- Volume 39, Issue 2, 1965
Volume 39, Issue 2, 1965
- Articles
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Streptomycin Action and Anaerobiosis
More LessSUMMARY:The question was asked whether the insensitivity to dihydrostreptomycin and streptomycin exhibited by facultatively anaerobic organisms growing strictly anaerobically could be due entirely to the lack of an anaerobic mechanism for the uptake of these antibiotics. A technique is described here which allows exposure of Escherichia coli b to dihydrostreptomycin, under conditions which are known to promote its intracellular accumulation, for a time, followed by further incubation in a growth medium under aerobic as well as under strictly anaerobic conditions, after removal of the extracellular antibiotic. With this technique it was possible to make a quantitative comparison of the inhibitory effects of dihydrostreptomycin on aerobic and anaerobic growth of E. coli b under conditions where no further uptake of the antibiotic could occur. The results show that for any given treatment of E. coli b with dihydrostreptomycin, subsequent aerobic and anaerobic growth rates are inhibited to exactly the same extent. It is concluded that the need for aerobic metabolism in the expression of antibiotic effect by dihydrostreptomycin is concerned only with its uptake into the organisms.
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Effects of Dihydrostreptomycin Treatment on the Growth of Escherichia coli after Removal of Extracellular Antibiotic
More LessSUMMARYThe growth characteristics of Escherichia coli b cultures treated with dihydrostreptomycin and then freed from extracellular antibiotic before growth had completely stopped, were examined. Growth, measured by extinction, proceeded exponentially, but at slower rates for a time, followed by gradual recovery. The degree of slowing of growth rate was a function of the duration of growth in the presence of a given concentration of dihydrostreptomycin. Comparison of viable colony count data and microscopic observation of such treated cultures showed that the majority of individuals in the populations must grow at the lower rates for two-three generations, after which some organisms cease to multiply and the rest recover. The proportion of organisms in treated populations which eventually ceased to grow was also a function of the duration of treatment. The amount of growth (cell synthesis), which had occurred at the time when onset of recovery became measurable, varied inversely with the % inhibition of growth rate. This suggests that recovery was due to some process not inhibited during the phase of inhibited exponential growth. It is concluded that intracellular dihydrostreptomycin consists of an ‘inhibitory fraction’ at the sites of inhibition, and a non-inhibitory ‘pool’ fraction; that the size of the latter varies between different individuals within a population and that transfer from ‘pool’ to inhibitory sites occurs by a process other than equilibration; e.g. that the factors which govern the uptake into these two phases must be, at least partly, independent. It is suggested that the degree of inhibition of growth rate reflects the extent of combination between antibiotic and inhibition sites at the time when extracellular dihydrostreptomycin is removed and no further uptake into the organisms can occur, and that the complex between dihydrostreptomycin and inhibition sites cannot dissociate to give active antibiotic which could re-enter the ‘pool’.
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A Biochemical Characterization of Histidine-Dependent Mutants of Staphylococcus aureus
More LessSUMMARY:Over 100 histidine-dependent mutants were isolated from strain 655 of Staphylococcus aureus. Paper chromatography was used to differentiate the mutants into classes according to their ability to accumulate Pauly-positive imidazoles. Classes E and G did not accumulate any detectable Pauly-positive imidazoles. Mutants of class A accumulated 5-amino-1-ribosyl-4-imidazolecarboxamide, mutants of class B accumulated imidazoleglycerol, and mutants of class C accumulated imidazoleacetol. Mutants of class D accumulated histidinol and were incapable of utilizing exogenous l-histidinol. A comparison of the accumulations obtained from mutants of S. aureus with accumulations obtained from previously characterized mutants of Salmonella typhimurium indicates that a similar, if not identical, pathway for the biosynthesis of l-histidine is used by these two species.
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Transduction Analysis of the Histidine Region in Staphylococcus aureus
More LessSUMMARYThe genetic control of histidine biosynthesis in Staphylococcus aureus was examined by an analysis of 66 histidine-dependent mutants by using stable and abortive transduction. These mutants, which previously had been differentiated into classes according to their biochemical characteristics, all occupied sites within a single linkage group, referred to as the histidine region. This region has been separated into six gene loci on the basis of complementation studies and the biochemical characteristics of the mutants. The order of genes within the histidine region, E, A, B, C, D, G, was determined from the results of reciprocal transductions to wild type and donor type. The order of mutant sites was also linearly related to the ability of these mutants to form histidine-independent transductants when infected with phage prepared on the parent strain. This observation, and the inequalities in reciprocal transduction frequencies obtained in inter-mutant transductions, support the hypothesis that all donor fragments which participate in transductions involving the histidine region are identical, and that the histidine region is located extremely close to one terminus of the donor fragment. Intergenic complementation occurred among mutants of all gene loci; in addition, three complementation units within the A gene and two complementation units within the G gene were detected.
