- Volume 84, Issue 1, 1974

SUMMARY: Parasitic development on rye of the ergots of two strains of Claviceps purpurea, only one of which produced alkaloid, was followed during each of two successive summers. Levels of carbohydrate, protein, lipid, nucleic acid, amino acid, phosphate, malate and alkaloid in the tissues were measured. Changes in the levels of these metabolites gave a clear indication of the transition from sphacelial to sclerotial growth form in both strains and the concentrations of the soluble components amongst these metabolites were also found to be sensitive to climatic conditions. These studies suggest that the synthesis of alkaloid was not accompanied or preceded by any significant changes in the pattern of identified metabolites, although the accumulation of an unidentified amino acid was correlated closely with alkaloid synthesis. Comparison of sclerotial metabolism with that of developing rye seeds demonstrated the superior metabolic activity of the parasitic fungus and its ability preferentially to divert host plant nutrients at the expense of adjacent uninfected florets.

SUMMARY: The properties of cells and mitochondria isolated from Candida lipolytica, a hydrocarbon-utilizing yeast, grown on ethanol, glucose and n-alkanes were examined. A comparative study was made of the properties of the whole cells and mitochondria of C. lipolytica and of those of Saccharomyces cerevisiae.

The reduced-minus-oxidized cytochrome spectrum of C. lipolytica grown on ethanol showed a much larger amount of cytochromes aa 3 and a very broad cytochrome b-type absorption band compared with S. cerevisiae. The relative amounts of cytochromes in C. lipolytica on hydrocarbons differed according to the growth phase but never reached the levels obsered in the cells grown on ethanol. Furthermore, as judged by the reduced-minus-oxidized cytochrome spectra, C. lipolytica was much less affected by glucose repression than was S. cerevisiae. The fatty acids of mitochondria isolated from ethanol-grown C. lipolytica consisted mainly of equal amounts of oleic (C18:1) and linoleic (C18:2) unsaturated fatty acids, each making up about 40% of the total. In contrast, S. cerevisiae mitochondria contained palmitoleic (C16:1) (approx. 45%) and oleic (approx. 35%) as the main fatty-acid components. There was an increase in the amount of oleic acid (60%) relative to linoleic acid (20%) when C. lipolytica was grown on even-numbered hydrocarbons and a progressive increase in the amount of heptadecenoic acid (C17:1) up to 75% when grown on odd-numbered n-alkanes of increasing carbon-chain length from C11 to C15. The changes in fatty-acid composition were correlated with changes in membrane fluidity as measured by differences in transition temperatures in Arrhenius plots of mitochordrial membrane-bound enzymes.

The ATPases of C. lipolytica and S. cerevisiae mitochondria were examined, and marked differences in specific activity (3- to 5-fold higher in S. cerevisiae), pH profile and oligomycin sensitivity were noted.

SUMMARY: Methods have been developed which permit the measurement of l-[U-14C]isoleucine incorporation into glutamine synthetase so that the rate of de novo enzyme synthesis can be measured during enzyme deactivation and reactivation. The results suggest that glutamine, a co-repressor of enzyme synthesis, affects enzyme translation. Pulse labelling experiments indicate that enzyme tetramers are not direct precursors of the active enzyme but instead represent a pool of ‘resting’ enzyme. ‘Fine’ control of activity is achieved by reversible aggregation of tetramers, with diminished biosynthetic activity, into active octamers. A number of negative and positive effectors can alter the equilibrium between the two enzyme forms. An earlier suggestion that the monomers produced by the irreversible dissociation of ‘tight’ enzyme tetramers cannot be re-utilized by the yeast has been confirmed. Proteolytic enzymes do not seem to be directly implicated in the destruction of the enzyme. A model is presented which incorporates all the available information on the control of glutamine biosynthesis in food yeast.

