- Volume 91, Issue 2, 1975
Volume 91, Issue 2, 1975
- Biochemistry
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Leghaemoglobin and the Supply of O2 to Nitrogen-fixing Root Nodule Bacteroids: Presence of Two Oxidase Systems and ATP Production at Low Free O2 Concentration
More LessSUMMARY: Studies of rates of consumption of dissolved O2 by suspensions of bacteroids (Rhizobium japonicum, strain cb1809) from soybean root nodules showed the presence of two different terminal oxidase systems. A high-affinity system, sensitive to inhibition by N-phenylimidazole and by carbon monoxide, was most active when the dissolved O2 was between 0·01 and 0·1 μ m. At 1 μ m-O2 or higher, this oxidase system had little activity and O2 was consumed largely by a low-affinity system insensitive to these inhibitors. At low concentrations of dissolved O2, bacteroid respiration rates appeared to be diffusion-limited. When purified oxyleghaemo-globin was added to such systems, this restriction was relieved and respiration was maintained to much lower concentrations of free dissolved O2 here nitrogenase activity was greatest.
Analysis of reactions which were terminated at various stages during the depletion of O2 from oxyleghaemoglobin showed that at low free O2 concentration, the high-affinity pathway produced up to five times greater bacteroid ATP concentrations than the low-affinity oxidase pathway operating about 1 μ m free O2 in the absence of leghaemoglobin. At intermediate free O2 concentrations, ocfnring during the later stages of deoxygenation of oxymyoglobin, intermediate concentrations of ATP were found in the bacteroids.
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Stability of Protein and Ribonucleic Acid in Bacillus stearothermophilus
More LessSUMMARY: The turnover of protein in a prototrophic strain of Bacillus stearothermophilus during exponential growth in a salts medium with glucose or succinate as carbon source was about 4 %/h and in a richer nutrient broth medium about 23 %/h. Protein degradation under non-growing conditions conformed to a similar pattern. The turnover of RNA (non-messenger) was about 1 %/h in salts medium and about 9 %/h in nutrient broth. The turnover of protein and RNA in the thermophile is thus moderate rather than massive. This conclusion was confirmed by measurement of the decay of a specific enzyme, isocitrate lyase, in the proto-troph and of the overall protein turnover in a non-prototrophic strain of B. stearothermophilus. The half-lives of a number of enzyme systems in intact cells of the prototrophic thermophile at its optimum growth temperature showed some variation but indicated a significant rate of inactivation. Such decay of protein in vivo apparently accounts for the moderate protein turnover observed during growth.
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Distribution of β-Lactamases A and B in some Groups of Yersinia enterocolitica and their Role in Resistance
More LessSUMMARY: Yersinia enterocolitica W222 (serological group 3) synthesized two different intracellular β-lactamases, called A and B. Enzyme B was more sensitive than A to inhibition by cloxacillin. The minimum inhibitory concentrations of various β-lactam antibiotics for strains of Y. enterocolitica of groups 3 and 9 and the effect of cloxacillin on these concentrations suggested differential roles for β-lactamases of types A and B in penicillin and cephalosporin resistance. Type B enzymes protected Y. enterocolitica against cephalothin and cephalosporin C, whereas type A enzymes protected very efficiently against carbenicillin. Protection against other β-lactam antibiotics was exerted by both enzymes. However, while both enzymes readily hydrolysed cephaloridine and showed no crypticity with this substrate, they only conferred a very weak protection against it. This may be because cephaloridine reached its target quickly, before it was degraded. The resistance of strains of Y. enterocolitica from groups 1, 2 and 16 was also explicable in terms of a two-enzyme system, whereas strains belonging to group 5b produced only a type B lactamase and were sensitive to carbenicillin.
