and composition differed between the strains: both contained an erythritol teichoic acid and strain CNRZ 925 also contained an N-acetylglucosaminylphosphate polymer. The erythritol teichoic acids differed in terms of the quality and quantity of substituents, but they both had N,N-diacetyl-2,3-diamino-2,3-dideoxyglucuronic acid in common.Abbreviations DNP dinitrophenyl - Ery erythritol - Gal galactose - GlcN glucosamine - GlcNAc N-acetylglucosamine - GlcUANAc₂ N,N-diacetyl-2,3-diamino-2,3-dideoxyglucuronic acid - Hex UANAc₂ N,N-diacetyl-2,3-diamino-2,3-dideoxyhexuronic - acid m-Dpm, meso-diaminopimelic acid - Mur muramic acid - MurNAc N-acetylmuramic acid     

Structural investigations on the cell surface of Erysipelothrix rhusiopathiae

The cell wall of Erysipelothrix rhusiopathiae was investigated. The corrected structure of the murein, which was published earlier, is reported here. The murein belongs to the B1delta type. It has L-alanine in position three of the peptide subunit and is crosslinked via a glycine-L-lysine-L-lysine interpeptide bridge. As expected by the complex serological situation in E. rhusiopathiae the carbohydrate moiety of the cell wall proofed to be very intricate. As accessory polymers polysaccharide(s) and teichoic acid(s) were identified. The teichoic acid(s) possess two novel polyols of which one could not be identified, the other one behaved like 6-O-methylgalactitol in gas liquid chromatography. Glucose and N-acetylgalactosamine were determined as substituents. The polysaccharide(s) is composed of mainly N-acetylfucosamine and smaller amounts of galactose, N-acetylglucosamine, and a further unidentified sugar appearing later than hexaacetylsorbitol in GLC.