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Volume 109,
Issue 2,
1978
Volume 109, Issue 2, 1978
- Biochemistry
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The Ammonia and Methylamine Active Transport System of Aspergillus nidulans
More LessConidia of Aspergillus nidulans germinated on glutamate have a single system for the active transport of ammonia and methylamine against a concentration gradient. The transport of methylamine is inhibited by azide, N-ethylmaleimide, valinomycin and anaerobiosis; it has a pH optimum of about 6·3 and a temperature optimum of 40°C. The affinity of the system for methylamine (K m 13 μ m) is about 20% of that for ammonia; the V max is also much lower. This transport system is present in methylamine-resistant mutants (meaA and meaB loci) and its formation is repressed in wild type and mutants by ammonia or a metabolite of ammonia. It is proposed that the system functions in the scavenging of low concentrations of ammonia during nitrogen starvation or during growth on other major nitrogen sources.
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Regulation by Glutamine of Ammonia Transport in Aspergillus nidulans
More LessConidia of Aspergillus nidulans had a very low activity of a high affinity ammonia transport system. During germination the activity increased, reaching a maximum soon after the onset of exponential growth. The activity in germinated conidia varied with the nitrogen source in the germination medium. Analyses of intracellular ammonia and amino acids revealed that a low level of ammonia transport activity was associated with a high intracellular concentration of glutamine and vice versa. The intracellular concentration of glutamine is probably involved in repression of synthesis of the ammonia transport system. It is suggested that glutamine and asparagine (rather than ammonia itself) regulate the pre-formed ammonia transport system by inhibition from within the intracellular pool. It is concluded that glutamine and glutamine synthetase may be more important in the regulation of some aspects of nitrogen metabolism rather than ammonia and glutamate dehydrogenase as has been previously suggested.
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Extracellular Glucan Containing (1→3)-β and (1→6)-β Linkages Isolated from Monilinia fructigena
More LessSummary: Monilinia fructigena produced more extracellular polysaccharide when grown in liquid shake culture than in stationary culture, with greater quantities of the polysaccharide being secreted if ascorbic acid was present in the cultures. The polysaccharide was subsequently degraded during autolysis, eventually disappearing completely. After partial purification, the carbohydrate content was 96% and gel chromatography indicated a molecular weight of about 70000. Strong acid hydrolysis of the polysaccharide produced only glucose. Infrared spectroscopy indicated that the anomeric configuration of its d-glucose units was of the β-linkage type only. Periodate oxidation, Smith degradation and enzymic analysis indicated that the polysaccharide was a (1→3)-β-glucan having single (1→6)-β-glucosyl side-chains on every second d-glucose unit.
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Isolation and Characterization of a Bacteriocin Produced by Pseudomonas solanacearum
More LessStrain b1, an avirulent non-slime-producing variant of Pseudomonas solanacearum k60, had greater bacteriocinogenic activity than did strain k60 both in liquid and on solid medium. The bacteriocin synthesized by strain b1 inhibited the growth of 43 of 51 P. solanacearum strains tested. It had no effect on strain b1, strain k60, or selected strains from 10 other bacterial species. Although the optimal growth temperature for strain b1 was 32 °C, the temperature most favourable for bacteriocin production was 30 °C. Bacteriocin titre could be increased by short exposure to ultraviolet light but not by chemical inducing agents.
The bacteriocin produced by strain b1 was purified from the culture supernatant by ammonium sulphate precipitation, anion exchange chromatography, membrane ultra-filtration and preparative electrophoresis. Purified bacteriocin, being non-sedimentable, thermolabile and sensitive to proteolytic enzymes, resembled the S-type bacteriocins produced by P. aeruginosa. As determined by sodium dodecyl sulphate-polyacrylamide gel electrophoresis, the purified bacteriocin had a molecular weight of approximately 65 000. It was insensitive to chloroform, nucleases and phospholipase. Although the bacteriocin present in culture supernatant was stable for 3 months at 4 °C, the activity of purified bacteriocin declined rapidly under a variety of storage conditions. Maximum pH stability was between 6 and 8.