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Properties of some Bacteriocins Produced by Rhizobium trifolii
More LessSUMMARY: Bacteriocins produced by six strains of Rhizobium trifolii were found to be of the relatively low molecular weight, non-phage type. The molecular weights ranged from approximately 1·8 105 to 2·0 × 105. All were of protein composition, as indicated by buoyant density (1·32 to 1·34 g/cm3) in CsCl and by sensitivity to proteolytic enzymes. They were resistant to RNAase but sensitive to DNAase. The six bacteriocins could further be separated into two subgroups on the basis of sensitivity to extremes of pH, binding to filter membranes, activity spectrum on sensitive strains of R. trifolii, and possibly mode of action on sensitive bacteria. Bacteriocin production ocfnred spontaneously during the early-to mid-exponential phase of bacterial growth in broth culture.
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- Development And Structure
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Enzyme Activity Changes during Cyclic AMP-induced Stalk Cell Differentiation in p4, a Variant of Dictyostelium discoideum
More LessSUMMARY: The P4 variant of Dictyostelium discoideum is characterized by the production of fruiting structures in which the overall proportion of stalk to spore material is increased, relative to the wild type. The altered morphology of the mutant is due to increased sensitivity to cyclic AMP which promotes stalk cell differentiation. In the presence of 10−4 m-cyclic AMP the entire population of P4 amoebae forms clumps of stalk cells on the surface of the dialysis membrane support.
Measurement of changes in activity of a range of developmentally-regulated enzymes during the development of P4 in the presence and absence of cyclic AMP has allowed us to identify three classes of enzyme: (i) Those, such as β-glucosidase II, trehalose-6-phosphate synthetase and uridine diphosphogalactose-4-epimerase, which are required for the production of spores, (ii) Enzymes, primarily but perhaps not exclusively, required during stalk cell formation. Typical of these are N-acetylglucosaminidase and alkaline phosphatase. (iii) General enzymes, such as threonine dehydrase, α-mannosidase and uridine diphosphoglucose pyrophosphorylase, which are present in both pre-stalk and pre-spore cells and appear to be necessary for the development of both cell types.
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Hyphal Wall Growth in Neurospora crassa and Geotrichum candidum
More LessSUMMARY: Growth of the walls of hyphae of Neurospora crassa and Geotrichum candidum was studied using longitudinal and serial transverse sectioning methods. Rigidification of the hyphal wall below the extension zone did not appear to involve the gross formation of a secondary wall since the transition from extensible to non-extensible wall was not associated with an increase in thickness. However, behind the extension zone the walls of leading hyphae of N. crassa increased in thickness until eventually they attained a thickness which was up to five times that of the tip wall. A hypothesis of hyphal wall growth is proposed.
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Morphology and Growth Kinetics of Hyphae of Differentiated and Undifferentiated Mycelia of Neurospora crassa
More LessSUMMARY: A comparison was made of the morphology and growth kinetics of hyphae of differentiated and undifferentiated mycelia of Neurospora crassa. Undifferentiated mycelia were formed during exponential growth on solid media or submerged culture. Hyphae at the margin of differentiated mycelia (colonies) differed from undifferentiated mycelia in diameter, extension rate, extension zone length, and intercalary and apical compartment length. The mean hyphal extension rate (E) of an undifferentiated mycelium was a function of the length of the mycelium’s hyphal growth unit (G) and the organism's specific growth rate (α). Thus, E=Gα.
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- Ecology
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Ureolytic Bacteria in Sheep Rumen
More LessEstimates were made of the numbers of viable bacteria in the rumens of sheep receiving different rations. Representative colonies were isolated and tested for urease production. Some urease-positive isolates were characterized and identified. The ureolytic activities of the urease-producing isolates were determined and compared with the activity of rumen fluid. The rations fed to the sheep did not exert a significant influence on the relative numbers of the urease-producing organisms in the rumen. No obligately anaerobic ureolytic bacteria were found. All urease-positive isolates were facultatively anaerobic, Gram-positive, catalase-positive cocci. Out of ten isolates, nine were identified as Staphylococcus saprophytics and one as Micrococcus varians. The total urease activity of the different isolates based on the lowest numbers in which they were present in the rumen, compared favourably with the urease activity of rumen fluid. The facultatively anaerobic Grampositive cocci were probably responsible for a large proportion of the urease activity of the rumen fluid. Conditions prevailing in the rumen were found to be conducive to urease production by the isolates tested.
