Flagellation of“Gluconobacter” melanogenus

Summary Electron-micrographs ofGluconobacter melanogenus strain AC 8 (formerlyG. liquefaciens), andG. melanogenus strain U 4, have conclusively confirmed the findings ofShimwell andCarr (1959),Stouthamer (1960), andLeifson (private communication), that these strains are not polarly flagellatedAcetomonas orGluconobacter strains, but peritrichously flagellated acetate-oxidisingAcetobacter ones. When transferred to the latter genus all evidence that these two genera are derived from a “common pool of ancestors”, as claimed byDe Ley (1961), disappears.     

Carbohydrate Metabolism by the Acetic Acid Bacteria

A general review of the acetic acid bacteria belonging to the intermediate type was accomplished physiologically, biochemically and morphologically. Conclusively, it was clarified that these were clearly a specific group and different from both Acetobacter and Gluconobacter, These were intermediate between lactaphilic and glycophilic, besides, on the carbohydrate oxidizability, these were intermediate between Acetobacter and Gluconobacter as mentioned previously.¹⁾ These showed the same result as Acetobacter on the vitamin requirement for the growth, but were closely related to Gluconobacter on the carbohydrate availability. And on the oxidative activity for amino acid, accompanying the deamination, these were also clearly distinguished from both Acetobacter and Gluconobacter, particularly these oxidized strongly l-serine. Differing from the observations by other investigators, these showed single flagellation, with the exception of multi-polar, but never multi-peritrichous.     

Threonine metabolism byPseudomonas aeruginosa

83 strains ofPseudomonas aeruginosa were unable to utilizel-threonine as carbon-energy source, although this compound served as sole nitrogen source. Auxotrophs ofP. aeruginosa 9-D2 that requiredl-serine or glycine for growth could grow in the presence ofl-threonine. Extracts ofP. aeruginosa 9-D2 grown in the presence ofl-threonine contained threonine dehydrogenase and alpha-amino beta-ketobutyrate: CoA ligase activities; threonine aldolase was not detectable. Cells grown in the absence ofl-threonine produced no detectable threonine dehydrogenase.l-Leucine neither stimulated nor repressed threonine dehydrogenase levels. Glycine, and to a lesser extentl-serine, repressedl-threonine-mediated threonine dehydrogenase synthesis. A mutant of strain 9-D2 was isolated that could utilizel-threonine as sole carbon-energy source. This strain produced elevated levels of threonine dehydrogenase, but only slightly higher levels of alpha-amino beta-ketobutyrate: CoA ligase activities.     

l-proline as a nitrogen source increases the susceptibility ofSaccharomyces cerevisiae S288c to fluconazole

Fluconazole inhibition ofSaccharomyces cerevisiae S288c growth was evaluated in media containing ammonia,l-proline orl-leucine as a nitrogen source. Growth inhibition by fluconazole was maximum whenl-proline was used as a nitrogen source, while rhodamine 6G accumulation and fluconazole resistance were the highest when ammonia was the sole nitrogen source.     

Metabolism of threonine by Fusaria growth on threonine as the sole carbon and nitrogen source

Three strains ofFusarium supporting aerobic growth onl-threonine as the sole source of energy and carbon and nitrogen, initially metabolised threonine to acetyl-CoA and glycine via induciblel-threonine:NAD⁺ dehydrogenase plus 2-amino-3-oxobutyrate:CoA ligase activities. Comparative enzyme induction patterns after growth of the three strains on a wide range of carbon sources indicated that the glycine produced by the NAD⁺ plus CoASH-dependent cleavage of threonine was subsequently utilised as an energy source and biosynthetic precursor via the glycine-serine pathway, pyruvate carboxylase, and ultimately the TCA cycle. Acetyl-CoA, the second initial C₂ threonine catabolism product, was subsequently assimilated via a combined TCA plus glyoxylate cycle.     

The Phylogeny of Acetic Acid Bacteria Based on the Partial Sequences of 16S Ribosomal RNA: The Elevation of the Subgenus Gluconoacetobacter to the Generic Level

