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Volume 35,
Issue 2,
1964
Volume 35, Issue 2, 1964
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A Method for the Control of Eh and pH during Bacterial Growth
More LessSummary: A method is described whereby pH and Eh of a medium can be closely controlled during the growth of Clostridium welchii type A for long enough to study the bacteriostatic effects of horse antiserum. The pH value was controlled manually to about ± 0·01 unit (s.d.), by adjustments in the CO2 tension of the gas above the medium. The Eh was controlled by the addition of oxygen to a system with an inherent tendency to become reduced. With manual control the Eh was maintained within ± 2 mV (s.d.); automatic control gave better regulation than this.
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Bacteriostatic Effects of Specific Antiserum on Clostridium welchii type A. The Role of Eh and pH of the Medium
More LessSummary: The bacteriostatic effect of specific antiserum on Clostridium welchii type A was profoundly influenced by the pH and Eh of the medium. With suitable concentrations of specific antiserum relatively high pH and Eh values led to a well-marked inhibition of growth accompanied by destruction of the bacteria. A relatively low pH or Eh led to a decrease or abolition of the inhibitory power of the specific antiserum. Neither complement nor properdin appeared to be involved in the bacteriostatic effect.
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Naturally Occurring Methicillin-Resistant Staphylococci
More LessSummary: Naturally occurring methicillin-resistant strains of Staphylococcus pyogenes isolated in hospitals in Britain, France and Denmark were studied. All strains belonged to a few closely related bacteriophage types and all behaved similarly in the presence of methicillin. The minimum inhibitory concentration of methicillin for the strains ranged from 5 to 100 μg./ml., but on ordinary nutrient agar growth in the presence of concentrations well below this was much less luxuriant than on control plates without antibiotic and tended to be confined to the site of heavy inoculum. Gramstained films from these cultures showed great irregularity in the size and staining of the cocci and many swollen forms were seen, suggesting that methicillin might be inhibiting cell wall synthesis without preventing multiplication. Further work showed that with addition of an excess of electrolytes (5% NaCl or 7·5% (NH4)2SO4) or decrease in agar concentration, growth in the presence of methicillin was almost equal to that on control plates. The addition of uracil to a partially defined medium had no significant effect. Initial incubation under anaerobic conditions also improved growth of these strains in the presence of methicillin.
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Reversion Rate in Continuous Cultures of an Escherichia coli Auxotroph Exposed to Gamma Rays
More LessSummary: Continuous cultures of the tryptophan-requiring strain of Escherichia coli known as wp2 were continuously exposed to gamma radiation for periods of several generation times. The frequency of prototrophic revertants increased steadily during irradiation at a rate which was proportional to the radiation dose rate. The change of revertant frequency per unit dose (rad) was equal to the induced reversion rate per mutable unit. This mutation rate (ρ) was independent of population density but slightly dependent upon the presence of supplements, such as nutrient broth, to the normal minimal medium with tryptophan. There was a marked dependence of ρ upon culture temperature, the values at 16°, 22° and 37° being 1·2 × 10−11, 2·4 × 10−11 and 3·9 × 10−11 per mutable unit per rad. It is probable that only a fraction of radiation-induced changes in the genetic material which could give rise to phenotypic reversions are actually expressed, this fraction being dependent on the post-irradiation temperature. The proportion of the population inactivated by radiation was intentionally kept at 10 % or less in order to avoid difficulties in the interpretation of results. Other possible sources of error in ρ have been reviewed.
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The Composition of Staphylococcus aureus in Relation to the Water Activity of the Growth Medium
More LessSummary: Staphylococcus aureus organisms, grown in basal medium of water activity (aw) 0·993 and in basal medium adjusted to several different aw values by addition of NaCl, were analysed for the following components: sodium, potassium, calcium, magnesium, total amino acids, inorganic phosphate, chloride, water, DNA, RNA, protein. Cell water decreased from 1·66 to 0·83 g./g. dry weight when aw was decreased from 0·993 to 0·90. Internal concentrations of solutes generally increased with decrease in aw, potassium and phosphate concentrations being greatest at 0·92 aw and sodium, chloride, magnesium, and amino acids at 0·90 aw, the lowest aw value studied. Increases in potassium and amino acid concentrations resulted largely from the decreased water contents of cocci grown at low aw values. Intracellular sodium and chloride concentrations were much lower than and proportional to the NaCl concentration in the medium. The predominant cell solute was potassium at 0·92 aw and above, and sodium below 0·92 aw. The data are discussed in relation to the inhibition of bacterial growth at low aw values.
