- Volume 95, Issue 2, 1976

SUMMARY: In Arthrobacter pyridinolis, a respiration-coupled transport system for l-rhamnose caused accumulation of free l-rhamnose, while a phosphoenolpyruvate: l-rhamnose phosphotransferase system caused accumulation of l-rhamnose l-phosphate ( Levinson & Krulwich, 1974 ). The pathways for subsequent metabolism of l-rhamnose and l-rhamnose i-phosphate have now been investigated. Arthrobacter pyridinolis contains an inducible l-rhamnose isomerase and l-rhamnulokinase, as well as a constitutive l-rhamnulose i-phosphate aldolase. Results with mutants which are unable to metabolize l-rhamnose suggest the presence of an l-rhamnose i-phosphate phosphatase, which forms free l-rhamnose by hydrolysis of l-rhamnose i-phosphate produced by the phosphotransferase system. Mutants which lack this enzyme exhibited severe inhibition of growth in the presence of l-rhamnose plus any of a variety of carbon sources. There is some evidence that this inhibition was due to accumulation of l-rhamnose i-phosphate at toxic concentrations within the bacteria. The metabolism of l-rhamnose transported by the phosphotransferase system therefore appears to occur by hydrolysis of l-rhamnose i-phosphate to free l-rhamnose by a phosphatase. Metabolism of the l-rhamnose thus produced, and of that accumulated by the respiration-coupled transport system, then proceeds by the sequence of reactions: l-rhamnose →l-rhamnulose → l-rhamnulose i-phosphate → dihydroxyacetone phosphate plus l-lactaldehyde.

SUMMARY: Transport of glutamine by the high-affinity transport system is regulated by the nitrogen status of the medium. With high concentrations of ammonia, transport is repressed; whereas with Casamino acids, transport is elevated, showing behaviour similar to glutamine synthetase. A glutamine auxotroph, lacking glutamine synthetase activity, had elevated transport activity even in the presence of high concentrations of ammonia (and glutamine). This suggests that glutamine synthetase is involved in the regulation of the transport system. A mutant with low glutamate synthase activity had low glutamine transport and glutamine synthetase activities, which could not be derepressed. A mutant in the high-affinity glutamine transport system showed normal regulation of glutamate synthase and glutamine synthetase. Possible mechanisms for this regulation are discussed.

SUMMARY: [14C] Glucose taken up by Epidinium ecaudatum caudatum was found in the pool, in the protozoal polysaccharide and in the bacteria associated with the protozoa. The amount incorporated into the polysaccharide depended on the square of the glucose concentration. Evidence was obtained that glucose was probably taken up initially into the pool unchanged, and then rapidly converted into glucose 6-phosphate and maltose which were subsequently hydrolysed to glucose. [14C]-Maltose was taken up at 20 to 30% of the rate of [14C] glucose, with 14C appearing initially in maltose and glucose 6-phosphate. 14C from 14C-labelled soluble starch appeared in the pool as maltose, glucose 6-phosphate and glucose in that order, but incorporation into protozoal polysaccharide was poor. Hexokinase, phospho-glucomutase, α-glucan and maltose phosphorylases, glucose 6-phosphatase and maltase activities were found in the protozoa.

Antisera have been raised in rabbits against three wall fractions from Neurospora crassa. Fractions were separated according to Mahadevan & Tatum (1965) , i.e. fraction I, glucan-peptide-galactosamine complex; fraction III, laminarin-like glucan; and fraction IV, chitin. Distinct patterns of immunofluorescent staining were obtained using an indirect staining method. Hyphae stained with antiserum to fraction I showed maximum fluorescence in the apical and/or subapical regions: in both cases, fluorescence showed a sharp decrease with distance behind the subapical region. Hyphae stained with antiserum to fraction III showed faintly fluorescent tips with fluorescence increasing with distance from the tip. Hyphae stained with antiserum to fraction IV showed faint fluorescence, equivalent to levels of autofluorescence, except at the sites of hyphal fractures. Antisera were also raised against whole walls from 24 and 120 h cultures. Hyphae stained with antisera against whole walls which had previously been absorbed to remove antibodies to fractions I, III and IV showed preferential staining of apices. The uncharacterized tip antigen(s) thus revealed was also demonstrated on immunodiffusion plates. This pattern of immunofluorescence was compared to the fluorescence of apices after staining with an optical brightener.

Enzymic dissection procedures did not generally give reliable results with apices from 24 h cultures. Untreated apices appeared amorphous, while a drastic chemical treatment revealed randomly oriented microfibrils which were shown to be α-chitin. The apical hyphal walls were significantly thinner than those from more mature hyphal regions.

SUMMARY: Axenic cultures of some commercial strains of the cultivated mushroom, Agaricus bisporus, produced primordia on malt agar medium. The number of primordia differed both between strains, and within each strain when inoculated on to replicate plates. Primordium formation was also dependent on the agar source; it was repressed by changes in the pH of the medium and its constitution, including the addition of further carbohydrate sources; and required translocation from a nutrient source and the presence of an air-agar interface. Two strains grown on malt agar formed complete sporophores, but only at a very low frequency. Attempts to produce mature sporophores from the primordia by using supplementary nutrients were unsuccessful.

Primordium formation was not stimulated by live suspensions or culture filtrates of Pseudomonas putida, suspensions of other bacteria, iron salts, iron-binding compounds or other iron-containing compounds. Cyclic AMP, theophylline and caffeine also had no effect.