Thirty-six strains of acetic acid bacteria classified in the genera Acetobacter, Gluconobacter, and Acidomonas were examined for their partial base sequences in positions 1220 through 1375, 156 bases, of 16S rRNA. The strains of the Q₁₀-equipped Gluconobacter species examined were divided into two subgroups, which included the type strains of Gluconobacter oxydans, the type species of the genus Gluconobacter, and of a second species, Gluconobacter cerinus, respectively. The base differences numbered four between the two type strains. The strains of the Q₉-equipped species examined classified in the type subgenus Acetobacter of the genus Acetobacter were not very distant phylogenetically from those of the genus Gluconobacter. The calculated number of base differences was 9–6 between the type strains of G. oxydans and G. cerinus and the type strains of Acetobacter aceti and Acetobacter pasteurianus. In contrast, the strains of the Q₁₀-equipped species examined classified in the subgenus Gluconoacetobacter of the genus Acetobacter were very distant phylogenetically from those of the Acetobacter and Gluconobacter species mentioned above. The number of base differences was calculated to be 14-8. Furthermore, the strains of the methanol-assimilating, Q₁₀-equipped species of the genus Acidomonas examined were located in phylogenetically isolated positions. The type strain of Acidomonas methanolica (≡ Acetobacter methanolicus), the type species of the genus Acidomonas, had 16–9 base differences. The data obtained here indicated that the members of the subgenus Gluconoacetobacter of the genus Acetobacter can be distinguished at the generic level. The new genus Gluconoacetobacter was proposed with the type species, Gluconoacetobacter liquefaciens, in recognition of the genus Acidomonas along with the genera Acetobacter and Gluconobacter in the classification of the acetic acid bacteria.     

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.     

Amino Acids as Nitrogen Sources for the Growth of Euglena gracilis and Astasia longa

SYNOPSIS. Euglena gracilis (bacillaris variety, strain SM-L1, streptomycin-bleached) used the following amino adds (10⁻³ M) as sole nitrogen source for growth on a defined medium: glycine, alanine, valine, leucine, isoleucine, serine, threonine, and glutamic acid. Aspartic acid was used at 10⁻² M. Glutamine and asparagine were used at 10⁻³ M and were better N sources than their parent dicarboxylic amino acids. Not used as sole N source for growth were phenylalanine, tyrosine, tryptophan, cysteine, cystine, methionine, proline, hydroxyproline, histidine, arginine, lysine, and taurine. Astasia longa (Jahn strain) was more restricted than Euglena and used only asparagine and glutamine as N sources for growth.     

Extracellular deamination of l-amino acids by Chlamydomonas reinhardtii cells

When grown in the light and in a Tris-acetate phosphate medium, cells of Chlamydomonas reinhardtii Dang. can use the following l-amino acids as a sole nitrogen source: asparagine, glutamine, arginine, lysine, alanine, valine, leucine, isoleucine, serine, methionine, histidine, and phenylalanine, whereas, in the absence of acetate, the cells only used l-arginine. The utilization system in the acetate medium consisted of an extracellular deaminating activity induced by l-amino acids; it took between 10 to 30 h before the system appeared in cells previously grown with ammonium. This deaminase activity was nonspecific, required an organic carbon source for its de-novo synthesis, and was sensitive to high ammonium concentration and light deprivation.Abbreviations HPLC high-performance liquid chromatography - TAP Tris-acetate-phosphate This work was supported by a grant of the CAICYT, Spain. The secretarial assistance of C. Santos and I. Molina is gratefully acknowledged.To whom correspondence should be addressed.     

Conversion of L-Hydroxyproline to glutamate by extracts of strains of Pseudomonas convexa and Pseudomonas fluorescens

Summary Three strains of Pseudomonas convexa and three strains of Pseudomonas fluorescens were found able to utilize L-hydroxyproline as sole source of carbon and nitrogen. Sonic extracts of these organisms converted L-hydroxyproline to glutamic acid.     

Control of l-amino acid oxidase in Neurospora crassa by different regulatory circuits

l-Amino acid oxidase is synthesized in Neurospora crassa in response to three different physiological stimuli: (i) starvation in phosphate buffer, (ii) mating, and (iii) nitrogen derepression in the presence of amino acids. During starvation in phosphate buffer, or after mating, l-amino acid oxidase synthesis occurred in parallel with that of tyrosinase. Exogenous sulfate repressed the formation of the two enzymes in starved cultures, but not in mated cultures. Sulfate repression was relieved by protein synthesis inhibitors, suggesting that the effect of sulfate required the synthesis of a metabolically unstable protein repressor. With amino acids as the sole nitrogen source only l-amino acid oxidase was produced. Under these conditions enzyme synthesis was repressed by ammonium and was insensitive to sulfate. Biochemical evidence suggested that the l-amino acid oxidase formed under the three different conditions was the same protein. Therefore, the expression of l-amino acid oxidase appeared to be under the control of least two regulatory circuits. One, also controlling tyrosinase, seems to respond to developmental signals related to sexual morphogenesis. The other, controlling other enzymes of the nitrogen catabolic system, is used by the organism to obtain nitrogen from alternative sources such as proteins and amino acids.     