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- Development And Structure
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A Mathematical Analysis of Dolipore/Parenthesome Structure in Basidiomycetes
More LessMeasurements were taken on the septal ultrastructure of 13 species of basidiomycete fungi from eight different orders. A continuum of dolipore size was found as opposed to the two size-types previously proposed. A cluster analysis was carried out on the measurements to suggest relationships between the species. Based on this and other published information on dolipores, possible evolutionary trends in the ultrastructure of the dolipore are discussed.
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- Ecology
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Pseudomonas savastanoi and Other Bacteria Colonizing the Surface of Olive Leaves in the Field
More LessAerobic, heterotrophic, mesophilic bacteria were isolated from olive leaves in January, April, July and October in each of three consecutive years. Phenetic data on the isolates and marker strains were collected and analysed using numerical taxonomic methods: 1744 of the 1789 isolates were recovered in phena that were equated with Pseudomonas savastanoi (67·86% of the isolates), Erwinia herbicola (8·50%), Bacillus megaterium (4·02%), Micrococcus luteus (3·63%), the Xanthomonas campestris group (3·35%), Arthrobacter globiformis (2·07%), Lactobacillus plantarum (1·45%), Klebsiella pneumoniae (1·40%), Serratia marcescens (1·34%), Acetobacter aceti (1·23%), Leuconostoc dextranicum (1·12%), Pseudomonas fluorescens (1·06%), Bacillus subtilis (0·34%) and Pseudomonas delafieldii (0·11%). There were characteristic seasonal fluctuations in the populations of most of the bacteria. The abundance of P. savastanoi on healthy leaves in April and October supports earlier suggestions that the phylloplane of the host may be an important source of readily available inoculum in the epidemiology of olive knot disease.
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- Genetics And Molecular Biology
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Linkage Analysis in Dictyostelium discoideum using Multiply Marked Tester Strains: Establishment of Linkage Group VII and the Reassessment of Earlier Linkage Data
More LessTo aid linkage analysis and mapping studies in Dictyostelium discoideum, we have constructed several tester strains with easily scored mutations characterizing the six currently identified linkage groups. Use has been made of conditionally lethal mutants unable to grow upon Bacillus subtilis, and the locus of the mutation involved (bsgA) has been assigned to linkage group III. The mutation cobA1, which confers resistance to cobaltous chloride, has been assigned to a previously unidentified linkage group (VII). The temperature-sensitive growth mutation tsgC7, previously reported to define linkage group V, has been reassigned to group III, leaving linkage group V presently unmarked. The further use of genetic tester strains is described.
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Thermosensitive H1 Plasmids Determining Citrate Utilization
More LessTwelve thermosensitive H1 plasmids from strains of Salmonella typhi that had caused outbreaks of chloramphenicol-resistant typhoid fever in Vietnam, Thailand and India mediated citrate utilization (Cit+) in a prototrophic Escherichia coli k12 strain but not in the S. typhi strains from which they were derived. Four H1 plasmids from a similar outbreak in Mexico differed from the Far Eastern plasmids in not mediating citrate utilization but in mediating mercury resistance. H1 plasmids resembling the Far Eastern and the Mexican plasmids in regard to citrate utilization and mercury resistance were found in sewage in Britain. Citrate utilization was transferred to eight pathogenic strains of E. coli and to one strain each of Shigella flexneri and Shigella sonnei. Cultures of Cit+ bacteria grew more rapidly in citrate media at 28 °C than at 37 °C. Plasmid mutants that were more efficient at utilizing citrate were present in all such cultures – they grew equally well or better at 37 °C than at 28 °C. None of 222 strains of E. coli or Shigella that contained a variety of different plasmids were able to utilize citrate. This property was not transferred to the prototrophic E. coli k12 strain from Citrobacter (3 strains), Salmonella (39 strains), Proteus (44 strains), Klebsiella pneumoniae (33 strains) or Pseudomonas aeruginosa (44 strains).