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The Transfer of Two Episomes, Colicinogenic Factor I and Resistance Transfer Factor, in Shigella flexneri Strains by Crosses Between Strains each Possessing a Single Episome
More LessSUMMARYIt was shown that bilateral transfer of two episomes col I and RTF, carried singly in different strains of Shigella flexneri, could occur at the same time. The transfer of col I occurred at a considerably higher frequeney than did the transfer of RTF. Whilst the presence of col I in the acceptor organism appeared not to affect the frequency of transfer of RTF it was observed that, in some cases at least, the presence of RTF in the acceptor organism lowered the frequency of col I transfer.
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‘Substrate-Accelerated Death’ of Aerobacter aerogenes
More LessSUMMARY‘Substrate-accelerated death’ ( Postgate & Hunter, 1963a , 1964 ) was observed with carbon-limited but not ammonium-, phosphate- or sulphate-limited Aerobacter aerogenes grown at 37° in defined medium and starved at 37° in aerated saline buffers containing the growth-limiting substrate. Carbon sources besides the one limiting growth increased the death-rate of starved mannitol-, glycerol-, galactose- and ribose-limited bacteria. Glycerol-accelerated death depended on the rate of oxidation of glycerol and the bacterial concentration; with bacteria fully adapted to glycerol, populations of less than 1–2 × 109 organisms/ml. died at a faster rate the denser the population and above this concentration the death-rate decreased with increasing bacterial concentration. Death was delayed when aerated bacterial suspensions containing glycerol were dialysed at 37° against saline buffer containing the substrate. Bacteria-free filtrates, from populations dying in the presence of glycerol, accelerated the death of fresh bacteria to a greater extent than did glycerol alone. In contrast, bacteria-free filtrates from dense populations surviving in the presence of glycerol partially protected fresh bacteria exposed to glycerol. Mg2+ abolished glycerol-accelerated death but not the lethal effect of filtrates from dying populations. Compared with its influence on glycerol-accelerated death, population density had much less influence on the death-rate of glycerol- or mannitol-limited organisms starved in the presence of glucose or mannitol. Irrespective of bacterial concentration, α-ketoglutarate had no effect and pyruvate, citrate, malate, succinate and oxalacetate had less effect than glycerol on the death-rate of starved glycerol-limited bacteria.
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Factors which Affect the Release of Newcastle Disease and Sendai Viruses from Infected Allantoic Cells
More LessSUMMARYSome aspects of the adsorption and release of Newcastle disease and Sendai viruses were studied in excised pieces of allantois of uniform size. About 40% of any saturating dose of either virus was adsorbed. The % adsorption was not influenced by treatment with neuraminidase. One-step growth curves indicated that the release process for both viruses was linear. The rate of release and the burst size were influenced by the composition of the medium used for maintaining the allantoic cells. Increases in the multiplicity of infection decreased the length of the latent period. Treatment of allantoic cells with suitable doses of actinomycin D or ultraviolet irradiation also affected the length of the latent period. These agents may increase the susceptibility of cells to infection by affecting DNA-directed mechanisms concerned in the control of nucleic acid synthesis.
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Relation of Biochemical Mutations to Actinomycin Synthesis in Streptomyces antibioticus
More LessSUMMARYBiochemical mutants of Streptomyces antibioticus were isolated and tested for their capacity to produce actinomycin. The yield of actinomycin produced in minimal medium plus the required amino acid, by strains requiring an amino acid not present in the molecule of actinomycin, was not significantly different from that obtained from the wild-type strain. On the other hand, all the strains which required an amino acid, which was also a precursor of the antibiotic, showed on minimal medium a drastic decrease in the production of actinomycin. The results have been interpreted by assuming a different utilization by the cell of exogenous and endogenous amino acid pools for antibiotic and protein synthesis.
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A Co-precipitation Method for the Preparation of Transforming DNA from Small Samples of Low Density Bacterial Cultures
More LessSUMMARYA method is described for the preparation of transforming DNA from 1 ml. samples of pneumococcal cultures of low density (e.g. about 107 bacteria). It seemed likely that the precipitation of very small amounts of transforming DNA from lysates of dilute pneumococcus cultures might result in loss of DNA unless a suitable co-precipitant was added. With high dilutions of transforming DNA, it was confirmed that such a loss was obtained. This loss was largely prevented by the addition of sodium hyaluronate in the presence of citrate. Dextran was not as efficient as hyaluronate as a co-precipitant.