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Activation of Soil Microflora by Fungus Spores in Relation to Soil Fungistasis
More LessSummary: Bacterial numbers in natural soil supplemented with living fungus spores or cell-free aqueous washings of spores, then incubated for 8 hr or more, were several-fold higher than those in non-supplemented soil. Rates of oxygen uptake in natural soil supplemented with washed or unwashed fungus spores, cell-free aqueous washings of spores, killed spores, or diluted nutrients, were several-fold higher than those in non-supplemented soil. Increased respiration occurred rapidly on addition of these supplements to soil. A single brief water washing of urediospores of Puccinia rubigo-vera or of conidia of Neurospora sp. extracted about 10% of the spore dry weight. Washed bacteria or Streptomyces spp., when incubated with fungus spores in absence of added nutrients, inhibited fungus spore germination, whereas sterile filtrates of bacteria were not inhibitory. The results indicate that fungus spores, by virtue of nutrients in their exudates, stimulate rapid activity of microbes in soil, and that the enhanced microbial activity causes inhibition of fungal spore germination.
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Identification of d-Alanine as the Auto-Inhibitor of Germination of Bacillus globigii Spores
More LessSummary: Thick suspensions of spores of Bacillus globigii did not germinate completely in nutrient media because the first spores to germinate released a substance which inhibited germination of the remainder. The inhibitory substance was identified as d-alanine by chromatography, high voltage electrophoresis, and its destruction by d-amino acid oxidase. When 14C-l-alanine was used to induce germination of B. globigii spores, the resulting inhibitory d-alanine was 14C-labelled with the same specific activity as the 14C-l-alanine used. Auto-inhibition of B. globigii spore germination is therefore due to racemization of exogenous l-alanine, which forms sufficient d-alanine to arrest the germination process in thick suspensions of spores.
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Enzymes of the Tricarboxylic Acid Cycle in Acetic Acid Bacteria
More LessSummary: The activities of seven enzymes which catalyse reactions of the tricarboxylic acid cycle were assayed in extracts of five lactaphilic and five glycophilic strains of acetic acid bacteria. Except for isocitrate dehydrogenase (which was not detected in glycophilic extracts) all other enzymes were found in all strains; but in general these enzymes were much more abundant in the lactaphilic extracts. In particular, extracts of glycophiles possessed only feeble citrate synthase, aconitate hydratase, fumarate hydratase and l-malate dehydrogenase activities and only their 2-oxoglutarate and succinate dehydrogenase activities were comparable to the corresponding activities in extracts of lactaphiles. Oxaloacetate decarboxylase activity was also greater in lactaphiles than in glycophiles. Two enzymes which oxidized l-malate were found: that in Acetobacter acidum-mucosum was an NADP-linked dehydrogenase, while the other more generally distributed enzyme required no added co-factors and may be cytochrome-linked. The evidence indicates that the tricarboxylic acid cycle may make a greater quantitative contribution to the metabolism of lactaphilic than to that of glycophilic organisms.
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Action of the Polyene Antibiotics Filipin, Nystatin and n-Acetylcandidin on the Yeast Cell Membrane
More LessSummary: The effects of the antifungal polyene antibiotics n-acetylcandidin, nystatin and filipin on the yeast cell membranes were compared with the reversibility by K+ and NH4 + of the inhibition of glycolysis caused by these antibiotics. The results confirm the existence of two functional classes of polyenes which correspond to the number of carbon atoms in the molecule; intermediate types of action can be illustrated within the group of large polyenes. The inhibition of yeast glycolysis by n-acetylcandidin, a large polyene, was annulled by K+ and NH4 +, even at high antibiotic concentrations; inhibition by filipin, a small polyene, was not so annulled. The inhibition of glycolysis by polyenes was associated with the loss of cell K+; filipin caused concurrent loss of inorganic phosphate and of accumulated sugar (l-sorbose) and initiated ATPase and pyruvate decarboxylase activity. Although n-acetylcandidin treatment did not produce loss of accumulated sorbose, added sorbose leaked into treated cells when uptake by way of the hexose transport system had been blocked (by glucose). Thus, definite membrane damage had occurred. Nystatin, a large polyene, was intermediate in effect: at low concentrations its action approached that of n-acetylcandidin; at high concentrations, its action was similar to that of filipin. It is suggested that the polyenes present a full spectrum of effects which relate to the degree of physical damage to the cell membrane. They range from filipin which destroys general structural integrity of the membrane even at the minimum concentration necessary to inhibit glycolysis (and growth) to n-acetylcandidin which produces only minimal damage so that relatively specific defects are observed, e.g. those related to K+ loss and sorbose leakage.
