- Volume 106, Issue 1, 1978

A membrane-bound, sulphide-linked nitrite reductase from Thiobacillus denitrificans was solubilized and after further purification its properties were examined. The purified enzyme, mol. wt 120 000, contained cytochromes c and d in the ratio of 1:1. Both cytochromes were reduced by sulphide and re-oxidized with nitrite or air. Oxidation by nitrite resulted in the appearance of an absorption peak at 572 nm. The kinetics of the reduction of the enzyme with sulphide indicated that cytochrome c was reduced before cytochrome d. The redox potential of cytochrome d was 22 mV more positive than that of cytochrome c. Cytochromes c and d were dissociated from the purified enzyme by treatment with sodium dodecyl sulphate. The purified nitrite reductase also had cytochrome oxidase activity and both the activities were stimulated by cytochrome c-551 isolated from T. denitrificans. Reduced cytochrome c-551 was an effective electron donor for the purified enzyme with either nitrite or air as the terminal electron acceptor. Neither cytochrome c-554 (also isolated from T. denitrificans) nor mammalian cytochrome c was effective as reductant for the enzyme. NO and N2O were identified as the products of nitrite reduction by the purified sulphide-linked nitrite reductase.

The arginine-specific carbamoyl-phosphate synthase of yeast was stabilized sufficiently to allow partial purification of the enzyme (30- to 40-fold). The synthase (mol. wt 115000) comprised two unequal subunits: a heavy subunit (mol. wt 80000) capable of catalysing synthesis of carbamoyl phosphate with ammonia as a nitrogen donor and a light subunit conferring upon the holoenzyme the ability to utilize glutamine. The enzyme had unusually high affinity for ATP (K m = 0.2 mM) and atypical negative cooperativity for glutamine binding ([S]0.5 = 0.25 mM). Glutamine activity was not modulated by possible effectors such as arginine, ornithine or N-acetylglutamate. Thus, although the yeast arginine enzyme physically and functionally resembles the single enteric synthase, the systems differ substantially both in kinetic properties and in regulation of activity.

Four temperature-sensitive mutants of Staphylococcus aureus with defects affecting DNA synthesis have been isolated and partially characterized. They fall into two groups: three have defects either in elongation of DNA or synthesis of its precursors; the fourth has properties inconsistent with a defect in either elongation or initiation. Transduction analysis indicated that the mutation in this fourth mutant is unlinked to the mutations in the other three, which are all clustered on one side of a gene conferring resistance to novobiocin.

When the Challis strain of Streptococcus sanguis was transformed by the 17 megadalton β plasmid from Streptococcus faecalis strain DS 5, the plasmid underwent a 1.5 megadalton deletion (LeBlanc & Hassell, 1976). Furthermore, the covalently closed circular (CCC) plasmid DNA isolated from Challis transformants was rapidly converted to a linear form which did not possess any detectable transforming activity. To obtain stable CCC plasmid DNA a competent culture of a Lancefield group F streptococcus, strain DL 8 (ATCC 12393), was used as a recipient of β plasmid DNA. The plasmid DNA isolated from group F transformants exhibited the same configuration and size characteristics as the DS 5 β plasmid, and the CCC configuration was stable upon storage. CCC plasmid DNA from a group F transformant was biologically active and, when added to competent cultures of strain DL8, transformed them at frequencies about 100-fold greater than did β plasmid DNA from DS 5. This suggests the existence of a restriction-modification system in strain DL 8.

Twenty-eight spontaneous auxotrophic aroP mutants with deletions in the azi-nadC-aroP-aceE-aceF-lpd region of the Escherichia coli K 12 chromosome were characterized genetically with respect to various azi, nadC, ace and lpd markers by P1-mediated transduction. One mutant (K ?18; aroP-lpd?) had a deletion which extended through the aceE and aceF genes to end within the lpd gene. The polarity of the ace operon (aceE to aceF) was confirmed. It was concluded that 10 out of 15 deletions generating a strict requirement for acetate terminated in the aceE gene. Of the ten, three mutants (K ?22, c?41 and c?42) synthesized detectable dihydrolipoamide acetyltransferase (the aceF gene product) and seven were assumed to possess deletions generating polar effects on aceF gene expression. Five deletions appeared to extend into the aceF gene. A further five deletions, which limited the expression of the ace operon without generating an Ace- phenotype or a complete Ace- phenotype, ended closest to the aroP-proximal aceE markers. The opposite ends of all these deletions appeared to terminate before (10), within (2) or extend beyond (9) the nadC gene. There was no obvious correlation between the deletion end-points and the corresponding lipoamide dehydrogenase activities, which ranged from 30 to 95% of parental levels in different deletion strains. The remaining seven deletions simply extended between the aroP and nadC genes (nad-aroP?) without affecting expression of the ace operon.

