- Volume 102, Issue 1, 1977

Cytoplasmic and membrane fractions were isolated from bacteroids of Rhizobium leguminosarum and R. lupini disrupted by lysozyme-induced lysis and sonication. The release of nitrogenase, glutamine synthetase (EC 6·3·1·2), glutamate synthase (EC 2·6·1·53) and glutamate dehydrogenase (EC 1·4·1·4) activities was compared with NADH oxidase and 3-hydroxybutyrate dehydrogenase (EC 1·1·1·30) activities as markers for membrane and cytoplasmic enzyme activities respectively. Nitrogenase was localized in the cytoplasm. Most of the glutamate dehydrogenase activity was released on lysozyme treatment of intact bacteroids although some was released from membrane fractions on sonication, indicating a very superficial localization. Glutamate synthase was present at very low levels in R. leguminosarum bacteroids; the level in R. lupini bacteroids was higher and the enzyme was probably weakly membrane-bound. About 95 % of the glutamine synthetase activity was in the nodule cytosol, and the supernatant of washed bacteroids had a high specific activity; the remaining activity was unevenly distributed between soluble and membrane fractions.

Rapidly growing liquid shake cultures of Monilinia fructigena secreted a polysaccharide which caused an increase in the viscosity of the nutrient liquor. Glucose/malt medium supported most rapid growth and greatest polysaccharide accumulation. Acetone precipitation and dialysis resulted in preparations which were essentially ash and nitrogen free. Gel chromatography indicated that the polysaccharide was polydisperse over the mol. wt range 80000 to 5000. Molecular size was positively correlated with viscosity and inversely correlated with aqueous solubility. Carbohydrate analyses by paper and gas-liquid chromatography indicated that glucose accounted for > 90% of the sugar residues present. Mannose and galactose were also detected. Charged groups were absent. Periodate oxidation revealed that the main linkage was 1 → 4, with a low proportion of 1 → 2 linkages. The degree of branching varied between every fifth and every tenth residue. Linkages of 1 → 3 type were virtually absent. Interaction with concanavalin A indicated the presence of non-reducing terminal α-d-glucopyranosyl or α -d-mannopyranosyl residues.

Zoospores of Phytophthora palmivora were induced to encyst synchronously and changes in morphology, particularly in surface structure, were examined by freeze-fracture and freeze-etching techniques.

A uniform ‘fuzzy coat’, 20 to 25 nm thick, covers the plasma membrane. This hitherto unknown structure surrounds the entire zoospore body and flagella. No structural correspondence between cyst wall microfibrils and plasma membrane particles was found. The pattern of distribution of plasma membrane particles remains unchanged during encystment.

Mutations defining three new loci, sapA, sapB and phoS, were detected by their ability to overcome the phosphatase-negative phenotype of early-blocked asporogenous mutants in sporulation conditions. Synthesis of alkaline phosphatase by Bacillus subtilis is subject to ‘vegetative’ and ‘sporulation’ controls. The phoS mutations resulted in constitutive production of alkaline phosphatase and so could be altered in either the ‘vegetative’ or the ‘sporulation’ control system. The sapA and sapB mutations only affected alkaline phosphatase formation in sporulation conditions, and were considered to be sporulation specific. They rendered ‘sporulation’ alkaline phosphatase formation independent of all the spo mutations tested, and so independent of the control of the dependent sequence of spo locus expression; as the enzyme was not formed constitutively, it remained subject to some other sporulation control.

The sapA and phoS loci were placed between argC4 and metC3 on the genetic map; the sapB locus was located close to purB6. The three loci mapped separately from all known spo loci.

Two plasmids from different sources, determining trimethoprim and streptomycin resistances, harbour transposons which we designate Tn71 and Tn72. These transposons are indistinguishable from Tn7 in the resistances determined, in their molecular masses and in the number and relative positions of their sites susceptible to the restriction enzymes EcoRI, HindIII and BamHI. We conclude that Tn7 has been naturally spread among plasmids.

