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.

     

Bacterial assimilation of d- and l-2-chloropropionates and occurrence of a new dehalogenase

The occurrence of a new bacterial dehalogenase acting on both the optical isomers of 2-halogenated alkanoic acids was demonstrated. When the haloalkanoic acid-utilizing bacteria were screened in a medium containing dl-2-chloropropionate as a sole carbon source, two types of bacteria were isolated: (1) a few strains utilizing both d- and l-isomers of 2-chloropropionate and (2) strains utilizing only the l-isomer. A dehalogenating enzyme was obtained from the cells of Pseudomonas sp. which is able to utilize both isomers. The crude enzyme catalyzed the dehalogenation of d- and l-2-chloropropionates to yield l- and d-isomers of lactate, respectively. The enzyme showed the same pH optimum and heat inactivation rate for the d- and l-isomers. Apparent K _m values for d- and l-2-chloropropionates were 4.5 and 1.0 mM, respectively. The enzyme acted specifically on 2-haloalkanoic acids. Activity staining of disc-gels electrophoresed witg the crude enzyme preparation showed that the dehalogenation of d- and l-2-chloropropionates, monochloroacetate, dichloroacetate, 2,2-dichloropropionate, and dl-2-chlorobutyrate is due to a single protein.Abbreviations MCA monochloroacetic acid - DCA dichloroacetic acid - TCA trichloroacetic acid - 2 MCPA 2-monochloropropionic acid - 22 DCPA 2,2-dichloropropionic acid - 3 MCPA 3-monochloropropionic acid - 2 MCBA 2-monochlorobutyric acid - 3 MCBA 3-monochlorobutyric acid - 4 MCBA 4-monochlorobutyric acid     

Plant regeneration from stem and petiole protoplasts of sweet potato (Ipomoea batatas) and its wild relative,I. lacunosa

A protoplast-to-plant regeneration system has been established for sweet potato (Ipomoea batatas (L.) Lam.) and its wild relative, I. lacunosa L. Viable protoplasts, isolated from preplasmolyzed stems and petioles of in vitro-grown plants, were cultured on liquid MS (Murashige & Skoog 1962) medium that supported cell division and colony formation. Embryogenic calli of sweet potato were induced on agar-solidified MS medium supplemented with 3% (w/v) sucrose, 50 mg l^-1 casamino acids, 0.2–0.5 mg l^-1 2,4-d, 1.0 mg l^-1 kinetin and 1.0 mg l^-1 ABA. On average, 3 plants were regenerated from a single sweet potato callus subcultured on semi-solid MS medium containing 3% (w/v) sucrose, 800 mg l^-1 glutamine, 2.0 mg l^-1 BA or 1.0 mg l^-1 kinetin and 1.0 mg l^-1 GA₃. Embryogenic calli of I. lacunosa L. were initiated on semi-solid MS medium containing 0.2–0.5 mg l^-1 IAA and 1.0–2.0 mg l^-1 BA. An average of 5 plants was regenerated from a single sweet potato callus subcultured on semi-solid MS medium containing 0.5 or 1.0 mg l^-1 GA₃.Abbreviations ABA abscisic acid - BA benzyladenine - 2,4-d 2,4-dichlorophenoxyacetic acid - GA₃ gibberellic acid - IAA indole acetic acid - MES 2-(N-morpholino)-ethane sulfonic acid - NAA -naphthaleneacetic acid     

Somatic embryogenesis and callus production from cotyledon explants of Eastern black walnut