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- Genetics And Molecular Biology
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Genetical and Biochemical Aspects of Resistance to p-Fluorophenylalanine in Saccharomyces cerevisiae
More LessSUMMARY: Growth of haploid yeast strains was inhibited by the phenylalanine (PA) analogue dl-p-fluorophenylalanine (FPA) in yeast extract media containing 0·2 mg PA/ml. Most strains had a maximum FPA tolerance of about 0·25 mg/ml when glycerol was the carbon source and 0·5 mg/ml in glucose medium. Spontaneous FPA-resistant mutants isolated on glucose medium showed little or no increase in FPA tolerance over that of the parent when metabolizing glycerol. Resistance was controlled by a different nuclear gene in each of four mutants analysed. In a proportion of the mutants the amount of FPA incorporated into cellular proteins in competition with PA was less than into the proteins of sensitive parental cells, whether glucose or glycerol was used as carbon source. This suggests that the mutational change allowed the cytoplasmic system to discriminate against the analogue without affecting its incorporation into mitochondrially-synthesized proteins. Although attempts to measure the latter were not made, the observed decrease in respiratory activity of cells grown in the presence of FPA suggests such incorporation. In other mutants showing resistance to FPA in glucose medium, the amount of FPA incorporated into cellular proteins varied with the carbon source, less analogue being incorporated in glucose medium than in glycerol medium.
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Analysis of an l-Histidinol-utilizing Mutant of Pseudomonas aeruginosa
More LessSummary: Transductional analysis was applied to the Pseudomonas aeruginosa mutant pa014 (hnc-1). This mutant can utilize l-histidinol as sole source of carbon and nitrogen and has a 60-fold increased histidinol dehydrogenase (HDH) content ( Dhawale, Creaser & Loper, 1972 ). Transductional analysis was carried out using 18 histidine-requiring mutants to see where the hnc-1 locus maps in relation to the structural genes of histidine biosynthesis. The hnc-1 marker cotransduced with group IV genes at 97 to 100 % and not at all with group I, which is known to be the structural gene for HDH.
The data obtained in the studies of K m (histidinol) and K m (NAD), and the effect of pH and temperature on the HDH activity from pa01 and pa014 are in full agreement with the genetic data that the hnc-1 mutation is not in the structural gene for HDH. It is suggested that hnc-1 may be a mutation in a regulatory gene affecting HDH synthesis in pa014 and may map close to his-IV whose function in histidine biosynthesis is not known.
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Hybridization and Selection for Increased Penicillin Titre in Wild-type Isolates of Aspergillus nidulans
More LessSUMMARY: Repeated hybridization and selection among wild-type isolates produced strains of Aspergillus nidulans with increased penicillin titre. Four independent selection lines were established, each originating from a sexual cross between two different heterokaryon-incompatible wild-type isolates. In each generation, two selected high-titre sister strains were crossed to produce the next generation. An initial increase in titre was obtained in each line, but after four or five generations of selection the genetic variation was considerably reduced and the rate of response to selection had decreased. From a base population of wild-type isolates with a mean titre of 8·6 units/ml the progeny mean titre was raised to between 16 and 20 units/ml in each line. The gradual nature of the response suggests that a number of genes determine penicillin titre in the wild-type isolates used. The gene action throughout the selection programme was predominantly additive.
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The Inheritance of Penicillin Titre in Crosses between Lines of Aspergillus nidulans Selected for Increased Productivity
More LessSUMMARY: Selection for increased penicillin production among the progeny of pairwise crosses between wild-type isolates of Aspergillus nidulans resulted in the production of high-titre strains. Three crosses were made between strains with increased titre derived from independent selection lines. Significant genetic variation was found among the progeny of each cross, indicating that different genes for increased titre had been selected in each line. Gene action was additive in each cross. Renewed selection from the progeny of one ‘between-line’ cross resulted in further increases in titre, over three successive generations.