Reversion studies with a variety of different lipoamide dehydrogenase mutants of Escherichia coli k12 led to the discovery of a class of partial revertant which no longer required succinate for aerobic growth on glucose but retained all the other nutritional characteristics of the Lpd− phenotype. These revertants were given the phenotypic designation Lpd−Sin+ to denote their succinate independence, and mutations in the gene(s) designated sin were considered responsible for generating the succinate-independent phenotype of lpd mutants. Enzymological and genetic studies confirmed that the revertants retained their lpd mutations. Moreover, all of the 24 independent revertants tested possessed sin mutations located in the gal region of the chromosome, presumably suppressing by an intergenic and indirect mechanism. The sin mutations exhibited no allele specificity for suppression and they were recessive to the wild-type sin + allele in merodiploid strains.

A common feature of the Lpd−Sin+ revertants (succinate-independent derivatives of lpd mutants) was a deficiency in succinate dehydrogenase. On their own, the sin mutations produced a phenotype analogous to that of sdh mutants. Furthermore, the probable identity of sin and sdh mutations was confirmed by reconstruction studies in which an lpd sdh double mutant was shown to exhibit an Lpd−Sin+ phenotype. It was concluded that the requirement for exogenous succinate by mutants lacking lipoamide dehydrogenase (and hence overall 2-oxoglutarate dehydrogenase complex activity) can be eliminated by inactivating succinate dehydrogenase because the latter enzyme depletes the intracellular succinate and succinylCoA pools by removing succinate faster than it can be supplied by other endogenous mechanisms. In fact, it would appear that the presence of active succinate dehydrogenase is responsible for the nutritional requirement for succinate of mutants lacking overall 2-oxoglutarate dehydrogenase complex activity during aerobic growth on glucose. A gene-dosage effect on the activities of the 2-oxoglutarate dehydrogenase complex was also demonstrated with corresponding merodiploid strains.

Mixing techniques, which kept killed or non-multiplying Tetrahymena pyriformis in fully randomized suspensions for short test periods (hours), were inadequate for ideal mixing of normal, untreated cells during long periods of growth (weeks). This poor mixing resulted in inhomogeneity in the cell suspension and invalidated calculation of cell multiplication rates in continuous-flow cultures. The development of such inhomogeneities was prevented by the combined use of large propeller blades for mixing and a growth-limiting substrate.

Azotobacter beijerinckii synthesized up to 70% of its dry weight as poly-β-hydroxybutyrate when grown in batch or oxygen-limited chemostat cultures on a glucose/ammonium salts medium. In a series of steady states during transition from oxygen to ammonium limitation and at different dilution rates in the chemostat, the poly-β-hydroxybutyrate content of the organism decreased to a minimum of 5 to 10 % of the dry weight at an oxygen inflow rate of 1·25 % (v/v) in 400 ml argon min−1. At higher dissolved oxygen tensions the polymer content increased to a new maximum of 20 to 40 % of the dry weight, depending upon the dilution rate, before declining to a negligible value. In contrast, nitrogen-grown organisms displayed a steady decrease in polymer content with increasing oxygen concentration. This difference in behaviour is attributed to the greater demand for reducing power and ATP by nitrogen-fixing cultures preventing the operation of respiratory control which, it is suggested, occurs in ammonium-grown cultures over a limited range of oxygen supply rates until respiratory protection and uncoupled electron transport intervene.

A fatty acid auxotroph of Candida albicans 6406, designated a′44 and originally isolated as an oleic acid requiring strain, has been shown to be a ∆9 desaturase mutant. Although lacking this step in fatty acid biosynthesis, it appears to retain the ability to desaturate mono-unsaturated fatty acids. The polyene sensitivity of the organism grown on different fatty acid supplements varied between 0·08 ± 0·02 and 1·20 ± 0·30 μg amphotericin B methyl ester ml−1 for exponentially growing cells. In spite of this variation, the sterol composition remained fairly constant, the major differences lying in fatty acid composition. Stationary-phase cells were more resistant to amphotericin B methyl ester, although again this change was not associated with changes in sterol content. The organism was most resistant when grown in the presence of oleic or linoleic acid. Protoplasts derived from resistant organisms grown on these two fatty acids were also resistant, indicating that the structure of the cell wall was less important than that of the plasma membrane in determining polyene sensitivity under these conditions.

