- Volume 130, Issue 12, 1984
Volume 130, Issue 12, 1984
- Physiology And Growth
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Mechanism of Mycobacteriocin and Tween 80 Mediated Antimycobacterial Activity
More LessRapidly growing mycobacteria (14 species, 67 strains) showed a highly significant correlation between their susceptibility to smegmatocin (a Tween-hydrolysing esterase of Mycobacterium smegmatis exhibiting antimicrobial activity in the presence of Tween 80) and sensitivity to oleic acid (a hydrolysis product of Tween 80). Polyoxyethylenesorbitan ether, the other product of Tween 80 hydrolysis, also showed some toxic effect on smegmatocin-sensitive mycobacteria. From Mycobacterium diernhoferi ATCC 19340, a representative smegmatocin-sensitive strain, mutant strains with smegmatocin-resistant phenotypes were isolated. These mutants were more resistant to oleic acid than the parent strain, although their oleic acid resistance was somewhat lower than their smegmatocin resistance.
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Factors Affecting the Biosynthesis of l-Tryptophan by Genetically Modified Strains of Escherichia coli
More LessDerivatives of Escherichia coli strain W3110 with increased tryptophan synthase (TS) activity were constructed. The biosynthesis of serine was shown to limit tryptophan production in minimal medium with indole as precursor. In the presence of serine and indole we obtained a correlation between the specific activity of TS and the specific productivity (q p) of tryptophan. Supplementation of the growth medium with glycine enhanced q p two-fold. In a strain with high serine hydroxymethyltransferase (SHMT) activity no such increase of tryptophan productivity was observed, although crude extracts from these cells were shown to produce tryptophan with indole, one-carbon units and glycine as precursors. Growth of the strain with high SHMT activity was inhibited in a medium with high glycine concentration. This inhibition could not be released by addition of isoleucine and valine. In a buffer system with permeabilized cells high in specific TS and SHMT activities we did not obtain any tryptophan production in presence of indole, glycine, one-carbon units and cofactors. On the other hand, in a buffer system with indole and serine as precursors we obtained high q p of tryptophan [13·3 g tryptophan (g dry wt cells)−1 h−1], which was correlated to the TS specific activity.
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Germination of Bacillus cereus Spores: Critical Control by DL-Alanine Racemase
More LessEffects of dl-alanine racemase, D-alanine, heat-activation and ammonia on germination of Bacillus cereus T spores were studied. Michaelis-Menten parameters for dl-alanine racemase activity in intact spores were: K m (l-alanine)=3·6 mm; K m (d-alanine) = 1·2 mm; V max (l- to d-alanine) = 1·6 μmol min−1 (mg dry spores)−1; V max (d- to l-alanine) = 0·53 μmol min−1 (mg dry spores)−1. Racemase activity was irreversibly inhibited with pseudo first order kinetics by incubating intact spores with 10 mm-d-cycloserine. (Aminooxy)acetate (50 μm) completely inhibited racemase activity with 100 mm-l-alanine as substrate. d-Cysteine was a moderately effective racemase inhibitor. Comparisons of the effects of racemase inhibitors, d-alanine and spore concentration on germination rates in 1 mm-l-alanine indicated that racemization of l-alanine accounted for nearly total inhibition of germination at spore concentrations exceeding about 50 μg ml−1, in the range conventionally used for spectrophotometric germination assays. Heat-activation decreased the inhibitory effect of d-alanine, increasing the germination rate in 2 mm-racemic alanine by a factor of 26. Using spores with an inhibited alanine racemase, germination rates in 1 mm-l-alanine were stimulated by 40 mm-NH4Cl to an extent comparable to the stimulation obtained by heat-activating the spores. Germination of unactivated spores in 1 mm-l-alanine + 40 mm-NH4Cl was completely inhibited by 1 mm-d-alanine. These results clarified the roles of d-alanine and dl-alanine racemase in controlling the germination of unactivated spores and suggested that the germinative functions of ammonia and the stereoisomers of alanine may be related.
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The Ability of Sordaria fimicola to Take up and Metabolize Glucose and Sucrose
More LessGlucose, fructose and trehalose, but not sucrose, supported the growth of Sordaria fimicola. Mycelial suspensions rapidly metabolized [14C]glucose, which was shown to be taken up by a mechanism saturated at 1 mm. Comparable experiments with [14C]sucrose were complicated by contaminating [14C]hexose but provided no conclusive evidence for sucrose uptake. Mycelial extracts hydrolysed sucrose at 7% of the rate found with trehalose. It is suggested that the fungus cannot grow on sucrose because it lacks a mechanism that permits uptake of sucrose carbon.
