Investigation of a killer strain of Zygosaccharomyces bailii.

The yeast Zygosaccharomyces bailii strain 412 was found to liberate a killer toxin (KT412) lethal to sensitive strains of Saccharomyces cerevisiae and Candida glabrata. Culture supernatants of the killer strain were concentrated by ultrafiltration and the extracellular protein was purified by gel filtration and ion-exchange chromatography. Gel filtration and SDS-PAGE of the electrophoretically homogeneous killer protein indicated an apparent molecular mass of 10 kDa. The killer toxin KT412 is probably not glycosylated since it did not show any detectable carbohydrate structures. KT412 was bound to sensitive but not to resistant yeast cells. The mannan, and not the glucan, fraction of the cell wall of the sensitive yeast was the primary target for the killer toxin binding. The killer strain Z. bailii 412 contained three double-stranded RNA plasmids of 1.9, 2.9 and 4.0 kb. Curing by cycloheximide resulted in the concomitant loss of killer activity and the 1.9 kb dsRNA species that is therefore regarded as equivalent to the killer-toxin-coding M-plasmids of S. cerevisiae.

Caseicin, a bacteriocin from Lactobacillus casei.

The intracellular bacteriocin caseicin 80 was purified from cell extracts of Lactobacillus casei strain B80. It is a thermolabile protein with an apparent molar mass of 42 kDa. As no plasmids were observed in the bacteriocinogenic strain it is assumed that caseicin is encoded by the bacterial chromosome. Using 14C-labelled precursors it was found that biosynthesis of DNA and proteins was influenced by caseicin but this inhibition is probably not the primary effect. The incorporation of fructose but not of glucose into cellular material was inhibited by caseicin.

Blockage of cell wall receptors for yeast killer toxin KT28 with antimannoprotein antibodies.

Binding of yeast killer toxin KT28 to its primary cell wall receptor was specifically blocked with polyclonal antimannoprotein antibodies which masked all toxin-binding sites on the surface of sensitive yeast cells. By indirect immunofluorescence, it was shown that KT28 binds to the cell wall mannoprotein and that the toxin resistance of mannoprotein mutants (mnn) of Saccharomyces cerevisiae was due to a lack of killer toxin-binding sites within the yeast cell wall. Structural analysis of acetylated mannoprotein from KT28-resistant mutant strains identified the outer mannotriose side chains as the actual killer toxin-binding domains.

Killer toxin of Hanseniaspora uvarum.

The yeast Hanseniaspora uvarum liberates a killer toxin lethal to sensitive strains of the species Saccharomyces cerevisiae. Secretion of this killer toxin was inhibited by tunicamycin, an inhibitor of N-glycosylation, although the mature killer protein did not show any detectable carbohydrate structures. Culture supernatants of the killer strain were concentrated by ultrafiltration and the extracellular killer toxin was precipitated with ethanol and purified by ion exchange chromatography. SDS-PAGE of the electrophoretically homogenous killer protein indicated an apparent molecular mass of 18,000. Additional investigations of the primary toxin binding sites within the cell wall of sensitive yeast strains showed that the killer toxin of Hanseniaspora uvarum is bound by beta-1, 6-D-glucans.

Molecular structure of the cell wall receptor for killer toxin KT28 in Saccharomyces cerevisiae.

The adsorption of the yeast killer toxin KT28 to susceptible cells of Saccharomyces cerevisiae was prevented by concanavalin A, which blocks the mannoprotein receptor. Certain mannoprotein mutants of S. cerevisiae that lack definite structures in the mannan of their cell walls were found to be resistant to KT28, whereas the wild-type yeast from which the mutants were derived was susceptible. Isolated mannoprotein from a resistant mutant was unable to adsorb killer toxin. By comparing the resistances of different mannoprotein mutants, information about the molecular structure of the receptor was obtained. At least two mannose residues have to be present in the side chains of the outer chain of the cell wall mannan, whereas the phosphodiester-linked mannose group is not essential for binding and the subsequent action of killer toxin KT28.

Effect of a killer toxin of yeast on eucaryotic systems.