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Chromosomal Location of the mop (groE) Gene Necessary for Bacteriophage Morphogenesis in Escherichia coli
More LessThe chromosomal location of a host gene, mop (groE), which is essential for the morphogenesis of several bacteriophages in Escherichia coli, was determined by two- and three-factor transductional crosses using phage P1. Cotransduction frequencies between mop and other markers were: aspA, 90%; ampA, 77%; frdA, 73%; mel, 24%. The sequence of markers in the corresponding segment (mel to purA; 91·5 to 93·5 min) of the E. coli linkage map was shown to be mel–aspA–mop(groE)–ampA–frdA–purA.
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Chloramphenicol Acetyltransferases Determined by R Plasmids from Gram-negative Bacteria
More LessThe mechanism of resistance to chloramphenicol specified by 18 plasmids from Gram-negative bacteria representing different incompatibility groups was investigated. Most determined the drug-inactivating enzyme chloramphenicol acetyltransferase. The enzymes were purified and their properties were compared with those of the previously characterized enzyme types specified by R429 (type I), s-a (type II) and R387 (type III). The type I enzyme was determined by plasmids representing incompatibility groups FII, C, S, I, H, L, O and Com9. Plasmids from incompatibility groups K, Iγ and the A–C complex specified the type III enzyme, while elements representing incompatibility groups V and W determined the type II variant.
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- Medical Microbiology
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Phenotypic Resistance to Amphotericin B in Candida albicans: the Role of Reduction
More LessThe development of resistance to amphotericin B methyl ester (AME), measured in terms of the amount (μg AME ml–1 = s.r.c.) of the antibiotic required to induce a standard rate of release of K+ from suspensions of washed organisms, has been followed in Candida albicans in starved cultures under controlled conditions of aeration, pH, stirring and temperature for periods up to 120 h, and the amino acid content of the pool and the carbohydrate content of the organisms have been determined. The soluble glucan fraction of the cells decreased during the first 24 h starvation but no significant changes were observed in other carbohydrate or lipid fractions. At pH 3, the glutamate content of the pool decreased rapidly during the starvation period but other amino acids showed a general pattern of increase during the first 24 to 72 h and decrease during the later stages of starvation. Increasing the glutamate content of the pool by growth in media supplemented with sodium glutamate delayed the onset of resistance, and, in all experiments, high resistance did not develop until the glutamate content of the pool had fallen below 10 nmol (mg dry wt organisms)–1. There was a highly significant correlation between the level of resistance and (i) the glutamate concentration in the pool and (ii) the rate of disappearance of glutamate from the pool.
Treatment of starved organisms with N-ethylmaleimide (NEM) increased the resistance, the increase ranging from 10-fold for cultures starved for 24 h to 2-fold or less for cultures starved for 120 h. The resistance of NEM-treated organisms increased with the time of starvation. Treatment of organisms with β-mercaptoethanol (ESH) decreased resistance ; for organisms after 24 to 96 h starvation, possessing moderate resistance (s.r.c. 15 to 20), treatment with ESH reduced this resistance to a value approximating to that of growing organisms (s.r.c. 0·1). In organisms starved for longer periods and with higher resistance, the effect of ESH was smaller and the level of ESH-insensitive resistance increased rapidly with time. Resistance thus developed in two stages; first, a stage where reduction of some components of the cell decreased resistance and, later, a stage of high resistance unaffected by reduction. Changes, similar in quality but smaller in quantity, were found in the development of resistance in organisms grown and starved at pH 7.
The activities of glutamate : NAD+ oxidoreductase (EC 1.4.1.2) and glutamate : NADP+ oxidoreductase (EC 1 .4.1.3) have been estimated in extracts from organisms during growth and starvation ; the NAD-linked enzyme activity remained relatively unchanged during the starvation period of 96 h while the NADP-linked enzyme activity decreased markedly during the first 24 h starvation. Estimation of the degree of reduction of the cellular NAD during the starvation period showed that AME resistance rose as the ratio of cellular NADH/NAD+ decreased.
It is suggested that the first stage of phenotypic resistance can be attributed to a reducible factor in the cell wall, and that the amount of this factor increases during starvation but that the developed resistance is determined by the degree of reduction of this factor. The evidence is not inconsistent with glutamate in the pool acting as a major source of reducing potential.
