- Volume 96, Issue 1, 1976

SUMMARY: Activity of diaminopimelate decarboxylase in Escherichia coli strain w, growing in an aerated fermenter, was only slightly (14%) repressed by 2 mm-lysine when approximately equimolar diaminopimelate was present in the medium. Lysine alone caused 78% repression. Diaminopimelate did not interfere with uptake of lysine by growing organisms. Organisms grown in medium containing diaminopimelate, without lysine, had a decarboxylase activity 24% higher than organisms from minimal medium. The extent of repression by pyridoxine (56% when added to minimal medium) was decreased (to 31%) when diaminopimelate was also present in the medium. A diaminopimelate-requiring mutant, with limited ability to take up diaminopimelate, formed almost three times less diaminopimelate decarboxylase than did a diaminopimelate-requiring second-stage mutant that had an increased rate of transport of this amino acid. The internal concentration of diaminopimelate thus probably regulates the activity of the decarboxylase by induction. Lysine might not directly repress the enzyme but might give an apparent repression by restricting the biosynthesis of diaminopimelate. This restriction is probably not caused only by inhibition or repression of aspartokinase. Lysine and threonine together, though not singly, almost completely inhibited aspartokinase in vitro but caused less apparent repression of diaminopimelate decarboxylase than did lysine alone. Lysine plus diaminopimelate strongly repressed the lysine-sensitive aspartokinase (85%) without much affecting diaminopimelate decarboxylase formation, and pyridoxine repressed the decarboxylase without affecting aspartokinase.

SUMMARY: Seven strains of viridans streptococci of the species Streptococcus sanguis, S. mutans and S. mitis were investigated for autolysis. The effect of pH, salt concentration and temperature on the autolytic process was studied in Na2HPO4/NaH2PO4 buffer. Whole cells and walls of all strains autolysed most rapidly at pH values above 7. Autolysis of whole cells of S. sanguis and one strain of S. mitis (atcc15909) was maximal in 0·05 to 0·2 m buffer, while the two S. mutans strains and S. mitis atcc15912 showed maximal autolysis in 0·5 and 1·0 m buffers. Cultures harvested in the stationary phase of growth possessed only slightly decreased autolytic activity compared with those from the exponential phase. Whole cells autolysed more rapidly at 37 °C than at 45C and 10C. Autolysis of isolated walls of three strains of S. mitis (atcc903, atcc15909 and atcc15912) was maximal at pH 7·0 and 7·5 and in 1·0 m buffers. Streptococcus mitis atcc15909 also showed maximal lysis in 0·01 m and 0·5 m buffers. An endopeptidase action of the autolytic system of S. mitis atcc15912 was indicated by the progressive release of soluble amino groups during autolysis of the walls. No release of reducing groups was observed. Several free amino acids were released during autolysis of these walls, alanine, lysine and glutamic acid being in greatest quantity.

SUMMARY: The interaction of amphotericin B methyl ester (AME) with protoplasts of Candida albicans was measured indirectly by following the incorporation of [U-14C]phenylalanine into the acid-insoluble material. The inhibitory effects of AME at the minimum inhibitory concentration were prevented by the addition of 85 mm-KCl and 45 mm-MgCl2, as shown by Liras & Lampen (1974) for Saccharomyces cerevisiae. In C. albicans, pretreatment of the yeast before antibiotic addition was unnecessary. KCl and MgCl2 did not prevent AME from binding to the protoplast membrane. This interaction was reversed by incubating the protoplasts in the presence of the protecting salts.

SUMMARY: A filtration technique is described whereby metabolically-active suspensions of Dasytricha ruminantium can be isolated from rumen contents with negligible contamination by bacteria or other protozoa. The effects of environmental factors and of the diurnal cycle of the rumen on the uptake and metabolism of soluble carbohydrates by these isolated cells were examined. The principal contribution of the protozoan metabolic end-products to the host ruminant is the supply of lactic, acetic and butyric acids during periods when soluble sugars are in excess.