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Mitochondrial Activities in the Methylotrophic Yeast Kloeckera sp. 2201 During Growth with Glucose and/or Methanol
Th. Egli and N.D. LindleyThe methylotrophic yeast Kloeckera sp. 2201 was grown in carbon-limited chemostat cultures with glucose and/or methanol as the carbon sources, to investigate the regulation of the synthesis of enzymes involved in the tricarboxylic acid (TCA) cycle and the electron transport chain.
Except for malate dehydrogenase, enzymes of the TCA cycle were repressed during growth with methanol to specific activities of 20–40% of those found in cells growing under identical growth conditions but with glucose as the carbon source. In methanol-grown cells, the specific activities of the enzymes of the respiratory chain were also repressed compared to cells grown with glucose [60–70% for NADH: cytochrome c (cyt c) oxidoreductase and cyt c oxidase; approximately 40% for succinate:cyt c oxidoreductase]. The measurement of cytochrome oxidized-minus-reduced spectra was impossible in methanol-grown cells because of interference of the chromophoric group of the peroxisomal enzyme alcohol oxidase under these growth conditions. The relative cytochrome c content of mitochondria from methanol-grown cells, separated from peroxisomes by density gradient centrifugation, was significantly lower (cyt aa 3:cyt b:cyt c = 1:1.24:0.86) than in mitochondria from glucose-grown cells (cyt aa 3: cyt b :cyt c = 1:1.27:2.20). The results are discussed with respect to the generation of energy for biosynthesis during growth with glucose and/or methanol.
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Protein Synthesis and Amino Acid Pool during Yeast-Mycelial Transition Induced by N-Acetyl-D-glucosamine in Candida albicans
More LessProtein synthesis at different stages of yeast-mycelial transition induced by N-acetyl-D-glucosamine in Candida albicans was evaluated by following incorporation of radioactive amino acids into the acid-insoluble cellular material. In passing from the early germ-tube formation (60–90 min) to the mature hyphal cell (240–270 min) there was a marked decrease in the capacity for protein synthesis. Apparently, this decrease was not due to a decreased amino acid uptake into the soluble cellular pool or to exhaustion of carbon/energy source in the inducing medium with consequent arrest of growth. Protein synthesis, however, did not decay when amino acids at high concentration were added to the medium fostering the yeast-mycelial transition and this effect was potentiated by glucose.
Analysis of the intracellular amino acid pool showed that both germ-tubes and hyphal cells were relatively depleted of several amino acids as compared to the yeast-form cells, whereas in the hyphae there was a higher concentration of glutamic acid/glutamine, the latter being the predominant component. These modulations in amino acid pool composition were not seen when yeasts were converted to hyphae in an amino acid-rich induction medium. This study emphasizes that yeast-form cells of C. albicans may efficiently convert to the mycelial form even under a progressively lowered rate of protein synthesis, and suggests that initiation of hyphal morphogenesis in the presence of N-acetyl-d-glucosamine is somehow separated from cellular growth.
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Substrate Specificities of the Soluble and Particulate Methane Mono-oxygenases of Methylosinus trichosporium OB3b
More LessThe intracellular location of methane mono-oxygenase (MMO) (soluble or particulate) in Methylosinus trichosporium OB3b is dependent on the availability of copper in the growth medium. Raising the Cu2+ concentration from 1 μm to 5 μm effected a transition from soluble to particulate MMO activity, and changes in major cell polypeptides were observed on SDS-polyacrylamide gels. Organisms containing soluble MMO oxidized a wide range of substrates including n-alkanes, n-alkenes, aromatic and alicyclic compounds. By contrast, organisms containing particulate MMO did not oxidize aromatic or alicyclic compounds. These observations provide further evidence that the two types of MMO are fundamentally different.
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- Systematics
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Discriminant Analysis of Volatile Fatty Acids Produced in Culture Medium: a Novel Approach to the Identification of Pseudomonas Species
More LessThe volatile fatty acids produced in culture medium by 357 Pseudomonas strains belonging to eight species were determined quantitatively by GLC. The resultant chromatograms were submitted to discriminant analysis. Stable discriminant functions were computed and included in a computerized identification system which also involved some distinctive volatile fatty acids regarded as two-state qualitative characters (presence or absence characters). Using a test group of 249 strains belonging to the studied species, more than 89% of the identifications made by this system agreed with those made by conventional biochemical methods despite the relatively poor differentiation between P. putida and P. fluorescens. When the individual species within the matrices were weighted with prior probabilities reflecting results given by two simple biochemical tests, 96% of the 249 strains were correctly identified.