SUMMARY: The release of acid phosphatase and polysaccharide-peptide complexes by hydrolytic enzymes from the surface of the blastospore and mycelial forms of Candida albicans has been examined in cells from 4 h and 18 h cultures and the results correlated with the appearance of the treated cells in the electron microscope. Treatment with dithiothreitol was necessary for the degradative action of the enzymes to occur. Material released by all the treatments used had a similar qualitative composition, but the proportions of mannan, glucan, peptide and acid phosphatase varied with different treatments and with the type of cell examined, 1,3-β-Glucanase was required for major changes in the cell wall to be effected, but a significant amount of material was released with a chitinase preparation containing some protease activity. Protoplasts were obtained from all types of cell using Cytophaga lytic enzyme L1 which had 1,3-β-glucanase and protease activity, but the purified 1,3-β-glucanase and protease prepared from Streptomyces violaceus cultures required the presence of a chitinase before protoplasts were released. The bonding association between the major components which comprise the cell wall, and the spatial distribution of these macromolecules, varies appreciably between the two dimorphic forms and with the age of the culture.

SUMMARY: Fertile cultures of Sordaria brevicollis have been shown to make incipient fruiting bodies (protoperithecia) only after contact with the edge of the containing vessel. Experiments with different sizes of Petri dish and different amounts of media confirmed that contact with the edge was the stimulus for fruiting. Mutants with an altered response to the edge were studied. A correlation between the glucose 6-phosphate concentration in the mycelium and the production and distribution of protoperithecia was demonstrated, but there was no evidence that this was causal.

SUMMARY: Dothistromin, an anthraquinone derivative produced by the pine-blight fungus, Dothistroma pini, inhibits the growth of Chlorella pyrenoidosa and Bacillus megaterium. At growth inhibitory concentrations, dothistromin strongly inhibits incorporation of [3H] uridine into RNA of both species. With B. megaterium, marked inhibition of [3H] uridine incorporation is apparent within 5 min of addition of dothistromin, but only a slight inhibition of [3H] thymidine incorporation into the DNA-containing fraction or of [14C] leucine incorporation into protein is detectable after 10 min.

SUMMARY: Phagocytes obtained from fresh human buffy coat (predominantly polymorpho-nuclear phagocytes) or from human buffy coat which had been incubated on a glass surface for 1 to 3 days (predominantly mononuclear phagocytes) were allowed to ingest gonococci, and then incubated with penicillin. More intracellular gonococci were killed at high than at low penicillin concentrations, indicating that penicillin penetrated the phagocytes. This was supported by autoradiography experiments with radiolabelled penicillin.

A pilated, small-colony-forming gonococcal strain survived and multiplied for at least 15 h in polymorphonuclear phagocytes which were incubated with penicillin at the optimum concentration for killing the extracellular bacteria but not the intracellular ones; whereas a non-pilated, large-colony-forming strain survived for only 10 h. The former strain survived for at least 6 h in similar experiments with mononuclear phagocytes. Intracellular survival and growth may be an important facet of the pathogenicity of gonococci.

SUMMARY: The apparent ATP requirement for N2 fixation in Klebsiella pneumoniae was high (the ATP/N2 molar ratio was 29 when estimated in anaerobic glucose-limited chemostat cultures) compared with that determined previously in Azotobacter chroococcum and in Clostridium pasteurianum. The high value was probably not due to unfavourable temperature, phosphate concentration or pH. The apparent ATP requirement for N2 fixation was probably no lower in O2-limited chemostat cultures than in anaerobic glucose-limited chemostat cultures. When hydrogen was introduced into the atmosphere over the anaerobic glucose-limited chemostat culture, there was an increase in the apparent ATP requirement for N2 fixation and in the activity of nitrogenase in intact organisms. A comparison of these increases suggests that some ATP is wasted by the ATP-dependent H2-evolving activity of nitrogenase.

SUMMARY: The quantity of glycogen that could be extracted from cultured dikaryotic mycelia of Coprinus cinereus increased during the first 5 days of growth but subsequently declined. The decline coincided with the main phase of removal of carbohydrate from the culture medium, with a major reduction in the level of soluble reducing sugars of the mycelium, and with the appearance of mature sclerotia. This result complements earlier electron microscope observations that intracellular accumulations of glycogen increase during sclerotium formation and decrease during sclerotium maturation. However, extractable glycogen reserves also accumulated and then dispersed under culture conditions which did not allow sclerotium formation and in strains unable to form sclerotia. It is concluded that glycogen also serves as a transient carbohydrate store for aspects of vegetative mycelial morphogenesis other than sclerotium production.

SUMMARY: Commitment of Bacillus megaterium cells to continue the sporulation process was tested at different times during the developmental period with respect to either addition of different carbon sources (sugars or amino acids) or dilution into media containing these. Organisms grown in minimal medium containing sucrose as sole carbon source were committed earliest with respect to aspartic or glutamic acid as sole carbon source, later with respect to fructose, glucose, glycerol or sucrose, and latest with respect to nutrient medium supplemented with casein hydrolysate. Addition of both aspartate and a carbohydrate resulted in later commitment than addition of either compound alone. The initial uptake rates of aspartate, glutamate, glucose and sucrose increased toward the end of growth in complex medium (but not in minimal medium for glucose and sucrose) and then decreased during the developmental period.