Starting at 8 weeks and continuing until 23 weeks (nut drop) after anthesis,1 m² explants from cotyledons of immature seeds were extracted from Juglans nigra fruits. Explants were placed on Woody Plant Medium with 1 g l^-1 casein hydrolysate and 30 g l^-1 sucrose. The explants remained in light for 4 weeks on primary media containing a 3×3 factorial of 0.05, 0.5, or 5.0 M thidiazuron (TDZ) and 0.1, 1.0, or 10.0 M 2,4-d. Explants were transferred to a secondary medium containing no plant growth regulators and incubated in darkness for 11 weeks. The greatest number of somatic embryos was produced 8, 10, and 12 weeks after anthesis from explants on media with 0.5 or 5.0 M TDZ and 0.1 or 1.0 M 2,4-d. Explants produced the greatest callus volume and dry weight 10, 12, and 14 weeks after anthesis. Throughout the study, callus generally increased with increasing concentrations of both TDZ and 2,4-d.Abbreviations BA 6-benzyladenine - captan 3a,4,7,7a-tetrahydro-2-[(trichloromethyl)thio]-1H-isoindole-1,3(2H)-dione - 2,4-d 2,4-dichlorophenoxyacetic acid - IBA indolebutyric acid - Physan n-alkyl- dimethyl-benzyl ammonium chlorides and n-alkyl-dimethyl-ethylbenzyl ammonium chlorides - TDZ-thidiazuron N-phenyl-N-1,2,3-thiadiazol-5-ylurea     

Syntheses of Diastereoisomers of the Recent Pyrethroids,Fenvalerate (S-5602) and Cypermethrin (NRDC-149) from (–)-3-Phenoxy-mandelic Acid and Determination of Their Absolute Configurations

Lipopeptin A is a selective inhibitor of in vitro peptidoglycan synthesis of E. coli Y-10. In the study here it inhibited the formation of lipid intermediates from UDP-[U-¹⁴C]GlcNAc and UDP-MurNAc-l-Ala-d-Glu-meso-DAP-d-Ala-d-Ala, but did not inhibit the formation of MurNAc-pentapeptide-p-p-lipid from UDP-MurNAc-l-Ala-d-Glu-[³H]meso-DAP-d-Ala-d-Ala. Lipopeptin A also did not have a significant effect on polymerase reaction. Therefore, the inhibition of the formation of GleNAc-MurNAc-pentapeptide-p-p-lipid from MurNAc-pentapeptide-p-p-lipid and UDP-GlcNAc is concluded to be the site of action.

Lipopeptin A inhibits fungal growth, causing swelling in mycelia. It did not significantly inhibit the incorporations of ¹⁴C-labeled glucosamine, thymidine, uridine, phenylalanine, and sodium acetate into TCA insoluble fraction of mycelial suspension of Piricularia oryzae. In in vitro test, however, it inhibited the transfer of mannose from GDP-[U-¹⁴C]mannose (ID_5O = 250 μg/ml) and GlcNAc from UDP-[U-¹⁴C]GlcNAc (ID₅₀ = 100 μg/ml) into proteoheteroglycan with a particulate enzyme of Piricularia oryzae. It also slightly inhibited chitin synthesis (ID₅₀ = 750 μg/ml) in the same enzyme system, but did not inhibit β-l,3-glucan synthesis.     

Induction of haploid callus and embryogenesis in in vitro cultured anthers of mulberry (Morus indica)

Anthers of Morus indica L., with microspores at the uninucleate stage were cultured; and the influence of temperature and kinetin pretreatment on induction of androgenic calluses was examined. The effects of various pretreatments revealed that 24 h cold pretreatment increased the percentage of cultures inducing callus. First microspore division was observed after 16 to 20 days of culture. Th anthers split and developed embryogenic calluses on MB medium supplemented with NAA (0.5 mg l^–1 and BA (1.0 mg l^–1)) using 8% sucrose. Rhizogenesis was induced on medium supplemented with NAA and BA (each 0.5 mg l^–1) with reduced myo-inositol (75 mg l^–1). Cytological study of induced roots confirmed the haploid nature of calluses. Different type of embryos were initiated upon transfer of calluses to medium supplemented with NAA, BA (each 0.5 mg l^–1), 2,4-d (1.0 mg l^–1) and PVP (600 mg l^–1). These embryoids further developed roots on removal of 2,4-d from the medium and developed precociously without developing cotyledons and formed elongated shoots.Abbreviations BA 6 benzylaminopurine - 2,4-d 2,4-dichlorophenoxyacetic acid - FAA formalin: Acetic acid: Alcohol - GA₃ gibberellic acid - IBA indole-3-butyric acid - MB modifed Bourgin (Qian et al., 1982) - NAA 1-naphthalene acetic acid - PVP polyvinylpyrrolidone - RFS-135 rainfed selection 135 - SE standard error     

