- Volume 106, Issue 2, 1978
Volume 106, Issue 2, 1978
- Biochemistry
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Mutants of Acinetobacter calcoaceticus NCIB 8250 Constitutive for the Mandelate Enzymes
More LessMethods for isolating mutants of Acinetobacter calcoaceticus NCIB 8250 constitutive for the mandelate enzymes were compared. Non-inducing substrates were not available. Continuous culture gave no stable constitutive mutants. Alternate culture in inducing (L-mandelate) and non-inducing (L-glutamate) media led to the isolation of mutants mesoconstitutive for L-mandelate dehydrogenase and phenylglyoxylate carboxy-lyase, and hyperinducible for benzaldehyde dehydrogenase I. Screening of possible anti-inducers showed that 2-phenyl-propionate was very effective; it was a competitive inhibitor of gratuitous induction by thiophenoxyacetate, and was used for the isolation of constitutive mutants. Some of the mutants were magnoconstitutive for L-mandelate dehydrogenase, phenylglyoxylate carboxylyase and benzaldehyde dehydrogenase I and could not be further induced; others had lower specific activities which could be increased by induction with phenylglyoxylate or thiophenoxyacetate. Similar constitutive mutants were derived from a mutant which lacked L-mandelate dehydrogenase.
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Nitrogen Regulation of Glutamine Synthetase in Neurospora crassa
More LessA higher activity of glutamine synthetase (EC 6.3.1.2) was found in Neurospora crassa when NH4 + was limiting as nitrogen source than when glutamate was limiting. When glutamate, glutamine or NH4 +were in excess, a lower activity was found. Immunological titration and sucrose gradient sedimentation of the enzyme established that under all these conditions enzyme activity corresponded to enzyme concentration and that the octamer was the predominant oligomeric form. When N. crassa was shifted from nitrogen-limiting substrates to excess product as nitrogen source, the concentration of glutamine synthetase was adjusted with kinetics that closely followed dilution by growth. When grown on limiting amounts of glutamate, a lower oligomer was present in addition to the octameric form of the enzyme. When the culture was shifted to excess NH4 +, glutamine accumulated at a high rate; nevertheless, there was only a slow decrease in enzyme activity and no modification of the oligomeric pattern.
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Aerobic and Anaerobic Metabolism of Trimethylamine, Dimethylamine and Methylamine in Hyphomicrobium X
More LessHyphomicrobium strain x was grown on trimethylamine and dimethylamine as the sole sources of carbon and energy under both aerobic and anaerobic conditions and the enzymes involved in the metabolism of these compounds were investigated. During aerobic growth of the organism on trimethylamine, accumulation and subsequent utilization of dimethylamine was observed. When the organism was grown on trimethylamine under anaerobic conditions in the presence of nitrate, a sequential accumulation and utilization of dimethylamine and methylamine was found. In cell-free extracts of Hyphomicrobium x grown on trimethylamine or dimethylamine under both aerobic and anaerobic conditions the following enzyme activities were detected: trimethylamine dehydrogenase, dimethylamine dehydrogenase, γ-glutamylmethylamide synthetase, N-methylglutamate dehydrogenase, methanol dehydrogenase, formaldehyde dehydrogenase, formate dehydrogenase and hydroxypyruvate reductase. Under neither growth condition were any of the following enzyme activities detected: trimethylamine mono-oxygenase, dimethylamine mono-oxygenase, trimethylamine-N-oxide aldolase (demethylase) and primary-amine dehydrogenase. Trimethylamine dehydrogenase and dimethylamine dehydrogenase were partially purified from bacteria grown on dimethylamine and the results suggest that in Hyphomicrobium x a novel enzyme, namely dimethylamine dehydrogenase, participates in the oxidation of dimethylamine.