Preliminary studies on the growth of Pseudomonas sp. BA2 and the production of L-aminoacylase

Summary The bacterial strain Pseudomonas sp. BA2 did not develop l-aminoacylase activity in the absence of N-acetyl-dl-alanine. The maximum growth rate and enzyme production were obtained when the acetylated amino acid was used as the sole carbon and nitrogen source. A maximum biomass of A₆₆₀=1.543, after 24 h of cultivation, was obtained. The l-aminoacylase activity reached the maximum value (5.6 U ml^–1 broth) in the stationary growth phase.     

Effect of amino acids on growth ofSphaerotilus discophorus

The effect of casein hydrolysate, of mixtures of amino acids and of individual amino acids on the growth of 4 strains ofSphaerotilus discophorus was determined. Growth was virtually completely inhibited by 1.0% Bacto Casamino Acids, 0.54% simulated casein hydrolysate and 0.2% of a uniform mixture of 18 amino acids. The latter were prepared withl amino acids except thatdl-serine,dl-valine anddl-threonine were present in the uniform amino acid mixture.Experiments designed to test the toxicity of the 18 individual amino acids at 0.018 – 0.36% concentration indicated that arginine, glutamic acid, leucine, lysine and proline were non-toxic. However, aspartic acid and methionine were moderately toxic; growth was greatly repressed at a concentration of 0.36%. The remaining 11 amino acids which included alanine, cystine, glycine, tyrosine, histidine, isoleucine, phenylalanine, serine, threonine, tryptophane and valine were the most toxic of the group. They prevented growth partially or completely, at a concentration of 0.18% or 0.36%.dl-Serine anddl-valine were especially toxic and prevented growth at a concentration of 0.018%. The toxicity of the individuall-amino acids can account for the toxicity of Casamino Acids and simulated casein hydrolysate. l-Methionine or cyanocobalamin (vitamin B₁₂) is required for the growth ofS. discophorus. Alsod- anddl-methionine can replace cyanocobalamin although they completely repress growth when used at the relatively high concentration of 200 µg per ml of medium.     

Characterization and physiological function of a soluble l-amino acid oxidase in Corynebacterium

A general l-amino acid oxidase (l-amino acid: oxygen oxidoreductase (deaminating), EC 1.4.3.2.) has been characterized in Corynebacterium. The enzyme is soluble (MW 130 000–140 000) and is active with most l-α-amino acids but not with aspartate, threonine, proline and glycine. It is subject to substrate inhibition. This amino acid oxidase is induced along with catalase by growth in the presence of amino acids as a nitrogen source and is repressed when ammonium ions are present in the medium. Its probable physiological function is to allow the utilization of amino acids as a nitrogen source.     

D-Amino acids of the amino acid pool and occurrence of racemase andD-amino acid oxidase activities inEscherichia coli B

Less than 20 % of the amino acid content of the amino acid pool ofEscherichia coli B exists in theD-form. Alanine, glutamic acid, and valine were shown by gas-chromatography to be partially in theD-form. OnlyD-alanine was formed by racemization in the crude extract of this organism. Alanine racemase was easily released from the membranes or vesicles butD-alanine oxidase activity remained firmly bound to the membrane. Most protein amino acids stimulated proline uptake into the vesicles, and the oxidative deamination activities were verified by the proline uptake stimulating amino acids. It is concluded that the obligatory pathway of L-amino acid -D-amino acid - oxo acid which exists in the oxidation ofL-alanine does not exist with otherL-amino acids. It is likely that otherD-amino acids in the pool are formed in the presence ofD-amino acid oxidase orD-amino acid aminotransferase.     

<Emphasis Type="SmallCaps">l</Emphasis>-Proline nutrition and catabolism in <Emphasis Type="Italic">Staphylococcus saprophyticus</Emphasis>

Staphylococcus saprophyticus strains ATCC 15305, ATCC 35552, and ATCC 49907 were found to require l-proline but not l-arginine for growth in a defined culture medium. All three strains could utilize l-ornithine as a proline source and contained l-ornithine aminotransferase and Δ¹-pyrroline-5-carboxylate reductase activities; strains ATCC 35552 and ATCC 49907 could use l-arginine as a proline source and had l-arginase activity. The proline requirement also could be met by l-prolinamide, l-proline methyl ester, and the dipeptides l-alanyl-l-proline and l-leucyl-l-proline. The bacteria exhibited l-proline degradative activity as measured by the formation of Δ¹-pyrroline-5-carboxylate. The specific activity of proline degradation was not affected by addition of l-proline or NaCl but was highest in strain ATCC 49907 after growth in Mueller–Hinton broth. A membrane fraction from this strain had l-proline dehydrogenase activity as detected both by reaction of Δ¹-pyrroline-5-carboxylate with 2-aminobenzaldehyde (0.79 nmol min⁻¹ mg⁻¹) and by the proline-dependent reduction of p-iodonitrotetrazolium (20.1 nmol min⁻¹ mg⁻¹). A soluble fraction from this strain had Δ¹-pyrroline-5-carboxylate dehydrogenase activity (88.8 nmol min⁻¹ mg⁻¹) as determined by the NAD⁺-dependent oxidation of dl-Δ¹-pyrroline-5-carboxylate. Addition of l-proline to several culture media did not increase the growth rate or final yield of bacteria but did stimulate growth during osmotic stress. When grown with l-ornithine as the proline source, S. saprophyticus was most susceptible to the proline analogues L-azetidine-2-carboylate, 3,4-dehydro-dl-proline, dl-thiazolidine-2-carboxylate, and l-thiazolidine-4-carboxylate. These results indicate that proline uptake and metabolism may be a potential target of antimicrobial therapy for this organism.     