SUMMARY: To obtain high yields of extracellular enzymes and toxins for immunological analysis, type culture collection strains of Clostridium perfringens types A to D and 28 fresh isolates of C. perfringens type A from humans were grown in fermenters under controlled conditions in a pre-reduced proteose peptone medium. The type culture collection strains all showed different characteristics with respect to growth rates and pH optima for growth. Production of phospholipase C (alpha-toxin), haemolysin and lethal activity varied considerably between the different types. Growth and extracellular protein production in fermenters with pH control and static or stirred cultures were compared. Production of all extracellular proteins measured was markedly improved by cultivation in fermenters with pH control.

Strain atcc13124 produced five times more phospholipase C than any of 28 freshly isolated strains of C. perfringens type A, grown under identical conditions. Haemolytic and lethal activities of the ATCC strain were equal or superior to the activities of any of the freshly isolated strains. There were no differences in the bacterial yields and in the production of extracellular toxins between type A strains isolated from clinical cases of gas gangrene and abdominal wounds, and those isolated from faecal samples from healthy persons.

SUMMARY: Observations on Dendryphiella salina have confirmed that bursting of hyphal apices is brought about by an excessive water potential gradient across the hyphal membrane. Low concentrations of the non-metabolizable sugars 3-O-methyl-glucose and l-sorbose are much more effective than metabolizable sugars in producing bursting. However, at high concentrations they have a protective effect by reducing the water potential gradient. These observations allow a reinterpretation of sugar transport kinetic data which suggested that the glucose transport system in filamentous fungi can exist in two states dependent on the repressive effect of glucose.

SUMMARY: Treatment with 60 % hydrofluoric acid (HF) removed most of the phosphorus and small amounts of mannan, glucan and protein from walls of two non-flocculent strains (ncyc366 and ncyc1004) and two flocculent strains (ncyc1005 and ncyc1063) of Saccharomyces cerevisiae. Organisms of all strains showed increased flocculating ability following HF treatment. Flocculation of untreated organisms of ncyc1005 and ncyc1063, and of HF-treated organisms of all four strains, declined appreciably when they were washed in deionized water, with or without EDTA, and the flocculation was measured in deionized water instead of in 0·05 m-sodium acetate containing Ca2+. Treatment with 1,2-epoxypropane also caused a decrease in the flocculating ability of these organisms. Extracting the lipids from organisms of strains ncyc366 and ncyc1004 had no effect on their flocculating ability, but decreased the flocculating ability of organisms of strains ncyc1005 and ncyc1063. pH-electrophoretic mobility curves of untreated and HF-treated organisms confirmed the loss of wall phosphate by HF treatment, and indicated that HF treatment had little effect on the content of protein carboxyl groups in the outer wall layers. Mannose at 0·22 m completely prevented floc formation by organisms of strain ncyc1063; but, even at 0·33 m, it had very little effect on floc formation by HF-treated organisms of strains ncyc366 and ncyc1063. Organisms of all four strains bound fluorescein-conjugated concanavalin A to the same extent after treatment with HF as before, but this treatment led to a greatly diminished binding of fluorescein-conjugated antiserum raised against organisms of strain ncyc366. The results indicate that phosphodiester linkages in yeast-wall mannan are not involved in bridge formation through Ca2+ during floc formation and that this arises principally through carboxyl groups.