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Isoelectric Focusing Studies on the ?-Fructofuranosidases and ?-Glucosidases of Streptococcus mitis
More LessExtracts of Streptococcus mitis ATCC 903 were analysed for ?-fructofuranosidase and ?-glucosidase activities by isoelectric focusing in thin-layer polyacrylamide gels combined with zymogram procedures. Three bands of activity were visualized in the gels after incubation with sucrose (pI 4.05, 4.25 and 4.85) and three other bands after incubation with p-nitrophenyl ?-D-glucopyranoside (pI 3.90, 4.45 and 4.65). The enzymes responsible for the reaction with sucrose were identified as ?-fructofuranosidases (EC 3.2.1.26) for the following reasons: identical enzyme bands were visualized in the gels after incubation with raffinose; no enzyme bands appeared in the gel after incubation with the ?-glucosides maltose, turanose, trehalose and melezitose; and the soluble fraction hydrolysed sucrose to equimolar amounts of glucose and fructose.
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- Ecology
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Inhibition as a Factor in the Maintenance of the Diversity of Microbial Ecosystems
More LessSimple models of growth, nutrient utilization, product formation and inhibitor action are used to show that two populations competing for a single nutrient which is limiting their rates of growth can coexist in an ideal chemostat if at least one of the populations produces an autoinhibitor. Production of a substance that is not autoinhibitory but inhibits instead the competitor population (antagonism) will not allow coexistence but can give a population a chance to exclude its competitor under conditions where it would otherwise be excluded by the competitor. These results show that production of autoinhibitors can play an important role in maintaining the species diversity of microbial ecosystems.
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- Genetics And Molecular Biology
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Genetic Evidence for a Plasmid Controlling Fertility in an Industrial Strain of Streptomyces rimosus
More LessA plasmid (SRP1) determines most of the genetic recombination which occurs between marked derivatives of our strain of Streptomyces rimosus. SRP1? strains occur with a frequency of about 0.3% amongst spores of SRP1 + strains. In SRP1 +xSRP1 ? crosses, SRP1 is transferred to more than 50% of the SRP1 ? parent. SRP1 +xSRP1 ? crosses are about 100 times more fertile than SRP1+xSRP1+ crosses (10?3 to 10?4 compared with 10?5 to 10?7). A very low frequency (about 10?8) of true recombinants occurs in the absence of SRP1.
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Genetic Factors Determining the Growth of Physarum polycephalum Amoebae in Axenic Medium
More LessEvidence is presented that in the true slime mould Physarum polycephalum the ability of the amoebal strain CLd-AXE to grow in axenic medium is determined by mutations in two genes axeA and axeB and that the axenic growth of RSD4-AXE amoebae is also under genetic control. Mutant amoebal strains are also able to grow on bacterial lawns and the ability to grow in axenic media persists during prolonged culture on bacteria. However, some of the mutant strains grow less well on bacteria than strains of similar genetic background which are unable to grow in axenic media. CLd-AXE has the same nuclear DNA content (0.59 pg per nucleus) as CLd, the strain from which it was derived. Amoebae able to grow in axenic media were also derived from strains E27 and LU858 which carry mutations for temperature sensitivity and leucine requirement expressed in the plasmodial phase. Tests in axenic media showed that these mutations were expressed in the amoebal phase. The elucidation of the genetic basis of axenic growth will allow the construction of a range of amoebal strains able to grow in axenic media and this will greatly facilitate the isolation and analysis of mutants in this organism.
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Specialized Transduction of a Cysteine Marker by Rhizobium meliloti Phage 16-3
More LessSpecialized transduction by phage 16-3 of one of the cys markers of Rhizobium meliloti is described. The frequency of transduction was 10?6 to 10?7 per plaque-forming unit. About two-thirds of the transductants were heterogenotes, unstable for the cys + marker, and yielded high frequency transducing lysates (10?2 per plaque-forming unit). In phage lysates, plaque-forming transducing particles were directly demonstrated while non-plaque-forming particles were only detected indirectly via complementation of defective lysogenic transductants. In the defective transductants, the two distal ends of the vegetative phage map were lacking. By plasmid transfer, the attachment site of phage 16-3 was located close to the cys-46 and met-5 markers. Transduction of the met-5 marker could not be demonstrated.