Nutritional requirements ofXanthomonas phaseoli var.fuscans

As found by Starr (1946),l-glutamic acid is necessary for the growth ofXanthomonas phaseoli var.fuscans. According to our results, the growth is stimulated byl-asparagine in the presence ofl-glutamic acid;l-asparagine itself, however, does not serve as a source of carbon and nitrogen.Xanthomonas phaseoli var.fuscans grew well in a medium containing tryptone. Some peptides of the acidic fraction isolated from tryptone affected the growth as much as tryptone itself. Vitamins and plant growth substances did not affect the growth of the bacteria; proteins appeared to be a poor carbon and nitrogen source. On substituting glucose in a glutamic acid-containing medium with another saccharide, the growth of the bacteria was found equal or better in media containing mannose, sucrose, fructose, maltose or starch. The bacteria grew less satisfactorily in media containing galactose and cellobiose as compared with media containing glucose.     

Lactobionic and cellobionic acid production profiles of the resting cells of acetic acid bacteria

Lactobionic acid was produced by acetic acid bacteria to oxidize lactose. Gluconobacter spp. and Gluconacetobacter spp. showed higher lactose-oxidizing activities than Acetobacter spp. Gluconobacter frateurii NBRC3285 produced the highest amount of lactobionic acid per cell, among the strains tested. This bacterium assimilated neither lactose nor lactobionic acid. At high lactose concentration (30%), resting cells of the bacterium showed sufficient oxidizing activity for efficient production of lactobionic acid. These properties may contribute to industrial production of lactobionic acid by the bacterium. The bacterium showed higher oxidizing activity on cellobiose than that on lactose and produced cellobionic acid.     

The effect of amino acids on the production of ochratoxin A in chemically defined media

The specific ability ofl-glutamic acid and ofl-proline to induce the formation of ochratoxin by a strain of the fungusAspergillus ochraceus in chemically defined media has been further confirmed. This induction was inhibited by 22 other amino acids as well as by analogues and antagonists of glutamic acid and proline. The inhibition could be reversed by increasing the concentrations of glutamic acid or proline and by adding lactic acid in low concentrations.     

Evaluation of alternative nitrogen and carbon sources for sugarbeet suspension culture platings in development of cell selection schemes

Summary Low molecular weight nitrogenous impurity compounds as well as raffinose are negative quality factors that interfere with efficient processing of sugarbeet (Beta vulgaris L.) for sucrose. In order to identify nutrient media for cell selection of biochemical mutants or transgenics that might have reduced levels of these processing impurities, the ability of 10 endogenous compounds to serve as sole nitrogen or carbon source for suspension plating and subculture callus growth was evaluated. The most productive concentrations of nitrate, ammonium, l-glutamine, l-glutamate, urea, and l-proline as sole nitrogen sources supported plating callus growth at 106, 159, 233, 167, 80, and 52%, respectively, as well as the historical 60 mM mix of nitrate and ammonium in Murashige-Skoog medium. Glycine betaine and choline did not support growth. d(+) Raffinose and d(+) galactose supported plating callus growth only 67 and 25%, respectively, as well as sucrose as sole carbohydrate source. No callus growth occurred on glutamine, glutamate, or glycine betaine as the sole carbon or carbon plus nitrogen source. Platings on either nitrate or ammonium as sole nitrogen source did not differ in sensitivity to the nitrate uptake inhibitor phenylglyoxal, suggesting that phenylglyoxal lacks the specificity for use in selection for mutants of nitrate uptake. The ability of raffinose to be used as the carbon source, and glutamine or glutamate as the nitrogen source, may preclude their use for selection of genetic variants accumulating less of these processing impurities. However, mutants or transgenics able to utilize either glutamine, glutamate, or glycine betaine might be selectable on media containing any one of these as carbon, nitrogen, or carbon plus nitrogen source, respectively, that is incapable of supporting wild-type cell growth.