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Mycoplasma cavipharyngis, a New Species Isolated from the Nasopharynx of Guinea-pigs
More LessTwo mycoplasma strains isolated from the nasopharynx of guinea-pigs in two separate colonies were biochemically and serologically identical, and distinct from 80 Mycoplasma and Acholeplasma spp. One of them, strain 117C (NCTC 11700), is designated the type strain of a new species Mycoplasma cavipharyngis.
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A Study by High-resolution Two-dimensional Polyacrylamide Gel Electrophoresis of Relationships between Neisseria gonorrhoeae and Other Bacteria
More LessHigh-resolution two-dimensional polyacrylamide gel electrophoresis was used to analyse the soluble proteins from seven strains of Neisseria gonorrhoeae, six strains of Neisseria meningitidis and one or two strains of twelve other species. Approximately 200 individual polypeptides could be visualized as Coomassie Blue stained spots on an electrophoretogram of N. gonorrhoeae and similar numbers were found for the other bacteria. Each species of bacterium had a distinctly different pattern of spots which could be recognized. Quantitative comparisons of 48 selected spots derived from one strain of N. gonorrhoeae with those of five other strains of gonococcus, three strains of N. meningitidis and one of Branhamella catarrhalis, showed relationships in agreement with their current taxonomic classification but with a higher level of discrimination than that of previously used methods. It was also possible to distinguish the individual gonococcal strains. It is suggested that the method could be useful for bacterial classification and identification.
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Some Biochemical and Morphological Characteristics of Serotypes of Verticillium albo-atrum from Hop
More LessSingle-spore strains of Verticillium albo-atrum from hop, which had previously been classified into three serotypes by immunoelectrophoretic and immunofluorescence methods, have been further characterized using a series of morphological and biochemical parameters. Serotype 1 strains were characterized by high sporulation and low polygalacturonase activity when cultured on hop root cell walls, the presence of certain protein bands on isoelectric focusing of mycelial extracts, and a characteristic dark-pigmented mycelial morphology on prune/lactose/yeast extract agar. It is suggested that the expression of serotype 1 characteristics is controlled by the previously reported hyl+ cytoplasmic factor. Serotypes 2 and 3 were both associated with low sporulation and high polygalacturonase activity and a fully hyaline fluffy mycelium, although many serotype 3 strains were only partially hyaline. Serotype 2 was associated with a characteristic pattern of mycelial proteins on isoelectric focusing gels: serotype 3 strains showed a heterogeneous series of patterns. None of the characters studied could be correlated with virulence for hop but the results do allow the selection for future studies of well-characterized strains which are extremely similar in many respects and yet still differ markedly in virulence.
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- Short Communication
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Use of a Synthetic Oligonucleotide to Identify a Chromosomal Gene for Chloramphenicol Acetyltransferase in a Plasmid-bearing Flavobacterium
More LessA micro-organism previously designated Flavobacterium sp. CB60 is resistant to chloramphenicol as a consequence of antibiotic acetylation by the enzyme chloramphenicol acetyltransferase and subsequent degradation of the acetylated product by co-metabolism. Although a 15·6 kb plasmid (pCB60) was demonstrated in this Flavobacterium strain, it did not appear to play a role in chloramphenicol acetylation. DNA hybridization was used to identify a fragment of DNA presumptively carrying the cat gene. The sequence of the synthetic probe was based upon known nucleotide sequences corresponding to the highly-conserved active site region of several chloramphenicol acetyltransferase variants. The structural gene for the enzyme in strain CB60 appeared to be chromosomal since the radioactive probe hybridized with a unique restriction fragment from chromosomal DNA but failed to do so with the DNA of plasmid pCB60.
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Genetic Analysis of Three Additional fla Genes in Salmonella typhimuvium
More LessIn Salmonella typhimurium, 27 fla responsible for formation of flagella have been identified and assigned to three regions on the genetic map, termed fla regions I to III. By genetic analysis of 1984 non-flagellate mutants obtained from a phase-1 stable strain of S. typhimurium, SJW 1103, three additional fla genes were identified; one, termed flaW, was assigned to fla region I and the other two, termed flaV and flaX, toflaA region III. By intergeneric complementation tests, the flaW, flaV and flaX genes were shown to be functionally homologous with flaS,flaC and flaP of Escherichia coli, respectively. Electron microscopy showed that flaW and flaV mutants carried hook-basal body structures.
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