Regulation of the synthesis of the pyruvate and ?-ketoglutarate dehydrogenase complexes was investigated in some of the parental and deletion strains under different physiological conditions including thiamin-deprivation. The results indicate that the syntheses of the two dehydrogenase complexes are independently regulated. Expression of the lpd gene appears to be coupled to complex synthesis but can be dissociated under some conditions. Mechanisms for regulating lpd gene expression are discussed and an autogenous mechanism involving uncomplexed lipoamide dehydrogenase functioning as a negatively acting repressor at the operator site of an independent lpd gene is proposed as the simplest mechanism which is consistent with all available information.

The high natural resistance of gonococci showing a characteristic ‘double highlight’ (DH) colonial morphology (Penn, Veale & Smith, 1977b) to intracellular killing by human phagocytes was markedly reduced by addition of rabbit antiserum to the phagocytosis medium or by preincubation of organisms with antiserum. Antisera raised to three different DH gonococcal strains showed a complex pattern of specificity in phagocytosis tests with the homologous organisms and three other DH strains. The effect of antiserum could be neutralized by adsorption with intact organisms or with extracts, prepared ultrasonically, of the homologous strain. Antiserum also promoted the intracellular killing of a strain which had a ‘single highlight’ colonial morphology (Penn et al., 1977 b) and a low natural resistance to phagocytic killing, but adsorption with this strain neutralized the antiserum less consistently than the DH strain. The neutralization of antiserum-mediated promotion of intracellular killing by extracts of organisms naturally resistant to such killing may provide an assay for the aggressins responsible for this resistance.

The contribution of phagocytes to protection against Listeria monocytogenes was analysed in outbred ddN mice. Most of the bacteria injected intravenously at a dose of 3 × 103 to 4 × 103 were trapped in the liver within 10 min. There was a transient 10-fold decrease in the number of bacteria by 6 h. Anti-listeria activity in the initial phase was resistant to X-irradiation but was inhibited by carrageenan, and was not influenced by immunization. The protection in this very early stage of infection seemed to be attributable to the function of fixed macrophages. Viable bacteria in the organs increased progressively but slowly from 6 h to 72 h to reach maximum numbers. Bacterial growth during this period was markedly enhanced by X-irradiation or treatment with carrageenan. Accumulation of free phagocytes seemed to suppress the bacterial growth in this phase. The number of bacteria began to decrease from day 4 and became undetectable by day 9. The suppressive effect on bacterial growth in this last phase may be dependent on immunologically activated macrophages and was reversed by X-irradiation and carrageenan. The course of local infection was similar to that of systemic infection except for the lack of initial decrease. We conclude that the course of infection with L. monocytogenes can be divided into three phases with regard to the roles of phagocytes in resistance.

Respiration rates, adenine nucleotide levels and heat production were measured during exponential asynchronous growth of Tetrahymena pyriformis and in synchronous cultures prepared by continuous-flow selection. In synchronous cultures rates of oxygen uptake rose to maxima three times in each cell cycle. The ATP pool oscillated in phase with respiratory activity. The ADP and AMP pools oscillated in phase with each other, but out of phase with ATP, and the rate of heat production also showed fluctuations. Values calculated for adenylate energy charge were low, increasing from less than 0.2 to more than 0.4 during the mid-exponential phase of growth in an asynchronous culture, and oscillating over a range from 0.34 to 0.47 in a synchronous culture.

Macrophages and certain batches of sera were essential for extracellular multiplication of Trypanosoma dionisii in vitro in medium 199 with 20% (v/v) calf serum at 37 °C. In mixtures of ‘good’ and ‘bad’ batches of sera, multiplication increased as the proportion of the former was increased. Mixtures of ‘bad’ and ‘intermediate’ sera permitted virtually no growth. Phagocytosis of parasites by macrophages was unaffected by different batches of sera, though reduced in medium 199 alone. Replacement of supernatant medium by medium containing ‘bad’ serum reduced the macrophage infection rate no more than did transfer to medium containing ‘good’ serum. Daily addition of medium conditioned by prior contact with macrophages in vitro to cultures of trypanosomes without macrophages resulted in growth at least as good as that in the presence of macrophages. Extracellular replication in medium 199 at 37 °C apparently required at least two factors: ‘M factor’ provided by macrophages, but not required at 28 °C; and ‘S+ factor’ present in some batches of calf serum, essential also at 28 °C but not required by intracellular parasites. Some sera appeared to contain an inhibitory ‘S-factor’.