SUMMARY: The uptake and synthesis of folate polyglutamates from [3H]pteroylglutamic acid and [14C]p-aminobenzoic acid was studied in Lactobacillus casei, Streptococcus faecalis, L. plantarum and Escherichia coli. The analytical techniques were based on oxidative or hydrolytic degradation of the folate compounds followed by chromatographic separation of the resulting mixtures of pteroylglutamate derivatives or p-aminobenzoylpoly-γ-l-glutamates and comparison with appropriate synthetic standards. All four organisms synthesized numerous polyglutamate derivatives from the precursors provided. The large majority (85 to 95%) of folates of lactobacilli were polyglutamates with more than seven glutamyl residues; L. casei formed several such unidentified long-chain components. In contrast, E. coli and particularly S. faecalis synthesized mainly polyglutamates with seven or less glutamate residues; tetra-, hexa- and heptaglutamates were tentatively identified in E. coli, and tri-, tetra- and pentaglutamates in S. faecalis. While L. casei appeared to synthesize a mixture of formyl- and methyltetrahydro derivatives, formyltetrahydro derivatives predominated in E. coli and S. faecalis.

SUMMARY: Fourteen of the sixteen nucleotides present in extracts of Dictyostelium discoideum were identified by thin-layer chromatography. Data are presented describing the incorporation of radioactive bases into the guanine nucleotide pools and into RNA. Studies with labelled guanine suggested that a small fraction of the RNA undergoes rapid turnover at the pre-culmination stage of differentiation. Analytical methods are described for determining flux rates in vivo and the percentage turnover of endogenous pools.

SUMMARY: Starvation of baker’s yeast, Saccharomyces cerevisiae, in 2% glycerol induces high levels of petite mutants. The rate of mutation is highest when exponential phase cells are used to inoculate the starvation medium. The starved cells are exceedingly sensitive to ethidium bromide mutagenesis. Cycloheximide and sodium nalidixate enhance starvation-induced mutation (although nalidixate reduces the ethidium bromide induction). Starvation-induced mutation is inhibited by rifampicin or erythromycin. The appearance of petite mutants is preceded by a fall in both Qo2 and mitochondrial DNA.

SUMMARY: Drug resistant mutants of the slime mould Dictyostelium discoideum useful for mitotic genetic analysis have been studied. Mutations to acriflavin resistance are recessive and have been assigned to two genes (acrA, acrB) located in different linkage groups. One of these (acrA) confers resistance to the unrelated compounds methanol and thiabendazole. Ability to grow axenically (in the absence of bacteria) is determined by genes on two linkage groups, one carrying acrA and the other an established temperature sensitive mutation. Preliminary results for two linkage groups show that the sequence of genes on a linkage group can be determined by analysis of homozygous drug resistant diploids, obtained by drug selection from diploids heterozygous for recessive drug resistance markers.

SUMMARY: The genes conferring white fruiting bodies (whi), growth-temperature sensitivity in strain x9 (tsgD12) and resistance to methanol or acriflavine (acrA1) are linked in a group designated chromosome II. A gene, present in strain ax2 and involved in the ability of this strain to grow axenically (axe2), has also been located on chromosome II. An analysis of diploid strains derived from a diploid initially heterozygous for all known genes of chromosome II has revealed that mitotic crossing-over occurs during the vegetative growth of diploid cells. An analysis of the classes of diploid thus isolated leads to a provisional ordering of the genes on chromosome II: centromere, whi, axe2, tsgD12, acrA1. The final element necessary to establish the existence of a parasexual cycle in Dictyostelium discoideum has thus been demonstrated.

SUMMARY: The effect of a mutation at acrA, which alters the bacterial membrane, on plasmid stability was studied. F-gal + residing in the mutant was remarkably unstable in the presence of acridine dyes or at high temperature (42°C) as compared with wild-type (acrA +) strains. Following suppression or back-mutation to acridine resistance, cells became capable of stably hosting the plasmid in the presence of acridine. Lipophilic substances, such as phenethyl alcohol and sodium dodecylsulphate, enhanced the curing-action of acridine in the F/acrA strain. Certain fatty acids promoted curing of the mutant bacteria in the absence of acridines. It is suggested that the stability of F-gal + may depend on some feature of the plasma membrane.

SUMMARY: A method for the selection of auxotrophic mutants in Aspergillus nidulans using N-glycosyl-polifungin has been developed. The method results in about 9000-fold enrichment. All types of auxotrophs were obtained. Under certain conditions preferential selection of nucleic acid hydrolysate-requiring auxotrophs was observed. The frequency of occurrence of these mutants was about 50% of all colonies tested. Cysteine auxotrophs of A. nidulans were obtained for the first time by means of this method.