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The Induction of Mutants of Acinetobacter calcoaceticus ncib8250 and their Selection by Vancomycin
More LessSUMMARY: The mutagenic and lethal effects of N-methyl-N′-nitro-N-nitrosoguanidine (NTG), ethyl methanesulphonate (EMS), ultraviolet light irradiation and near-ultraviolet light irradiation with 8-methoxypsoralen on the bacterium Acinetobacter calcoaceticus ncib8250 were examined. The production of auxotrophic mutants was used as a measure of mutagenic efficiency. Under appropriate conditions all four agents were mutagenic. EMS and NTG although more effective than irradiation, did not cause such a high frequency of mutation as has been observed with other bacteria. A combination of vancomycin and penicillin V gave enrichment of non-metabolizing bacteria and optimum conditions were found for the use of these compounds in a selection technique.
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Mutagenic DNA Repair in Escherichia coli: Conditions for Error-free Filling of Daughter Strand Gaps
More LessSUMMARY: Two situations have been observed in which daughter strand gaps in DNA synthesized after exposure of excision-deficient Escherichia coli to ultraviolet light are filled but in which no mutations are formed as judged by loss of photoreversibility: (i) during the first 20 min of growth after u.v. irradiation, and (ii) when repair is allowed to ocfn in buffer. We suggest as an explanation that the majority of daughter strand gap-filling is error free and that mutations arise through a minor error-prone repair pathway which is inoperative under these conditions.
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The Molecular Relatedness of R Factors in Enterobacteria of Human and Animal Origin
More LessSUMMARY: The molecular length and DNA homology of R factors isolated from enterobacteria of human and animal origin have been examined. DNA from plasmids of the same compatibility group, whether of human or animal origin, is indistinguishable, after allowance has been made for the regions coding for different antibiotic resistances. These results indicate that there is a common pool of R factors in man and animals.
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- Medical Microbiology
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Protein Reagent Modification of Cholera Toxin: Characterization of Effects on Antigenic, Receptor-binding and Toxic Properties
More LessSUMMARY: The effects of protein modification procedures on the biologically most important properties of cholera toxin, i.e. the toxic activity, the GM1 receptor-binding capacity and the antigenic (antibody-fixing) properties, have been studied quantitatively using microgram amounts or less of toxin protein.
Most of the 24 group-specific reagents used had either no inhibitory effect on the toxic or the combination of G m1-binding and antibody-fixing properties of cholera toxin, or they had a concomitant inhibitory effect on these activities. Separate testing of G m1 -and antibody-binding revealed a close, but not absolute, structural association between these properties. Amino group reactive substances were particularly effective in decreasing the G m1-binding activity, while leucine aminopeptidase had no effect. This suggests that lysine residues may be involved in binding toxin to the acidic G m1 receptor.
Sodium dodecylsulphate and mercaptoethanol, which caused dissociation of the subunits of cholera toxin as indicated by polyacrylamide gel electrophoresis, abolished toxicity without inhibiting the concomitant G m1- and antibody-binding properties of the toxin. Similar differential effects were also obtained with three reagents which did not seem to change the aggregation state of the toxin. These substances all had specificity for arginine, suggesting that arginyl residues of the toxin molecule may be involved in a ‘toxic site’ distinct from the receptor-binding site(s). A selective effect on the toxic site was also found by treating the toxin with carboxypeptidase or trypsin in the presence of urea; in the absence of urea no enzymic effect on any toxin property was noted.
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- Physiology And Growth
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The Inhibitory Action of Fatty Acids on the Growth of Escherichia coli
More LessSUMMARY: The effect of fatty acids on Escherichia coli k12 was dependent on the source of the inoculum, the growth phase and the washing of the bacteria. The effects of saturated fatty acids from C4 to C16 and oleic acid at two concentrations (0·1 and 0·4%, w/v) were determined on E. coli k12/154 growing exponentially in five different culture media. Depending on the media, 0·1 % fatty acids increased the doubling times of the cultures by up to 96 %. Fatty acids of medium chain length (C6 to C11) at 0·4 % produced a decrease in cell concentration, nonanoic and decanoic acids being the most effective. A correlation was found between the decrease in cell concentration and the loss of viability of the cultures after addition of 0·4 % decanoic acid, with stationary-phase bacteria being affected more than those from exponential-phase cultures. Experiments carried out with E. coli B and c gave results similar to those obtained with E. coli k12/154.