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The Germination of Sporangiospores of Rhizopus arrhizus; Spore Swelling and Germ-Tube Emergence
More LessSummary: The germination of sporangiospores of Rhizopus arrhizus was investigated, and differing requirements found for the initiation of germination, spore swelling and germ-tube emergence. The initiation of germination, as indicated by the commencement of swelling and by the spores becoming permeable to methylene blue, requires the presence of glucose or fructose. Maximal spore swelling requires in addition the presence of a nitrogen source, PO4 3− and K+ or Na+. If these requirements are satisfied, the increase in spore diameter with time is approximately linear for 8 hr, implying the maintenance of a constant rate of water uptake per unit area of spore surface for this period. If germinating spores are transferred to a medium lacking glucose, swelling soon ceases. The rate of swelling is identical at widely differing osmotic pressures. It is suggested that water uptake by the germinating spore is an active process, requiring energy. Germ-tube emergence from some spores can be obtained with glucose alone, but for germ-tube production from all spores other nutrients must also be supplied. If glucose is present, spores can take up sufficient nutrients in 1–2 hr to permit complete germination in the absence of exogenous nutrients several hours later. Depending on conditions, germ-tube production can occur after either slight or massive spore swelling. The effect of anaerobic conditions on germination was also examined and found to permit only partial spore swelling and greatly diminished germ-tube production.
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Some Properties of a New Bacteriocin Formed by Bacillus megaterium
More LessSummary: A new bacteriocin, megacin C, was recognized in certain strains of Bacillus megaterium; it was initially observed as the agent responsible for killing sensitive organisms in mixed culture. The bacteriocin was liberated during growth of cultures; but later, after reaching a maximum value, it disappeared from the culture media. Megacin C was produced by strains already known to produce another bacteriocin, megacin A. The activity spectra of the two megacins differed markedly and non-megacin A (MA −) mutants still produced megacin C. A preliminary grouping of megacin types is proposed. Megacin A is comparable to the original megacin first studied by Ivánovics & Alföldi (1954). Megacin A produced inhibition zones surrounding colonies on solid medium but its formation in liquid medium required induction with ultraviolet (u.v.) radiation. Megacin B, whose activity spectra differed from those of megacins A and C, can be detected only on a solid medium; it is not formed in liquid culture even after u.v.-irradiation. Megacin C can be detected only on a particular solid medium and its formation in liquid media is not induced by u.v.-irradiation.
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Bacteriophage Sensitivity and Biochemical Group in Xanthomonas malvacearum
More LessSummary: Isolates of Xanthomonas malvacearum, of which 18 representative cultures were examined in detail, were classified into two groups differing in: capacity to oxidize lactose, colony form on first isolation, proteolytic activity, and lysis by bacteriophages. The seven group 1 isolates examined in detail gave confluent lysis at routine test dilution by 7 of 13 phages isolated from diseased cotton plant material, and were weakly proteolytic and did not oxidize lactose in common with other group 1 isolates. All but one of the 11 group 2 isolates gave confluent lysis at RTD by the 6 phages to which the group 1 isolates were resistant but not by the other 7 phages. All isolates of group 2 oxidized lactose and were relatively strongly proteolytic. Phages active against X. malvacearum were detected in 87 out of 127 collections of infected leaves examined, and there was some correlation between the presence of groups 1 or 2 xanthomonads and the group-specificity of the phage isolated from the same material. An isolate of group 2 X. malvacearum which was resistant to some of the group 2-specific phages was apparently lysogenic.
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Temperature Sensitivity of Mutants of Bacillus anthracis Caused by a Block in Thymine-Nucleotide Synthesis
More LessMutants were isolated from the non-capsulated strain Vollum of Bacillus anthracis which were unable to grow above 34° in the absence of certain pyrimidines. At elevated temperatures, one of the mutants, YC–TdR–, was found to be dependent on thymidine while the other, VC–T–, required thymine. Both mutants, however, grew normally in the absence of pyrimidines at near to room temperature. A relatively high concentration of thymine was needed in order to overcome the thymidine requirement of mutant YC–TdR– at 37°, whereas a combination of a low concentration of thymine with different deoxyribosides (deoxyadenosine, deoxyguano-sine, deoxycytidine) gave good growth of the mutant. This observation is suggestive of the presence of a particular enzyme, trans-N-deoxyribosylase. in the mutant VC–TdR–, an enzyme which appears to be of limited distribution in nature. The second mutant. VC–T–, utilized added thymine readily at 37° and the base could not be substituted by its nucleoside, thymidine. In fact, thymidine and deoxyribonucleosides inhibited the growth of mutant VC–T– in the presence of thymine.
Both mutants also grew well at 37° in the presence of thymidine-5-phosphate, which indicated that the de novo pathway of pyrimidine synthesis is blocked above 34° somewhere in the pathway between deoxycytidine-phosphate and thymidine-5-phosphate. This block in the pyrimidine synthesis occurring at elevated temperatures caused an unbalanced synthesis of macromolecules accompanied by an abnormal cell-wall formation. At 37°, germinated spores showed an abnormal elongation of the initial cell concomitant with a gradual loss of viability. At this temperature cell-wall formation was also abnormal at limiting concentrations of pyrimidines and minor deficiencies in cell-wall structure of the mutants were still apparent even in the presence of a large excess of pyrimidine. This, however, did not involve any change in virulence of mutant VC+TdR– in the homoiothermic mouse.