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Properties of a Temperature-sensitive Mutant of Staphylococcus aureus Defective in DNA Replication and Cell Division and Replication of Plasmids in the Mutant
More LessThe properties of a temperature-sensitive mutant (ts39) of Staphylococcus aureus NCTC 8235 are described. After transfer to the restrictive temperature (42 °C), absorbance increased 10-to 20-fold but DNA content did not increase beyond 150 to 200% and cell division continued at a greatly reduced rate. On transfer back to the permissive temperature, both cell division and DNA synthesis resumed if the transfer occurred after less than 120 min at 42 °C. Resumption of DNA replication was blocked by chloramphenicol (100 ?g ml?1). The results are discussed with reference to possible defects in DNA replication.
Replication of the plasmids pI258 and pT10501 and the chromosome were affected to a similar extent in ts39. Growth at 42 °C resulted in the appearance of an increased amount of pI258 DNA in a form that sedimented slowly in a sucrose gradient.
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- Immunology
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A Polysaccharide Antigen from the Gram-positive Organism Eubacterium saburreum Containing Dideoxyhexose as the Immunodominant Sugar
More LessA highly active surface antigen, reacting by precipitation and complement fixation, has been isolated from Eubacterium saburreum strain L32. The antigen is a polysaccharide polymer built up of galactose, ribose and dideoxyhexose. The dideoxyhexose is the immunodominant sugar.
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Purification and Partial Characterization of the Serologically Active Substance from Absidia cylindrospora
More LessAffinity chromatography with a concanavalin A-Sepharose column was successfully used to separate the serologically active substance of Absidia cylindrospora from the supernatant of disrupted mycelium fluid, the phenol extract of intact washed mycelium and the culture filtrate. The active substance, a fucomannan-peptide, was isolated homogeneously by means of zone electrophoresis using Pevikon. It was composed of fucose and mannose (94.6%, w/w) in the ratio 1:2.2 and a small amount of protein (1.8%, w/w). It may be bound loosely on the surface of the mycelium.
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- Physiology And Growth
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Effect of Oxygen Concentration, Temperature and Combined Nitrogen on the Morphology and Nitrogenase Activity of Rhizobium sp. Strain 32H1 in Agar Culture
More LessCultures of Rhizobium sp. strain 32H1 grown in a layer of soft agar on top of a layer of harder agar developed maximum nitrogenase activity [80 to 90 nmol acetylene reduced h?1 (mg protein)?1] after 12 d in air at 28 °C. Electron microscopy of sections of cores of the soft agar layer showed differences in the morphology of rhizobia in colonies growing at the surface, in the middle or at the bottom of the layer. The properties of rhizobia growing in the colonies in the middle of the soft agar layer suggested that only these contained nitrogenase. These rhizobia were present as a distinct band of cells passing through individual colonies at a constant depth in the agar. They were larger than normal vegetative Rhizobium, were pleiomorphic and were similar in morphology to the nitrogen-fixing bacteroids formed by strain 32H1 in cowpea root nodules. Their location within the soft agar layer changed with the O2 concentration in the atmosphere above the agar during growth, and they were not found in cultures showing little or no nitrogenase activity. Loss of nitrogenase activity occurred when cultures were grown at 33 °C, in a O2/Ar (1:4, v/v) atmosphere, or when the concentration of combined nitrogen in the agar medium was increased from 2 to 20 mM-N.
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Protein Synthesis, Cell Division and the Cell Cycle in Saccharomyces cerevisiae following a Shift to a Richer Medium
More LessWhen both haploid and diploid cells of the yeast Saccharomyces cerevisiae were shifted to media supporting faster growth rates, rate maintenance of cell division was observed. Both protein synthesis and cell division displayed a variable period of rate maintenance prior to achieving new rates after shift-up; however, the interval between the increase in rate of protein synthesis and the increase in rate of cell division remained constant. These results are consistent with a model of the cell division cycle in which there is an expandable G1 phase during which growth to some critical size must be attained. Once cell division cycle events are initiated they proceed at a constant rate regardless of the overall population growth rate.