Theoretical models of nitrification were tested using an experimental model consisting of a column of glass beads inoculated with nitrifying bacteria, through which was passed a constant flow of substrate at non-limiting concentration. Effluent was analysed for conversion products. Both zero-order and first-order rate constants for substrate conversion decreased by 1·4% for each 1 ml h−1 decrease in flow rate presumably because of the development of oxygen limitation. Logistic growth kinetics described growth adequately and bacteria were distributed evenly throughout the column after establishment of steady states. Bacterial concentrations of 1·4 × 109 ml−1 and conversion rates of 5·62 × 10−9 p.p.m. N as NO2 − h−1 organism−1 were obtained for Nitrobacter and 6·47 × 106 ml−1 and 3·75 × 10−7 p.p.m. N as NH4 + h−1 organism−1 for Nitrosomonas. During periods of oxygen starvation a lag was observed following increases in flow rate producing undershoots in product concentrations before establishment of new steady states.

A mathematical model of nitrification in a column is developed in which time (t) and distance are considered independently, using a finite difference approach incorporating the following equations for removal of substrate (S) and growth of biomass (m):

where μ is the maximum specific growth rate, K s the saturation constant for growth, k a growth limiting factor and D the dilution rate. This model gives new qualitative and quantitative predictions and in particular predicts overshoots and undershoots in conversion product concentrations following decreases and increases in dilution rate. The model was tested using an experimental system consisting of a column of glass beads, inoculated with Nitrobacter, through which was passed a constant flow of medium containing 1 p.p.m. N as NO2 −; effluent was analysed for nitrite and nitrate.

Short undershoots, lasting several hours, were due to reactivation of bacteria following nutrient starvation. Expected overshoots and undershoots lasting several days were also observed. However, these were not asymmetrical, as predicted, since washout of cells as a limiting growth factor and delay of adjustment to growth rate to different substrate concentrations were not considered.

Complex changes in the activities of cytochrome c oxidase, succinate dehydrogenase, catalase, alkaline phosphatase and acid hydrolases, and in the levels of cytochromes were observed during the growth cycle of Acanthamoeba castellanii in a proteose peptone/glucose/yeast extract medium. A transition occurred at the mid-exponential phase of growth without any change in the growth rate. A second transition corresponded with the cessation of growth; the specific activities of cytochrome c oxidase, succinate dehydrogenase and alkaline phosphatase declined during the stationary phase of growth whereas the specific activities of catalase and the acid hydrolases increased. Glucose was not utilized during the first 30 h of growth and the respiration rate was low, AMP levels were high and some AMP appeared extracellularly. After 30 h, values for the adenylate charge rose from less than 0·1 to more than 0·7, although ATP levels per cell did not vary. When cells were grown in conditioned medium (obtained by removing cells from a 48 h culture) the characteristics of a normal culture observed before 30 h were lost.

A simple method for studying the chemotactic responses of the plasmodium of Physarum polycephalum was designed. A positive chemotactic response was detected towards the sugars d-glucose, lactose, d-maltose and d-galactose, the amino acids l-leucine, l-serine and dl-phenylalanine, and the nucleotide uracil. The significance of chemotaxis in Physarum is discussed.

An axenic and continuous culture of a blue-green alga, Spirulina platensis, was established in a Roux bottle. The opalescent plate method was used to measure the light energy absorbed by the cells and to assess the cell growth yield, Y kcal i.e. the amount of dry algal cells harvested per kcal light energy absorbed. Values of Y kcal calculated ranged from 0·01 to 0·02 g cell kcal−1.