SUMMARY: Phosphoglycerate mutase has been purified from methanol-grown Hyphomicrobium x and Pseudomonas ami by acid precipitation, heat treatment, ammonium sulphate fractionation, Sephadex G-50 gel filtration and DEAE-cellulose column chromatography. The purification attained using the Hyphomicrobium x extract was 72-fold, and using the Pseudomonas ami extract, 140-fold. The enzyme purity, as shown by analytical polyacrylamide gel electrophoresis, was 50% from Hyphomicrobium x and 40% from Pseudomonas ami. The enzyme activity was associated with one band. The purified preparations did not contain detectable amounts of phosphoglycerate kinase, phosphopyruvate hydratase, phosphoglycerate dehydrogenase or glycerate kinase activity. The molecular weight of the enzymic preparation was 32000 ± 3000. The enzyme from both organisms was stable at low temperatures and, in the presence of 2,3-diphosphoglyceric acid, could withstand exposure to high temperatures. The enzyme from Pseudomonas ami has a broad pH optimum at 70 to 76 whilst the enzyme from Hyphomicrobium x has an optimal activity at pH 73. The cofactor 2,3-diphosphoglyceric acid was required for maximum enzyme activity and high concentrations of 2-phosphoglyceric acid were inhibitory. The K m values for the Hyphomicrobium x enzyme were: 3-phosphoglyceric acid, 6·0 × 10−3 m; 2-phosphoglyceric acid, 69 × 10−4 m; 2,3-diphosphoglyceric acid, 80 × 10−6 m; and for the Pseudomonas ami enzyme: 34 × 10−3 m, 37 × 10−4 m and 10 × 10−6 m respectively. The equilibrium constant for the reaction was 11·3 ± 2·5 in the direction of 2-phosphoglyceric acid to 3-phosphoglyceric acid and 0·09 ± 0·02 in the reverse direction. The standard free energy for the reaction proceeding from 2-phosphoglyceric acid to 3-phosphoglyceric acid was −5·84 kJ mol−1 and in the reverse direction +5·81 kJ mol−1.

SUMMARY: Two mutants which lacked both capsular and lipopolysaccharide O-antigen polysaccharides were isolated from Klebsiella aerogenes serotype 2 by phage selection; these were designated rough mutants. The polysaccharide fractions solubilized by partial acid hydrolysis of the lipopolysaccharide from both the wild type and mutants were chromatographed on Sephadex G-50. Analysis of the fractions obtained confirmed that the rough mutants lacked the galactan portion of the molecule, which is analogous to the Salmonella O-antigen polysaccharide.

Membranes prepared from wild-type K. aerogenes, from a non-mucoid strain (lacking capsule only), and from one of the rough mutants were used in incubation mixtures to compare the biosynthesis of polysaccharides by these organisms. The incorporation of sugar nucleotides into both lipid intermediates and polymer was followed. Results show that the transferases were apparently present in all membranes, while the polymerases were absent in both the non-mucoid and rough mutants.

SUMMARY: ε-Aminocaproic acid (EACA) inhibited the development of Dictyostelium discoideum strain ax2 after the aggregation stage. Biochemical changes that occurred early in development (loss of cellular protein, RNA and carbohydrate; increase in the specific activity of β-N-acetylglucosaminidase, a-mannosidase, threonine deaminase and leucine aminopeptidase) were not affected by concentrations of EACA which blocked development; but biochemical changes that occurred later (synthesis of carbohydrate, increase in the specific activity of UDP-glucose pyrophosphorylase) were inhibited. Spores from fruiting bodies formed in the presence of low concentrations of EACA were larger, more spherical and less able to survive heat treatment than spores from fruiting bodies of control (no EACA) cells.

SUMMARY: The differentiation of filaments of a continuous culture of Anabaena cylindrica after removal of fixed nitrogen has been examined. Rapid development of proheterocysts (4·5 h) and mature heterocysts (14 h) in an ordered sequence was observed; the development of the latter was concomitant with the onset of nitrogenase activity. Commitment times of 2·3 h (proheterocyst) and 5·0 h (heterocyst) were measured. Evidence is presented that shows that heterocyst development, rather than any product of heterocyst function in nitrogen fixation, is responsible for the imposition of the pattern of heterocysts in a filament of vegative cells. Both phycocyanin content and carbon dioxide fixation declined markedly throughout the filament during the early stages of heterocyst development, indicating that all the cells of the filament are involved in the initial stages of the differentiation process. Heterocysts do not evolve oxygen in the light, but do respire. Nitrogenase activity in isolated heterocysts and in intact filaments was stimulated by phosphoenolpyruvate, ATP and dichlorophenolindophenol-ascorbate.