Isolation and chemical characterization of lipopolysaccharides from four Mycoplana species (M. bullata,M. segnis,M. ramosa and M. dimorpha)

Lipopolysaccharides (LPS), isolated from four Mycoplana species, i.e. the type strains of M. bullata, M. segnis, M. ramosa and M. dimorpha, were characterized onto their chemical composition and their respective lipid A-types. Those of M. bullata and M. segnis showed on DOC-PAGE an R-type character and had lipid A's of the Lipid A_DAG-type which exclusively contained 2,3-diamino-2,3-dideoxy-d-glucose as lipid A sugar. LPS's of M. ramosa and M. dimorpha showed, although only weakly expressed, ladder-like patterns on DOC-PAGE indicating some S-type LPS's and lipid A of the d-glucosamine type (Lipid A_GlcN). M. bullata LPS contained mannose and glucose in major amounts and additionally l-glycero-d-mannoheptose, whereas M. segnis LPS was composed of rhamnose, mannose and glucose together with both, d-glycero-d-manno- and l-glycero-d-manno-heptoses in a molar ratio of 1:2. All LPS's contained 2-keto-3-deoxy-octonic acid (Kdo), phosphate and an unidentified acidic component X. In addition to X, M. segnis LPS contained glucuronic and galacturonic acids, whereas M. ramosa LPS contained only galacturonic acid. Acetic acid hydrolysis of the LPS resulted in splitting off lipid A moieties, very rich in 3-hydroxy fatty acids, in particular in 3-OH-12:0 (in Lipid A_DAG), or in 3-OH-14:0 (in Lipid A_GlcN). Analysis of the 3-acyloxyacyl residues revealed major amounts of amide-linked 3-OH(3-OH-13:0)12:0 in lipid A of M. bullata and 3-OH(12:0)12:0 in lipid A of M. segnis. The rare 4-oxo-myristic acid (4-oxo-14:0) was observed only in M. bullata LPS, where it is ester-linked. Amide linked diesters could not be traced in M. ramosa and M. dimorpha. All four lipid A's lacked erster-bound acyloxyacyl residues.Non-standard abbreviations DAG 2,3-diamino-2,3-dideoxy-d-glucose - Kdo 2-keto-3-deoxy-octonate - LPS lipopolysaccharide - PITC phenyl isothiocyanate - NANA N-acetyl neuraminic acid     

Lipopolysaccharides of Thiobacillus species containing lipid A with 2,3-diamino-2,3-dideoxyglucose

Lipopolysaccharides were isolated from two strains of Thiobacillus ferrooxidans and one strain each of Thiobacillus thiooxidans, Thiobacillus novellus and Thiobacillus sp. IFO 14570. Neutral sugars, 2-keto-3-deoxyoctonate, fatty acids and the rare 2,3-diamino-2,3-dideoxyglucose were detected in all lipopolysaccharides. Lipopolysaccharides of both T. ferrooxidans strains contained l-glycero-d-manno-heptose, whereas that of T. thiooxidans contained both l-glycero-d-manno-heptose and d-glycero-d-manno-heptose. On the other hand, heptoses were absent in lipopolysaccharides of T. novellus and Thiobacillus sp. IFO 14570. Lipid A of T. ferrooxidans and T. thiooxidans contained both glucosamine and 2,3-diamino-2,3-dideoxyglucose, in contrast, lipid A of T. novellus and the Thiobacillus sp. IFO 14570 most likely contain only 2,3-diamino-2,3-dideoxyglucose as backbone sugar. Deoxycholate polyacrylamide gel electrophoresis revealed S-type character for all lipopolysaccharides studied. The significance of the lipopolysaccharide composition for taxonomic and phylogenetic questions with regard to thiobacilli is discussed.Abbreviations DAG 2,3-diamino-2,3-dideoxyglucose - DOC sodium deoxycholate - GC gas-liquid chromatography - GC/MS gas-liquid chromatography/mass spectrometry - d,d-Heptose d-glycero-d-manno-heptose - l,d-Heptose l-glycero-d-manno-heptose - KDO 2-keto-3-deoxyoctonate - LPS lipopolysaccharide - 3-OH-14:0 3-hydroxy-tetradecanoic acid - PAGE polyacrylamide gel electrophoresis - PCP phenol-chloroform-petroleum ether     