The Saccharomyces cerevisiae killer toxin KT 28, which inhibits sensitive yeasts, was shown to have no effect on several pathogenic fungi or on the protozoan Trichomonas vaginalis. At concentrations of about 0.1 mg/ml, a partial inhibition of the skin pathogenic fungi Trichophyton rubrum and Microsporum canis was observed at pH 6.5. No pharmacological activity was detected in various tests with several animal organs.

Mannoprotein of the yeast cell wall as primary receptor for the killer toxin of Saccharomyces cerevisiae strain 28.

The killer toxin KT 28 of Saccharomyces cerevisiae strain 28 is primarily bound to the mannoprotein of the cell wall of sensitive yeasts. The mannoprotein of S. cerevisiae X 2180 was purified; gel filtration and SDS-PAGE indicated an estimated Mr of 185,000. The ability to bind killer toxin KT 28 increased during purification of the mannoprotein. Removing the protein part of the mannoprotein by enzymic digestion or removing the alkali-labile oligosaccharide chains by beta-elimination did not destroy the ability to bind killer toxin KT 28. However, binding activity was lost when the 1,6-alpha-linkages of the outer carbohydrate backbone were hydrolysed by acetolysis. The separated oligomannosides of the side chains also failed to bind toxin, indicating that the main mannoside chains were essential for the receptor activity. The reversible adsorption of killer toxin to mannoprotein was demonstrated by linking it covalently to Sepharose and using this material for affinity chromatography. A 90-fold increase in the specific activity of a preparation of killer toxin KT 28 was achieved in this way.

[High performance liquid chromatographic determination of organic acids, sugars, glycerin and alcohol in wine on a cation exchange resin]. / Hochleistungsflüssigchromatographische Bestimmung von organischen Säuren, Zuckern, Glycerin und Alkohol im Wein an einer Kationenaustauschersäule.

The main constituents of wine (ethanol, glycerol, glucose, fructose, tartrate, malate, lactate, succinate, acetate and citrate) were separated by HPLC in one run and determined quantitatively in a time of 25 min. The stationary phase was the cation exchange resin HPX 87H and the mobile phase was dilute sulfuric acid. The results of the HPLC analysis of several wines are presented. Good agreement is observed with the results obtained by conventional methods.

The metabolism of several carboxylic acids by lactic acid bacteria.

The anaerobic metabolism of citrate, fumarate, gluconate, malate, 2-oxoglutarate and pyruvate by 137 strains of 23 species of lactic acid bacteria was investigated. The bacteria were from various sources (plant material, meat and dairy products, dough and wine) and belonged to the genera Lactobacillus, Leuconostoc, Pediococcus, and Streptococcus. The ability of metabolize the acids was determined by thin layer chromatography or by enzymatic analysis after growth of the strains in a glucose-containing medium. All strains metabolized pyruvate and only 12 mainly heterofermentative strains were malate negative. These strains were also unable to decompose citrate. This acid was fermented by 23 strains, all of which metabolized malate. Many lactic acid bacteria reduced 2-oxoglutarate to hydroxyglutarate. The strains of Lactobacillus plantarum did not metabolize 2-oxoglutarate whereas all strains of Leuconostoc oenos decarboxylated this acid and formed 4-hydroxybutyrate and succinate. Gluconate was fermented by 52 mainly heterofermentative strains. No correlation was observed between the ability to ferment citrate, malate or gluconate.

Comparison of the killer toxin of several yeasts and the purification of a toxin of type K2.

A total of 13 killer toxin producing strains belonging to the genera Saccharomyces, Candida and Pichia were tested against each other and against a sensitive yeast strain. Based on the activity of the toxins 4 different toxins of Saccharomyces cerevisiae, 2 different toxins of Pichia and one toxin of Candida were recognized. The culture filtrate of Pichia and Candida showed a much smaller activity than the strains of Saccharomyces. Extracellular killer toxins of 3 types of Saccharomyces were concentrated and partially purified. The pH optimum and the isoelectric point were determined. The killer toxins of S. cerevisiae strain NCYC 738, strain 399 and strain 28 were glycoproteins and had a molecular weight of Mr = 16,000. The amino acid composition of the toxin type K2 of S. cerevisiae strain 399 was determined and compared with the composition of two other toxins.