SUMMARY: The Flac factor showed unstable maintenance in Salmonella typhimurium dnaC MP10LT2. The properties of a more stable lac + derivative (sd-i) are described. sd-1 was ts and carried the fi + property and the ability to transfer the lac + character. It contained a large plasmid of molecular weight about 129 x 106 daltons. The properties of sd-1 and its derivatives suggested that the large plasmid was a cointegrate of Flac and the MP10LT2 plasmid. Lac + transfer was efficient from sd-i to m799 MP10LT2 and one lac + exconjugant contained the intact cointegrate. The cointegrate was not successfully transferred to strains lacking MP10LT2. It dissociated into apparently unaltered Flac and MP10LT2 plasmids, but the deletion of small parts of one or both plasmids during cointegrate formation could not be ruled out. Cointegrate dissociation was more marked in m799 than in sd-1 especially during growth in glucose-Casamino acids minimal medium. In the presence of R1drd19, the cointegrate (like the MP10LT2 plasmid) was stably maintained in the dnaC strain; maintenance of Flac was, however, unstable. It seems likely that replication of the cointegrate was controlled by the MP10LT2 plasmid constituent.

SUMMARY: Using an enrichment procedure, mutant strains of Agrobacterium tumefaciens were isolated that lacked the ability to utilize octopine as a nitrogen source. Of 55 such isolates, 44 were unable to utilize several amino acids; the remaining 11 strains were altered solely in their ability to utilize octopine, octopinic acid and lysopine. It is concluded that only the latter were plasmid mutations. Among them, there was a high, but no absolute, correlation with avirulence. All strains contained the T1 plasmid. All virulent strains showed active transport of octopine when they had previously been grown in medium containing octopine, whereas the avirulent strains failed to show such transport. All the virulent mutants induced tumours containing octopine. The results are discussed in relation to the hypothesis that the genes which code for the octopine synthesizing enzymes in the tumour are of bacterial origin.

SUMMARY: Effects of alkalinity and hypertonicity on the motile behaviour of Leptospira interrogans (biflexa) b16 were observed, quantified, and compared with effects previously shown by similar factors on the motility of eubacteria. Leptospira interrogans tolerated relatively high concentrations of hydroxide ions. Motility similar to that in controls was observed at pH values up to 9·8; but at pH 10·0 motility declined sharply with time of exposure, and there was structural alteration, visible as a blebbing of the cell envelope. Unlike the behaviour of eubacteria, immobilization of L. interrogans induced by hydroxide ions could not be reversed by lowering the pH. It is suggested that by restricting entry of hydroxide ions, the cell envelope protects its motility apparatus from adverse effects.

Leptospira interrogans was completely immobilized in 0·5 m and 1·0 m-sucrose solutions. Unlike the eubacteria, leptospires were incapable of spontaneous reversion to motile forms and resumption of motility was dependent on both concentration and time of exposure to sucrose. Deuterium oxide did not affect movement, suggesting that even though leptospire endoflagella and the exoflagella of eubacteria are analogous, the motile behaviour of L. interrogans is significantly different from that of eubacteria.

SUMMARY: Respiration in well-fed and starved Podophrya fixa was measured by means of Cartesian diver microrespirometry at 5 °C intervals between 10 °C and 30 °C. At 10C encystment occurred. The optimum temperature for respiration was 25C where a rate of 41·3 × 10−6 μl h−1 per individual was recorded in well-fed animals. Mean cell size also reached a maximum at 25 °C in well-fed individuals. Both respiration rate and mean cell size decreased with starvation, as did the rate of metabolism. A table of daily respiratory energy losses in well-fed and starved Podophrya is presented. The results and their implications are discussed in relation to other protozoa.