Utilization of d-amino acids by dadR mutants of Salmonella typhimurium

Utilization of d-amino acids being substrates of d-amino acid dehydrogenase of Salmonella typhimurium was examined. The experiments were done with wild type strains and the mutants dadA missing the enzyme activity and dadR in which its synthesis is released from catabolite repression. Growth on d-tryptophan, d-histidine and d-methionine used as precursors of the l-amino acids was faster when the respective auxotrophs carried dadR mutations. The dadR mutants grew faster when d-or l-alanine was present as a sole source of nitrogen. Experiments with d-amino acid dehydrogenase in vitro provided evidence that d-tryptophan is its substrate with a very low affinity to the dehydrogenase.     

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.

     

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

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.     

Development and evaluation of a multiplex real-time PCR assay for the detection and differentiation of Moraxella bovis, Moraxella bovoculi and Moraxella ovis in pure culture isolates and lacrimal swabs collected from conventionally raised cattle

Aim: To develop a multiplex real‐time PCR assay for the detection and differentiation of Moraxella bovis (M. bovis), M. bovoculi and M. ovis. Methods and Results: The multiplex real‐time PCR assay was validated on three reference strains, 57 pure culture isolates and 45 lacrimal swab samples. All reference strains were identified correctly with no cross‐reactions between species. Sequencing of 53 of the 57 culture isolates confirmed the results obtained with the multiplex real‐time PCR, and the assay had 96·5% (55/57) concordance with a Moraxella spp. multiplex conventional PCR assay on the isolates. Among the lacrimal swab samples, the concordance between the multiplex real‐time PCR and culture was 86·7% (39/45) for M. bovoculi and 75·6% (34/45) for M. bovis. Conclusions: The multiplex real‐time PCR assay is specific and sensitive and can be used directly on lacrimal swab samples. Significance and Impact of Study: The lack of a rapid, specific and sensitive detection method is a barrier for determining the roles of M. bovis, M. bovoculi and M. ovis in infectious bovine keratoconjunctivitis cases, and the developed PCR assay will contribute to improved understanding of the epidemiology and pathogenesis of these three Moraxella species.     

Chemical composition of Eubacterium nodatum cell wall peptidoglycan

The structure of Eubacterium nodatum cell wall peptidoglycan was investigated. The peptide subunit of E. nodatum peptidoglycan has the following structure: L-Ala-D-Glu (Gly)-L-Orn-D-Ala. The carboxyl group of alanine occupying position 4 is attached to the -amino group of ornithine of an other subunit by the cross-linking bridge L-Ala-L-Ala-L-Orn. All glycine molecules are connected with the -carboxyl group of glutamic acid with the ratio being 0.5–1. The hydrolysis of E. nodatum peptidoglycan by the S. albus G enzyme proceeds primarily due to the activity of alanyl-alanine endopeptidase, ornithyl-ornithine endopeptidase, ornithyl-alanine endopeptidase, N-acetyl-muramyl-alanine amidase, N-acetylmuramidase and N-acetylglucosaminidase.     