The glucose-dependent transport of L-malate in Zygosaccharomyces bailii.

Zygosaccharomyces bailii possesses a constitutive malic enzyme, but only small amounts of malate are decomposed when the cells ferment fructose. Cells growing anaerobically on glucose (glucose cells) decompose malate, whereas fructose cells do not. Only glucose cells show an increase in the intracellular concentration of malate when suspended in a malate-containing solution. The transport system for malate is induced by glucose, but it is repressed by fructose. The synthesis of this transport system is inhibited by cycloheximide. Of the two enantiomers L-malate is transported preferentially. The transport of malate by induced cells is not only inhibited by addition of fructose but also inactivated. This inactivation is independent of the presence of cycloheximide. The transport of malate is inhibited by uranyl ions; various other inhibitors of transport and phosphorylation were of little influence. It is assumed that the inducible protein carrier for malate operates by facilitated diffusion. Fructose cells of Z. bailii and cells of Saccharomyces cerevisiae do not contain a transport system for malate.

Partial purification and characterization of succinyl-CoA synthetase from Saccharomyces cerevisiae.

Succinyl-CoA synthetase from Saccharomyces cerevisiae was partially purified (20-fold) with a yield of 44%. The Michaelis-Menten constants were determined: Km (succinate) = 17 mM; Km (ATP) = 0.13 mM; Km (CoA) = 0.03 mM. The succinyl-CoA synthetase has a molecular weight of about 80000 dalton (as determined by polyacrylamide gradient gel electrophoresis). The pH optimum is at 6.0. During fermentation the activity of succinyl-CoA synthetase is lower than in aerobically grown yeast cells. The presence of succinyl-CoA synthetase in fermenting yeasts may be regarded as an indication for the oxidative formation of succinate. In fermenting yeast cells succinyl-CoA synthetase is repressed by glucose if ammonium sulphate serves as nitrogen source. This catabolite repression is not observed with disaccharides or when amino acids are used as nitrogen source.

Malolactic enzyme of Lactobacillus plantarum. Purification, properties, and distribution among bacteria.

The malolactic enzyme of Lactobacillus plantarum was purified from 5.5 units/mg to a specific activity of 265 units/mg of protein. The enzyme has an isoelectric point of pH 4.4. The molecular weight is Mr = 140,000 as determined by gradient gel electrophoresis. The enzyme consists of two probably identical subunits (Mr = 70,000) that were observed after treatment with sodium dodecyl sulfate. Malolactic enzyme catalyzes the NAD- and manganese-dependent reaction L-malate leads to CO2 + L-lactate. Therefore, this enzyme can be distinguished from the well known malic enzymes (L-malate: NAD+ oxidoreductase, oxalacetate-decarboxylating EC 1.1.1.38 or 1.1.1.39). Malolactic enzyme is found in most lactic acid bacteria (Lactobacteriaceae); it has not been detected in other bacteria.

The anaerobic metabolism of malate of Saccharomyces bailii and the partial purification and characterization of malic enzyme.

1. The main pathway of the anaerobic metabolism of L-malate in Saccharomyces bailii is catalyzed by a L-malic enzyme. 2. The enzyme was purified more than 300-fold. During the purification procedure fumarase and pyruvate decarboxylase were removed completely, and malate dehydrogenase and oxalacetate decarboxylase were removed to a very large extent. 3. Manganese ions are not required for the reaction of malic enzyme of Saccharomyces bailii, but the activity of the enzyme is increased by manganese. 4. The reaction of L-malic enzyme proceeds with the coenzymes NAD and (to a lesser extent) NADP. 5. The Km-values of the malic enzyme of Saccharomyces bailii were 10 mM for L-malate and 0.1 mM for NAD. 6. A model based on the activity and substrate affinity of malic enzyme, the intracellular concentration of malate and phosphate, and its action on fumarase, is proposed to explain the complete anaerobic degradation of malate in Saccharomyces bailii as compared with the partial decomposition of malate in Saccharomyces cerevisiae.