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Growth and Characterization of the Virus of Bovine Malignant Catarrhal Fever in East Africa
More LessSUMMARYA strain of bovine malignant catarrhal fever virus (MCF) recovered from the blood of a blue wildebeest was developed by passage in vitro to a stage where it could be propagated serially in primary thyroid cell cultures by inoculation of cell-free fluids. Released virus titres ranged from 103·8 to 105·8 50% tissue culture infectious doses/ml. This virus still caused fatal disease when inoculated to cattle, and was neutralized by antibody that appeared in the sera of cattle recovering from experimental infection. The principal cytopathic effects of the virus were the development of DNA-containing intranuclear inclusions and syncytia; the inclusions became increasingly basophilic as they matured. The cytopathic effects were inhibited in the presence of 5-iodo-2′-deoxyuridine (IUDR), and infectivity of the virus was abolished by treatment with ether or chloroform. Electron microscopy of inoculated cell cultures showed intranuclear, cytoplasmic and extracellular herpes-like virus particles. Suspensions of cell-free virus examined by negative-stain electron microscopy contained some particles of diameter 140 mμ–220 mμ, comprising an outer envelope and a central body or capsid; others consisted of only a naked capsid about 100 mμ in diameter. MCF virus is evidently a member of the herpes group, and has particular affinities to a subgroup which contains the agents of varicella, herpes zoster and the cytomegaloviruses.
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Comparative Counts of Bacterial Nuclei Made Visible by Four Different Techniques
More LessSUMMARYFour different methods were used to make visible the nuclei of Escherichia coli strains b and wp 2, and Bacillus subtilis. They were: (1) Giemsa staining after acid hydrolysis; (2) fluorescence microscopy after acridine orange treatment; (3) acriflavine staining and phase-contrast microscopy; (4) phase-contrast observations with high refractive index mounting medium (polyvinylpyrrolidone). Statistical analyses showed small but significant differences in nuclear counts between the methods. Methods 1 and 2 were somewhat preferable to methods 3 and 4 in that 1 and 2 were easier to score and there was less difference between observers. It was calculated that a single mean nuclear count, by any observer or method, based on 200 scored bacteria should 19 times out of 20 estimate the ‘true’ mean nuclear number of E. coli wp 2 to within about ± 7.5% and of B. subtilis and E. coli b to within about ± 12%. By increasing the number of bacteria counted and the number of methods used these limits could be appreciably decreased.
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The Effect of Chloramphenicol on the Growth of Scenedesmus quadricauda
More LessSUMMARYThe effects of different concentrations of d (–) threo and l (+) threo-chloramphenicol on the growth of Scenedesmus quadricauda have been studied. The results indicate: (a) both isomers caused an increase in the total population largely independent of concentration; (b) at concentrations between 100–200 μg./ml. both isomers gave a secondary increase in growth after a secondary lag period of 4–6 days; (c) both isomers inhibited the growth rate at concentrations comparable with those which inhibit bacterial growth and protein synthesis. The l (+) threo isomer was twice as effective as the d (–) threo isomer, though it did not give any inhibition below 22 μg./ml. (d) above 50 μg./ml. the d (–) threo isomer gave a significantly greater lag period than below this concentration. The l (+) threo isomer did not affect the lag period below 25 μg./ml., but above this concentration, an increase in the concentration lengthened the lag period at the same rate as with the d (–) threo isomer above 50 μg./ml.
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Uptake of 3H-Griseofulvin by Micro-organisms and its Correlation with Sensitivity to Griseofulvin
More LessSUMMARYA correlation was observed between the sensitivity of different microorganisms to griseofulvin and their ability to take up the antibiotic, especially into their nucleic acid and protein fractions. The insensitive yeasts Candida albicans and Saccharomyces cerevisiae and the bacterium Escherichia coli did not bind an appreciable amount of [4-methoxy-3H] griseofulvin. The poorly sensitive filamentous fungi Aspergillus niger and Neurospora crassa accumulated a considerable quantity of antibiotic, mostly in the water-soluble pool. This was in contrast to the highly sensitive dermatophytes Microsporum gypseum, Trichophyton mentagrophytes and T. persicolor in which the nucleic acid and protein fraction contained about half of the total bound griseofulvin (as relatively stable complexes). It is proposed that uptake of griseofulvin is essential for antibiotic action and that the degree of sensitivity shown by an organism is dependent upon the tendency of its macromolecules to complex with the accumulated griseofulvin. Griseofulvin was not degraded by the insensitive or the poorly sensitive organisms tested. Metabolic products of griseofulvin were detected, however, in culture fluids of the sensitive dermatophytes.
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