Purification and characterization of myo-inositol 6-O-methyltransferase from Vigna umbellata Ohwi et Ohashi

The cyclitol 1d-4-O-methyl-myo-inositol (d-ononitol) is accumulated in certain legumes in response to abiotic stresses. S-Adenosyl-l-methionine:myo-inositol 6-O-methyltransferase (m6OMT), the enzyme which catalyses the synthesis of d-ononitol, was extracted from stems of Vigna umbellata Ohwi et Ohashi and purified to apparent homogeneity by a combination of conventional chromatographic techniques and by affinity chromatography on immobilized S-adenosyl-l-homocysteine (SAH). The purified m6OMT was photoaffinity labelled with S-adenosyl-l-[¹⁴C-methyl]methionine. The native molecular weight was determined to be 106 kDa, with a subunit molecular weight of 40 kDa. Substrate-saturation kinetics of m6OMT for myo-inositol and S-adenosyl-l-methionine (SAM) were Michaelis-Menten type with K _m values of 2.92 mM and 63 M, respectively. The SAH competitively inhibited the enzyme with respect to SAM (K _i of 1.63 M). The enzyme did not require divalent cations for activity, but was strongly inhibited by Mn²⁺, Zn²⁺ and Cu²⁺ and sulfhydryl group inhibitors. The purified m6OMT was found to be highly specific for the 6-hydroxyl group of myo-inositol and showed no activity on other naturally occurring isomeric inositols and inositol O-methyl-ethers. Neither d-ononitol, nor d-3-O-methyl-chiro-inositol, d-1-O-methyl-muco-inositol or d-chiro-inositol (end products of the biosynthetic pathway in which m6OMT catalyses the first step), inhibited the activity of the enzyme.Abbreviations DTT dithiothreitol - m6OMT myo-inositol 6-O-methyltransferase - SAH S-adenosyl-l-homocysteine - SAM S-adenosyl-l-methionine We are greatful to Professor M. Popp (University of Vienna) for helpful discussion and comment. This work was supported by Grant P09595-BIO from the Austrian Science Foundation (FWF).     

Production of <Emphasis Type="SmallCaps">d</Emphasis>-lactic acid by <Emphasis Type="Italic">Corynebacterium glutamicum</Emphasis> under oxygen deprivation

In mineral salts medium under oxygen deprivation, Corynebacterium glutamicum exhibits high productivity of l-lactic acid accompanied with succinic and acetic acids. In taking advantage of this elevated productivity, C. glutamicum was genetically modified to produce d-lactic acid. The modification involved expression of fermentative d-lactate dehydrogenase (d-LDH)-encoding genes from Escherichia coli and Lactobacillus delbrueckii in l-lactate dehydrogenase (l-LDH)-encoding ldhA-null C. glutamicum mutants to yield strains C. glutamicum ΔldhA/pCRB201 and C. glutamicum ΔldhA/pCRB204, respectively. The productivity of C. glutamicum ΔldhA/pCRB204 was fivefold higher than that of C. glutamicum ΔldhA/pCRB201. By using C. glutamicum ΔldhA/pCRB204 cells packed to a high density in mineral salts medium, up to 1,336 mM (120 g l⁻¹) of d-lactic acid of greater than 99.9% optical purity was produced within 30 h.     

Plant Growth Inhibitory Compounds from Aqueous Leachate of Wheat Straw

When seedlings of lettuce, cress, rice and wheat were incubated with the leachate of wheat straw, the roots growth of lettuce and garden cress were particularly inhibited. The leachate of wheat straw (100 g eq./l) showed 80.5 and 79.4% inhibition for lettuce and cress roots, respectively. The inhibitory activity was stronger as the concentration of wheat straw leachate was greater. This result indicates that allelochemical(s) inhibiting the roots growth of lettuce and cress are leached from the wheat straw into the water. Two potent compounds were isolated from the leachate of the wheat straw and identified as syringoylglycerol 9-O-β-d-glucopyranoside and l-tryptophan by spectral analyses. Syringoylglycerol 9-O-β-d-glucopyranoside inhibited the roots growth of lettuce and cress at concentrations greater than 0.1 and 10.0 μM, respectively. On the other hand, l-tryptophan inhibited the roots growth of lettuce and cress at concentrations greater than 0.1 and 1.0 μM, respectively. The content of syringoylglycerol 9-O-β-d-glucopyranoside and l-tryptophan in the leachate of wheat straw (100 g eq./l) was 18.4 ± 0.7 and 6.2 ± 0.6 μM, respectively. Syringoylglycerol 9-O-β-d-glucopyranoside (18.4 μM) showed 21.5 and 13.5% inhibition in the lettuce and cress roots assay, respectively. On the other hand, 6.2 μM of l-tryptophan showed 47.5 and 35.0% inhibition in the lettuce and cress roots assay, respectively. These results suggested that l-tryptophan may be a major contributor to the allelopathy in aqueous leachate of wheat straw and syringoylglycerol 9-O-β-d-glucopyranoside may be a minor contributor.     