D-Malic enzyme of Pseudomonas fluorescens.

By the enrichment culture technique 14 gram-negative bacteria and two yeast strains were isolated that used D(+)-malic acid as sole carbon source. The bacteria were identified as Pseudomonas putida, Pseudomonas fluorescens, Pseudomonas aeruginosa and Klebsiella aerogenes. In cell-free extracts of P. fluorescens and P. putida the presence of malate dehydrogenase, D-malic enzyme (NAD-dependent) and L-malic enzyme (NADP-dependent) was demonstrated. D-Malic enzyme from P. fluorescens was purified. Stabilization of the enzyme by 50 mM ammonium sulphate an 1 mM EDTA was essential. Preparation of D-malic enzyme that gave one band with disc gel electrophoresis showed a specific activity of 4-5 U/mg. D-Malic enzyme requires divalent cations. The Km values were for malate Km = 0.3 mM and for NAD Km = 0.08 mM. The pH optimum for the reaction was found to be in the range of pH 8.1 to pH 8.8. D-Malic enzyme is partially inhibited by oxaloacetic acid, meso-tartaric acid, D-lactic acid and ATP. Determined by gel filtration and gradient gel electrophoresis, the molecular weight was approximately 175 000.

Bacterial 2,3-butanediol dehydrogenases.

Enterobacter aerogenes, Aeromonas hydrophila, Serratia marcescens and Staphylococcus aureus possessing L(+)-butanediol dehydrogenase produced mainly meso-butanediol and small amounts of optically active butanediol; Acetobacter suboxydans, Bacillus polymyxa and Erwinia carotovora containing D(-)-butanediol dehydrogenase produced more optically active butanediol than meso-butanediol. Resting and growing cells of these organisms oxidezed only one enantiomer of racemic butanediol. The D(-)-butanediol dehydrogenase from Bacillus polymyxa was partially purified (30-fold) with a specific activity of 24.5. Except NAD and NADH no other cofactors were required. Optimum pH-values for oxidation and reduction were pH 9 and pH 7, respectively. The optimum temperature was about 60 degrees C. The molecular weight was 100000 to 107000. The Km-values were 3.3 mM for D(-)butanediol, 6.25 mM for meso-butanediol, 0.53 mM for acetoin, 0.2 mM for NAD, 0.1 mM for NADH, 87 mM for diacetyl, 38 mM for 1,2-propanediol; 2,3-pentanedion was not a substrate for this enzyme. The L(+)butanediol dehydrogenase from Serratia marcescens was purified 57-fold (specific activity 22.3). Besides NAD or NADH no cofactors were required. The optimum value for oxidation was about pH9 and for reduction pH 4.5. The optimum temperature was 32-36 degrees C. The molecular weight was 100000 to 107000. The Km-values were 5 mM for meso-butanediol, 10 mM for racemic butanediol, 6.45 for acetoin, 1 mM for NAD, 0.25 mM for NADH, 2.08 mM for diacetyl, 16.7 mM for 2,3-pentanedion and 11.8 mM for 1,2-propanediol.

[On the metabolism of amino acids by lactic acid bacteria isolated from wine (author's transl)]. / Uber den Aminosäurestoffwechsel von Milchsäurebakterien aus Wein

The changes in the concentrations of amino acids in the culture medium of lactic acid bacteria were determined by ion exchange chromatography after growth of 105 strains that were mainly isolated from wine. After growth of lactic acid bacteria a small but not significant decrease in the concentration of most amino acids is observed. There is apparently no difference between essential or not essential amino acids. Certain bacterial strains decompose the amino acids arginine, glutamic acid, histidine, and tyrosine completely. Other amino acids (tryptophane, aspartic acid, threonine, isoleucine, and phenylalanine) are only partially metabolized. Among 28 strains of Pediococcus cerevisiae only one strain was able to decarboxylate histidine to histamine. This was the only strain found to have this ability. Several strains of Lactobacillus brevis were able to form 4-aminobutyric acid and ornithine from glutamic acid and arginine respectively.