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- Physiology And Growth
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The Use of Step Enzymes as Markers During Meiosis and Ascospore Formation in Saccharomyces cerevisiae
More LessThe activities of ornithine aminotransferase, sucrase and acid and alkaline phosphatases have been studied throughout sporulation in Saccharomyces cerevisiae. The same enzymes were monitored during synchronous vegetative growth. Each of these enzymes has been demonstrated to increase in a ‘step’ manner during both growth and sporulation. Alkaline phosphatase increased in a two-step manner whereas the others increased in a single step. The times of increase of these enzymes formed a similar sequence during both sporulation and growth. It has been proposed that these enzymes are under a common mechanism of control during growth and sporulation and that the sequence of enzyme appearance may be used as markers of the sporulation process.
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Physicochemical and Biological Properties of Mycobacteriocin M12 Produced by Mycobacterium smegmatis ATCC 25855
More LessA mycobacteriocin (M12) produced by Mycobacterium smegmatis ATCC 25855 was partially purified by ammonium sulphate precipitation followed by DEAE-cellulose chromatography and Sephadex G100 chromatography. Production of M12 was maximal when bacteria were harvested after 3 d cultivation in liquid medium and disrupted by sonication. The molecular weight of M12, estimated by Sephadex G100 chromatography, was about 85000. M12 was sensitive to proteolytic enzymes but resistant to DNAase and RNAase, and was relatively stable to heat treatment, sonication, ultraviolet irradiation and pH over the range 4 to 8. When sensitive bacteria were exposed to the mycobacteriocin, the number of viable cells began to decrease after about 6 h incubation. The killing curve of M12 thus appeared to be a multiple-hit curve. Electron microscopic observation revealed that the mycobacteriocin induced morphological changes in the cells: these were partial loss of ribosomes, enlargement of lipoidal inclusion bodies and thickening of the cell envelope. The activity spectrum of M12 was restricted to the genus Mycobacterium.
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Influence of Cultivation Conditions on the Production of Staphylocoagulase by Staphylococcus aureus 104
More LessHigh yields of staphylocoagulase from Staphylococcus aureus 104 were obtained in a simple salts medium supplemented with glycerol, casein hydrolysate and three vitamins. Conditions of oxygen-limitation (dissolved oxygen concentration ≪2%), a pH of 7·4, a temperature of 35°C and a 1 in 10 inoculum of overnight culture were required for optimal yields of staphylocoagulase.
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The Uptake of Choline by Streptococcus pneumoniae
More LessSummary: Uptake of choline, a structural component of pneumococcal C- and F-teichoic acids, into bacteria growing in a defined medium was very efficient with an uptake constant ([S]0·5) of 3·2 μ m. It was inhibited by iodoacetate, dinitrophenol and oligomycin but not by structural analogues of choline. Ethanolamine, however, was transported in the absence of choline but with a reduced affinity ([S]0·5 71·4 μ m). The same constitutive system was probably used by both ethanolamine and choline. It is suggested that this system required ATP and probably involved choline kinase.
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The Relationship between Energy-dependent Phagocytosis and the Rate of Oxygen Consumption in Tetrahymena
More LessThe induction of high rates of food vacuole formation in Tetrahymena pyriformis increased the rate of respiration in exponentially growing cells by 17% and in starving cells by 47·5 %. The increased rate of oxygen uptake was caused by phagocytosis itself, as shown by comparing the rates of respiration of a Tetrahymena mutant exposed to particles at the permissive or restrictive temperatures for food vacuole formation. During cell division, heat-synchronized cells in rich, particle-supplemented medium showed a significant decrease in the rate of respiration. Furthermore, dimethyl sulphoxide, in concentrations sufficient to block food vacuole formation, suppressed the rate of respiration to a level similar to that of starved cells. Cytochalasin B, however, did not reduce the rate of oxygen uptake despite the inability of the cells to complete the formation of food vacuoles during treatment; a possible explanation for this finding is discussed. There was a strong correlation between formation of food vacuoles and a high metabolic rate in Tetrahymena.
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- Short Communication
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