SUMMARY: R factors of the compatibility class P were transferred between strains of Escherichia coli k12 and Rhizobium leguminosarum. These R factors were stable in R. leguminosarum and conferred similar levels of antibiotic resistance to those in the corresponding R+ E. coli k12 hosts, with the exception of carbenicillin resistance which was greatly reduced. Transfer between R. leguminosarum strains was by conjugation and was stimulated by conditions favouring spheroplast formation. R factor mediated recombination could not be demonstrated.

SUMMARY: R906, a plasmid derived from a wild strain of Bordetella bronchiseptica, was transferred to Escherichia coli k12. In this host it is stably heritable, replicating as a DNA molecule with a molecular weight of 34.6 megadaltons. There are approximately three copies of the plasmid per chromosome. It is a member of compatibility group P. In E. coli, 906 confers resistance to ampicillin, streptomycin, sulphonamides and mercury salts. Resistance to ampicillin is determined by an oxacillin-hydrolysing β-lactamase whereas the resistance to streptomycin is not determined by either of the previously described streptomycin-inactivating enzymes but by some other mechanism.

SUMMARY: All of 108 strains of Escherichia coli that synthesized K88 antigen caused mannose-resistant and eluting (m.r.e.) haemagglutination of guinea-pig erythrocytes in a microhaemagglutination test; none of 23 representative strains did so in a tile haemagglutination test which requires firmer binding. It was concluded that the K88 antigen was the m.r.e. haemagglutinin since (i) only K88-positive strains caused m.r.e. haemagglutination (ii) K88-positive strains grown at 18°C failed to produce both haemagglutinin and K88 antigen (iii) haemagglutinating activity was not detected in K88-negative mutants of a K88-positive enteropathogenic strain, and (iv) extracts of K88 antigen possessed haemagglutinating activity which could not be separated from the K88 antigen by the fractionation and serological procedures examined. Haemagglutination appears to resemble the attachment of K88-positive bacteria to the gut wall in enteric disease and the haemagglutination test may assist in characterizing this mechanism.

SUMMARY: Aliphatic dicarboxylic acids, homologous series C4 to C10 (except pimelic acid, C7), were utilized as sole carbon and energy sources by Pseudomonas azelaica, wild strain. A spontaneous Ps. azelaica Pma+ mutant, able to utilize pimelic acid, was isolated from the wild strain on pimelate agar medium. Utilization of pimelic acid was the only character that differentiated the wild strain from the mutant. Experiments with Tween 80 showed that the ability to utilize pimelic and suberic acids by both strains of Ps. azelaica depended on cell permeability. These results suggest that the aliphatic dicarboxylic acids are metabolized by inducible enzymes and that the induction is not specific.

SUMMARY: Clostridium perfringens type A (atcc13124) was grown in bottles (static culture) and in fermenters (batch and continuous culture) in a proteose peptone medium supplemented with phosphate, cysteine, vitamins and different carbohydrates. The lag phase was shorter and the growth rate higher when the medium was pre-reduced. The final yields of bacteria, phospholipase C, and theta-haemolysin were also significantly higher in the pre-reduced medium. The optimum pH for maximum bacterial yield was 7·0 and the optimum temperature for growth was 37°C. The formation of phospholipase C was optimum between pH 6·0 and 7·0 and for theta-haemolysin between pH 7·0 and 7·5. The optimum temperaure for synthesis of both phospholipase C and theta-haemolysin was 37°C. Culture under controlled conditions gave more reproducible production of these two proteins than experiments in bottles. The cultures reached the stationary phase before phospholipase C activity started to decline, while theta-haemolysin activity was stable. The strain was also grown in continuous culture at a dilution rate of 0·4 h−1, at pH 7·0 and 37°C, with a yield of 1·2 mg dry wt/ml in the complex medium supplemented with glucose (5 g/l). After the continuous feed was started the activity of phospholipase C declined very rapidly to zero, while theta-haemolysin continued to be produced at a constant level. Thus continuous culture offered no benefit over batch culture for reproducible laboratory-scale production of phospholipase C.

SUMMARY: Under diminished oxygen tension in a semisolid medium staphylococci and some enterobacteria circumvented the metabolic block caused by sulphamethoxazole and trimethoprim.