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Studies on the Rumen Flagellate Neocallimastix frontalis
More LessSUMMARY: The vast increase in the population density of the rumen flagellate Neocallimastix frontalis shortly after the host animal has commenced eating is caused by stimulation of a reproductive body on a vegetative phase of the organism to differentiate and liberate the flagellates. The stimulant is a component of the host's diet. The vegetative stage of N. frontalis bears a strong morphological resemblance to that of certain species of aquatic phycomycete fungi, and consists of a reproductive body borne on a single, much branched rhizoid. The flagellates liberated in vivo within 15 to 45 min of feeding lose their motility within 1 h and develop into the vegetative phase, thus producing a rapid decrease in population density of the flagellates. Conditions for maximum flagellate production are similar to those ocfnring in the rumen: pH 6·5, 39 °C, absence of O2, presence of CO2. Differentiation of the reproductive body is inhibited by compounds affecting membrane structure and function, but not by inhibitors of protein synthesis. The organism was cultured in vitro in an undefined medium in the absence of bacteria or other flagellates.
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A Timing Control of Cell Division in Escherichia coli
More LessSUMMARY: The effect of heat treatment at 42 °C on a thermosensitive division-defective strain of Escherichia coli k12, mac1, has been studied under conditions which support a generation time of about 50 min. Synchronous cells gained simultaneously the ability to divide at 42 °C and to divide in the presence of nalidixic acid or chloramphenicol, 20 min before physical separation of daughter cells. When synchronous cells of different ages (between 0 and 20 min after elution from an absorbent membrane) were subjected to a heat shock, division always took place 55 to 60 min after the shock. A similar treatment of an exponential culture resulted in synchronous cell division after a lag of 55 to 60 min during which no division ocfnred. Division is probably controlled for 40 to 45 min by the gene mutated in maci. Thus maci cells of different ages appear to return to the same point of their division cycle when they are heated at 42 °C. We propose that the gene mutated in maci has a role in the timing control of E. coli cell division. Progress to division appears to require a fixed period in which the function controlled by the gene is performed; this period ends, under physiological conditions, when division does not require further protein or DNA synthesis.
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Control of Isocitrate Lyase in Nocardia salmonicolor (ncib9701)
More LessSUMMARY: Nocardia salmonicolor, grown on acetate, commercial d,l-lactate or hydrocarbon substrates, has high isocitrate lyase activities compared with those resulting from growth on other carbon sources. This presumably reflects the anaplerotic role of the glyoxylate cycle during growth on the former substrates. Amongst a variety of compounds tested, including glucose, pyruvate and tricarboxylic acid cycle intermediates, only succinate and fumarate prevented an increase in enzyme activity in the presence of acetate. When acetate (equimolar to the initial sugar concentration) was added to cultures growing on glucose, there followed de novo synthesis of isocitrate lyase and isocitrate dehydrogenase, with increases in growth rate and glucose utilization, and both acetate and glucose were metabolized simultaneously. A minute amount of acetate (40 μ m) caused isocitrate lyase synthesis (a three-fold increase in activity within 3 min of addition) when added to glucose-limited continuous cultures, but even large amounts added to nitrogen-limited batch cultures were ineffective. Malonate, at a concentration that was not totally growth-inhibitory (1 mm) prevented the inhibition of acetate-stimulated isocitrate lyase synthesis by succinate, but fumarate still inhibited in the presence of malonate. Phosphoenolpyruvate is a noncompetitive inhibitor of the enzyme (apparent K i 1·7 mm).
The results are consistent with the induction of isocitrate lyase synthesis by acetate or a closely related metabolite, and catabolite repression by a C-4 acid of the tricarboxylic acid cycle, possibly fumarate.
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Volumes and issues
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Volume 170 (2024)
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