It is assumed that the mutants produce an altered enzyme protein corresponding to a block in de novo synthesis of pyrimidine, or that an inhibitor is produced at high temperatures which diminishes and finally prevents the action of the normal enzyme.
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Lysozyme Lysis of Gram-Negative Bacteria without Production of Spheroplasts
More LessSummary: Cells of Gram-negative bacteria undergo lysis when treated with lysozyme in the presence of ethylenediaminetetraacetic acid (EDTA) and tris buffer, as shown by Repaske. However, contrary to the prevalent assumption, lysis is not necessarily preceded by formation of a spheroplast as the cell wall is damaged. Treatment of Escherichia coli and Pseudomonas aeruginosa with the lytic system was shown to cause the formation of osmotically fragile rods, rather than spheres. The extent of destruction of the cell walls of Gram-negative bacteria by lysozyme in this system is, at least in some cases, less than has been generally supposed.
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Regulatory Mechanisms Governing Synthesis of the Enzymes for Tryptophan Oxidation by Pseudomonas fluorescens
More LessSummary: Both co-ordinate and sequential inductions govern the synthesis of the enzymes required for the oxidative dissimilation of l-tryptophan by a strain of Pseudomonas fluorescens. The first two enzymes of the sequence, tryptophan pyrrolase and formylkynurenine formamidase, are induced co-ordinately by l-kynurenine, the product of their successive action on l-tryptophan; l-trytophan itself is not an inducer. Both these enzymes are present at low concentrations in uninduced organisms, so that inducer is generated endogenously when such organisms are exposed to an exogenous supply of l-tryptophan. l-Kynurenine also induces formation of the third enzyme of the sequence, kynureninase, which is not detectable in uninduced bacteria. Although it is elicited by the same inducer, synthesis of kynureninase is not co-ordinate with the syntheses of pyrrolase and formamidase; the induction of kynureninase can accordingly be considered as the first sequential inductive step in the pathway. It is immediately followed by another sequential inductive step: the synthesis of anthranilic acid oxidase, elicited by anthranilic acid, which is formed from kynurenine through the action of kynureninase.
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Persistence of Staphylococcus aureus in Penicillin in vitro
More LessSummary: After exposure of Staphylococcus aureus strain Oxford (h) to penicillin in vitro under conditions leading to the death of at least 99·9% of the exposed population, the offspring of surviving cocci (‘persisters’) showed the same sensitivity as the original population. Successive exposure of the offspring of persisters did not increase the proportion of survivors nor their resistance. Persisters were not the most resistant cocci in the original population; their offspring showed the same distribution of cocci resistance as the parent culture. The number of survivors, about 1 in 2500 cocci, was proportional to the original number when exposed to penicillin in a small volume (10–20 ml.), but not in a larger volume (40-200 ml.). Inocula of less than 10 staphylococci allowed to multiply to 103/ml. before addition of penicillin yielded persisters. Populations greater than about 2 x 108/ml. were not acted upon by penicillin, even when the majority of the cocci were only 4 hr old, unless the mixtures were shaken. The proportion of survivors at 37° from equal volumes of penicillin broth containing 105–108 organisms/ml. was not affected by the age and growth phase of the exposed bacteria, conditions of previous storage of the inoculum, initial exposure at low temperature, type of container, aeration by shaking, clumping of cocci, a 1000-fold increase in the dose of penicillin, or addition of streptomycin. When removed from penicillin, persisters and their offspring multiplied at the same rate as the parent culture without requiring a recovery period. The persisters were neither spheroplasts nor L forms. Indirect evidence indicates that persisters survived because at the time of first contact with the penicillin they were in a state unfavourable to initiation of division or cell wall synthesis.
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Cellular Factors Affecting Nitrogen Fixation in the Blue-Green Alga Chlorogloea fritschii
P. Fay, H. D. Kumar and G. E. FoggSummary: Synchronous cultures of a nitrogen-fixing blue-green alga, hitherto known as Chlorogloea fritschii but more probably an anomalous species of the genus Nostoc, were obtained by a combination of light and temperature treatments. Variation in dimensions, dry weight, pigment content and total nitrogen content of cells was followed during the development of synchronous cultures. The nitrogen-fixing activity was greatest in the small-celled filaments which develop from the endospores and predominate during exponential growth of the alga in cultures of limited volume. Strains of the alga produced by repeated exposure to X-rays, ultraviolet radiation, or sublethal concentrations of colchicine or urethane, were found to have lower rates of nitrogen fixation per unit dry weight than the original strain but liberated relatively more extracellular nitrogenous products.
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