Carbohydrate metabolism in lactic acid bacteria

The term “lactic acid bacteria” is discussed. An overview of the following topics is given: main pathways of homo- and heterofermentation of hexoses, i.e. glycolysis, bifidus pathway, 6-phosphogluconate pathway; uptake and dissimilation of lactose (tagatose pathway); fermentation of pentoses and pentitols; alternative fates of pyruvate, i.e. splitting to formate and acetate, CO₂ and acetate or formation of acetoin and diacetyl; lactate oxidation; biochemical basis for the formation of different stereoisomers of lactate.     

cDNA cloning and sequencing of tarantula hemocyanin subunits

Summary Tarantula heart cDNA libraries were screened with synthetic oligonucleotide probes deduced from the highly conserved amino acid sequences of the two copper-binding sites, copper A and copper B, found in chelicerate hemocyanins. Positive cDNA clones could be obtained and four different cDNA types were characterized.     

Evaluation of the extent of heterogeneity in the Glycera dibranchiata monomer haemoglobin fraction by the use of n.m.r. and ion-exchange chromatography.

The coelomic haemoglobin of Glycera dibranchiata is known to be separable into monomeric and higher-Mr fractions. Although exhibiting homogeneity with respect to Mr, the extent of haemoglobin heterogeneity for the monomer fraction has never been adequately assayed. In the present paper we demonstrate that there exists in the monomer haemoglobin fraction reproducibly detectable heterogeneity regardless of the presence or absence of proteinase inhibitors during the isolations. These results show that, considered on the same time scale as previous preparations used for amino acid sequencing, crystallography and kinetics, the monomer haemoglobin fraction is highly heterogeneous. Application of ion-exchange chromatography and ion-filtration methods resulted in the isolation of four resolvable haem protein components from the Glycera monomer haemoglobin fraction. Three of these components were isolated in sufficient quantity to employ proton n.m.r. as a successful analytical tool for discriminating the individual haemoglobins. These results are not surprising. Several previous studies indicated less extensive heterogeneity in the monomer fraction. Moreover, the ability of the Glycera monomer haemoglobin to bind oxygen at even quite low partial pressures has been attributed to functional diversity originating in multiple haemoglobin components. The present work reveals the extent of the haemoglobin heterogeneity. The results show that it is more extensive than previously believed. Examination of this monomer fraction is particularly important, since crystallography indicates that one of the components of the monomer fraction lacks the E-7 (distal) histidine residue. As a consequence, the identification of such extensive heterogeneity is important to many previously published ligand-binding studies.     

Die Beeinflussung der Aminosäuresequenz des serinhaltigen Mureins von Staphylococcus epidermidis Stamm 24 durch die Nährbodenzusammensetzung

Zusammenfassung Beim Wachstum von S. epidermidis, Stamm 24, in Hefe-Dextrose-Bouillon weist das Murein folgende Molverhältnisse auf (auf- bzw. abgerundete Zahlen): Mur-GlcNH₂:Ala:Glu:Lys:Gly:Ser=1:1:2,4:1:1:4,2:0,6. Die Glutaminsäure ist amidiert.Durch Isolierung und Identifizierung der Peptide des Partialhydrolysates des Mureins wurde die Aminosäuresequenz bestimmt. Die an die Muraminsäure gebundene Peptiduntereinheit (l-Ala-d-GluNH₂ -l-Lys-d-Ala) stimmt mit der von S. aureus, Copenhagen bzw. S. epidermidis, Stamm 66, überein. Bei knapp einem Drittel der Peptiduntereinheiten ist das C-terminale d-Alanin der Mureinvorstufe nicht abgespalten, so daß diese noch als Pentapeptide vorliegen. Dies konnte aus dem Verhältnis l-Ala/d-Ala (1:1,3), dem Ergebnis der Hydrazinolyse und der Isolierung der Muropeptide nach Spaltung der Zellwände mit Lysozym geschlossen werden.Bei etwa der Hälfte aller aus 5 Glycinresten aufgebauten Interpeptidketten ist ein Glycinrest durch l-Serin ersetzt. Die genaue Position des Serins konnte nicht bestimmt werden. Serin ist sicher nicht direkt an die -Aminogruppe des Lysins gebunden.In selteneren Fällen kann Lysin mit l-Alanin substituiert sein, das N-terminal vorliegt und nicht der Quervernetzung dient.Die Dinitrophenylierung des Mureins ergab, daß in etwa 3,5% der Fälle die Interpeptidketten fehlen und rund ein Drittel der Interpeptidketten nicht quervernetzt ist.Bei Wachstum in einem halbsynthetischen, glycinarmen Medium (Minimal, medium) nimmt der Glycinanteil des Mureins um rund 40% ab, während l-Alanin zunimmt. Es konnte gezeigt werden, daß rund 15% des Mureins ein an die -Amino-gruppe des Lysins gebundenes l-Alanin enthalten, das aber im Unterschied zu S. epidermidis, Stamm 66, nicht mit Glycin substituiert ist, sondern N-terminal bleibt und nicht zur Quervernetzung benützt werden kann. Weiterhin liegen hier rund 35% des Lysins unsubstituiert vor, und nur etwa 50% der Peptiduntereinheiten weisen eine Pentaglycyl-Interpeptidkette auf. Die Quervernetzung des Mureins ist bei den in Minimalmedium gewachsenen Zellen nur zu rund 30% durchgeführt. Bei Zusatz von Glycin zum Minimal-Nährboden wird der Glycingehalt im Murein erhöht, während der extra Alaninanteil praktisch verschwindet. Serinzusatz erhöht nicht nur den Serin-, sondern auch den Glycinanteil. Bei Alaninzusatz dagegen wird der Alaningehalt im Murein etwas erhöht und der Glycingehalt weiter erniedrigt.Die Ergebnisse dieser Untersuchungen wurden mit entsprechenden, vorläufigen Versuchen bei S. epidermidis, Stamm 66 und S. aureus, Stamm Copenhagen, verglichen. Es zeigt sich, daß trotz starker modifikativer Veränderungen der Mureinzusammensetzung eindeutige genetische Unterschiede zwischen diesen drei Stämmen vorliegen.