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Synchronization of Cell Division in the Fission Yeast Schizosaccharomyces pombe by Ethylenediaminetetra-acetic Acid
More LessWhen cultures of Schizosaccharomyces pombe growing exponentially in a minimal medium were treated with 50 mM-EDTA for 1 h, nucleic acid synthesis was inhibited but protein synthesis was not. On transfer to fresh medium, 50% of the cells divided synchronously. These phenomena may be explained on the basis of reduced availability of Mg2+ following chelation with EDTA.
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Fluoroacetate Metabolism in Gloeocapsa sp. LB795 and its Relationship to Acetylene Reduction (Nitrogen Fixation)
More LessSodium fluoroacetate (1 mM) caused an accumulation of citrate and altered the lipid composition in cells of Gloeocapsa sp. LB795. It also inhibited acetylene reduction (nitrogen fixation) by the alga - markedly under aerobic conditions, but much less so in the absence of oxygen. This inhibition is largely the result of the conversion of fluoroacetate to fluorocitrate which, by inhibiting aconitate hydratase (EC 4.2.1.3), interrupts the synthesis of the 2-oxoglutarate required for the assimilation of NH4 +. The consequent accumulation of NH4 + within the cells of Gloeocapsa sp. inhibits nitrogenase synthesis and, since oxygen rapidly inactivates pre-existing nitrogenase, nitrogen fixation by Gloeocapsa sp. decreases under aerobic conditions.
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In vivo Regulation of NADP-specific Glutamate Dehydrogenase by L-Amides in Stemphylium botryosum
More LessThe activity of NADP-specific glutamate dehydrogenase (NADP-GDH; EC 1.4.1.4) increased at a linear rate of 2.1 x 103 units h?1 (g fresh wt)?1 following the transfer of Stemphylium botryosum mycelium grown in L-asparagine medium to nitrate medium. The maximum enzyme activity was reached after 5 h. De novo synthesis was demonstrated by density labelling of the enzyme with deuterium and inhibition of NADP-GDH synthesis by cycloheximide, p-fluorophenylalanine or 6-methylpurine. L-Asparagine or L-glutamine could serve as a corepressor of NADP-GDH synthesis whereas the D-isomers were ineffective. Of the various amide derivatives tested, only L-asparagine tert-butyl ester could mimic the effect of L-asparagine. Enzyme repression was not correlated with the internal pool of L-amides. After NADP-GDH had been induced to the maximum level, the addition of L-asparagine and cycloheximide resulted in a decrease of activity with half-lives of 4.5 h and 8 h respectively. The mean half-life, as measured by following the decay in specific radioactivity of the enzyme in nitrate medium after administration of 35SO4 2-, was 7 h. Mycelium starved of carbon and nitrogen sources showed a slow decrease (half-life of 17 h) in NADP-GDH activity. Depletion of energy by carbon starvation or the presence of sodium azide did not prevent the decrease in enzyme activity caused by L-asparagine. The decrease in NADP-GDH activity mediated by L-asparagine was inhibited by cycloheximide or ?-iodoacetamide. Sodium azide inhibited the decrease in enzyme activity caused by cycloheximide.
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Origins of Fermentation Products Formed during Growth of Bacteroides ruminicola on Glucose
More LessBacteroides ruminicola grown on complex medium with glucose as carbon source gave acetate, CO2, formate and succinate as main fermentation products. No evidence was found for significant glucose catabolism by pathways other than the Embden-Meyerhof sequence. However, [U-14C]glucose fermentation gave products whose specific radioactivities were much lower than expected. There appear to be two main causes. Firstly, a rapid exchange occurred between metabolic intermediates and CO2, probably due to reversibility of the pathway between phosphoenolpyruvate and fumarate. Secondly, non-glucose precursors, mainly peptides and acetate, added to the medium as growth factors, also gave rise to the above end-products. The distortions that such reactions introduce into measurements of ATP molar growth yields based on product analyses and measurements of carbon flux based on radioactivity recovered in products are discussed.
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- Short Communications
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