Chemical and biochemical studies for the differentiation of coagulase-positive staphylococci

The cell wall composition, the configuration of lactic acid produced from glucose under anaerobic conditions, the occurrence of fructose-1,6-diphosphate (FDP) activatedl-lactate dehydrogenase (l-LDH), and the esterase pattern were determined from more than 80 strains of coagulase-positive staphylococci isolated from man and animal. Strains isolated from man, swine, bovines and hares form a rather homogencous group. They exhibit a similar cell wall composition, produce predominantlyd,l-lactate and have a characteristic and simple esterase pattern. Coagulasepositive staphylococci isolated from dogs, horses, minks and pigeons are quite distinct from typicalStaphylococcus aureus strains. They exhibit a different cell wall composition, produce onlyl-lactate, possess anl-LDH which is specifically activated by FDP, and have a quite complex esterase pattern.List of Abbreviations BBP bromphenol blue - FDP fructose-1,6-diphosphate - d-LDH d-lactate dehydrogenase - l-LDH l-lactate dehydrogenase - NAD nicotinamide adenine dinucleotide     

Inhibitory effect of ethanol on growth and solute accumulation by Saccharomyces cerevisiae as affected by plasma-membrane lipid composition

Incorporation of ethanol (1.0 or 1.25 M) into exponential-phase cultures of Saccharomyces cerevisiae NCYC 366 growing anaerobically in a medium supplemented with ergosterol and an unsaturated fatty acid caused a retardation in growth rate, which was greater when the medium contained oleic rather than linoleic acid. Ethanol incorporation led to an immediate drop in growth rate, and ethanol-containing cultures grew at the slower rate for at least 10 h. Incorporation of ethanol (0.5 M) into buffered (pH 4.5) cell suspensions containing d-[6-³H] glucose, d-[1-¹⁴C] glucosamine, l-[U-¹⁴C] lysine or arginine, or KH₂ ³²PO₄ lowered the rate of solute accumulation by cells. Rates of accumulation of glucose, lysine and arginine were retarded to a greater extent when cells had been grown in the presence of oleic rather than linoleic acid. This difference was not observed with accumulation of phosphate. Ethanol was extracted from exponential-phase cells by four different methods. Cells grown in the presence of linoleic acid contained a slightly, but consistently, lower concentration of ethanol than cells grown in oleic acid-containing medium. The ethanol concentration in cells was 5–7 times greater than that in the cell-free medium.     

Methylglyoxal Bypass Identified as Source of Chiral Contamination in l(+) and d(−)-lactate Fermentations by Recombinant Escherichia coli

Two new strains of Escherichia coli B were engineered for the production of lactate with no detectable chiral impurity. All chiral impurities were eliminated by deleting the synthase gene (msgA) that converts dihydroxyacetone-phosphate to methylglyoxal, a precursor for both l(+)- and d(−)-lactate. Strain TG113 contains only native genes and produced optically pure d(−)-lactate. Strain TG108 contains the ldhL gene from Pediococcus acidilactici and produced only l(+)-lactate. In mineral salts medium containing 1 mM betaine, both strains produced over 115 g (1.3 mol) lactate from 12% (w/v) glucose, >95% theoretical yield.