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The effect of nutrition on the amino acid sequence of the serine containing murein of Staphylococcus epidermis strain 24

Summary The murein (peptidoglycan) of S. epidermidis strain 24 contains Mur GlcNH₂, Ala, Glu, Lys, Gly, Ser at a molar ratio of about 1:1:2.4:1:1:4.2:0.6 when grown in a yeast extract dextrose medium. Glutamic acid occurs as an amide.The amino acid sequence was determined by analysing the oligopeptides from partial acid hydrolysate. The tetrapeptide bound to the muramic acid (l-Ala-d-Glu-NH₂-l-Lys-d-Ala) is identical with those found in S. aureus and S. epidermidis strain 66. About 1/3 of the muropeptides is still present as pentapeptides, since the second d-alanine of the muramyl pentapeptide precursor is not split off. This fact is indicated by the ratio of l-Ala/d-Ala of 1:1.3, the isolation of muropentapeptides from the lysozyme lysates and by the result of the hydrazinolysis.About 50% of the pentaglycine interpeptide chains contain one mole of l-serine. The exact position of l-serine could not be determined. However, it could be shown, that serine is never bound to the -amino group of lysine. In very rare cases, the -amino group of lysine is substituted by l-alanine which remains N-terminal and can not be used for crosslinkages.As shown by dinitrophenylation, about 3.5% of the -amino groups of lysine is free and about 50% of the interpeptide chains are not cross-linked.If the organism is grown in a glycine deficient minimal medium, the glycine content of the murein drops by 40%, while l-alanine increases. Here, about 15% of the -amino groups of lysine is substituted by l-alanine, which again is not used for cross-linkages. Another 35% of the -amino groups of lysine remain free. From the existing interpeptide chains 30% are not cross-linked.The addition of glycine to the minimal medium causes an increase of the glycine content in the murein, however, the extra alanine protion nearly disappears. The addition of serine leads to an increase of not only the serine portion but also the glycine portion in the murein. However, when alanine is added the alanine portion of murein is slightly increased and the glycine portion further decreased.The results of these experiments were compared to corresponding preliminary experiments with S. epidermidis (strain 66) and S. aureus (strain Copenhagen). In spite of modificative changes in the murein composition, clear genetical differences between the 3 strains were obvious.

     

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.

     

On the specificity of the uridine diphospho-N-acetylmuramyl-alanyl-D-glutamic acid: Diamino acid ligase of Bifidobacterium globosum

The peptidoglycan of Bifidobacterium globosum contains ornithine and lysine alternately in the same position of the peptide subunit. The uridine diphospho-N-acetylmuramyl-alanyl-D-glutamic acid: diamino acid ligase of this organism was purified 700-fold. Since the activities for the incorporation of ornithine and lysine into uridine diphospho-N-acetylmuramyl-tripeptide did not separate during purification and since the incorporation of ornithine is competitively inhibited by lysine and vice versa, both ornithine and lysine are assumed to be incorporated by one single enzyme. Studies on the specificity of the ligase toward analogs of ornithine have shown that the enzyme requires a diamino, monocarboxylic acid with 4–6 carbon atoms. Methylation of the -amino group or hydroxylation of the -carbon atom of lysine decreases the competitive properties of the analog, whereas the substitution of the -methylen group by sulfur (S-2-aminoethyl cysteine) results in a highly competitive compound.Abbreviations BSA bovine serum albumine - MurNAc N-acetyl-muramyl - DA diamino acid - Ala-DGlu--L-DA-DAla-D-Ala pentapeptide - Ala-DGlu--LDA tripeptide - Ala-DGlu dipeptide - DSM Deutsche Sammlung von Mikroorganismen - CEM clostridial enrichment medium     

The amino acid sequence of the peptide moiety of the pseudomurein from Methanobacterium thermoautotrophicum

The amino acid sequence of the peptide subunits of the peptide moiety of the sacculus polymer (pseudomurein) of Methanobacterium thermoautotrophicum was elucidated by analysing overlapping peptides obtained from partial acid hydrolysates of isolated sacculi. It is suggested that the peptide subunits are attached to glycan strands via one of their glutamyl residues. Another glutamyl residue may crosslink two adjacent peptide subunits to form a dimer. The calculated molar ratios of the amino acids and the percentages of the N-or C-terminal amino acid residues of the supposed dimers are compatible with those actually found in the sacculus polymer.