The rumen holotrich protozoa Isotricha intestinalis and I. prostoma showed chemotaxis to sucrose, glucose and fructose. They attached themselves, by means of an organelle on the anterior cell surface, to particulate sources of these carbohydrates provided soluble protein was present in the medium. The concentration of protein eliciting attachment varied with the species and the state of nutrition of the cell, but was between 20 and 150 ?g ml-1. Attachment occurred only if the concentration of carbohydrate, at its source, exceeded the chemotaxis threshold concentration (50 ?M for sucrose) and if it was less than 1 mM. At concentrations exceeding 1 mM, indiscriminate attachment to gas-liquid and solid-liquid interfaces occurred, provided the protein concentration was high enough to elicit attachment. In the rumen, soluble carbohydrates diffusing from food particles may attract the protozoa which attach themselves to the particles in the presence of soluble plant protein at > 20?g ml-1; these conditions exist in the host animal soon after feeding when fed infrequently. The attachment mechanism may confer an ecological advantage on the Isotricha spp. over other rumen organisms dependent on soluble carbohydrates as energy and carbon sources.

A single-substrate three-compartment model for the growth of a mushroom crop is constructed. The model describes the growth of the mycelium, and the initiation and growth of sporophores, up to the end of the first flush. The growth of mycelium and sporophores is controlled by the substrate density in the storage component of the mycelium, and initiation of sporophores is modelled by assuming the existence of a threshold substrate density, below which initiation cannot take place. When the substrate density exceeds the threshold density, the rate of initiation is assumed to be proportional to the difference between these two densities.

Parameter values are given which lead to a solution of the model which agrees reasonably well with observed data. Various aspects of the solution are examined, and the important parameters are identified. The parameter controlling the rate of initiation of sporophores has little effect on either the number of sporophores initiated or the duration of initiation.

When a growing culture of Cryptococcus albidus was shifted from 27 to 37 °C, growth ceased abruptly and the cells died rapidly (over 90% after 4 h). The temperature shift resulted in a rapid cessation of [3H]uridine incorporation (as perchloric acid-insoluble material). DEAE-Sephadex chromatography of crude extract from C. albidus cells revealed the presence of two major enzyme fractions with RNA polymerase activity. The fractions were insensitive to α-amanitin, and both synthesized RNA efficiently at 23 °C but not at 37 °C. Neither proteolytic digestion of the enzymes nor ribonuclease degradation of the RNA products could account for the observed decline in RNA synthesis at the elevated temperature.

Sordaria fimicola on 0.02% malt extract agar displayed spiralling of the colony in a clockwise direction (viewed from above the agar plate). Only hyphae at the agar surface contributed to this spiral aspect. The direction of curvature of individual hyphae was related to their supporting surface; hyphae curved to the right as they advanced over the surface. This sustained departure from straight growth is attributed to a displacement of the plastic apex of the hypha to the right as a result of its own clockwise axial rotation (as viewed tipwards from within the hypha). Attempts to observe directly the rotation of the apex failed. Calculations show that the rate of angular rotation of an individual leading hypha was of the order of 500° per millimetre of extension, a rate similar to that in several other cells which spiral as they grow. However the angle of spiral growth was very small (about 0.7°). Out of 157 isolates of fungi tested, 21 showed pronounced spiralling of their colonies on 0.02% malt extract agar and 39 showed weak spiralling, whence it is inferred that spiral growth of individual vegetative hyphae is of widespread occurrence amongst fungi.

The genus Erwinia is heterogeneous with respect to the presence of the ammonia assimilating enzymes glutamine synthetase (GS), NADP-linked glutamate synthase (GOGAT) and NADP-linked glutamate dehydrogenase. Three major groups were distinguished; one showed activity of all three enzymes, the second only GS and GOGAT activity and the third only GS activity. Aspartase did not appear to be significant in ammonia assimilation in any group.

The intracellular concentrations of adenylates and the value of the adenylate energy charge in Schizosaccharomyces pombe have been determined. Dilution of extracts before enzymically converting AMP and ADP to ATP was necessary for the quantitative measurement of all three adenylates. Using trichloroacetic acid or perchloric acid extracts of exponentially growing cultures, the energy charge was calculated to be 0.8 to 0.9; the value rose to about 0.95 in the stationary phase of growth. Lower energy charge values (0.6 to 0.64) were obtained using chloroform extracts. During the last 2.5 h of a 4 h treatment of an exponentially growing culture with 2?-deoxyadenosine, and during the induced synchronous growth that followed subsequent removal of the inhibitor, the pool sizes of all three adenylates oscillated. Minimal ATP/ADP ratios (and energy charge values) occurred concurrently with maximal rates of respiration that were relatively insensitive to stimulation by carbonyl cyanide m-chlorophenylhydrazone. Cultures not treated with 2?-deoxyadenosine but centrifuged and resuspended in fresh medium, showed a transient extensive increase in the ATP/ADP ratio 1 to 2 h after resuspension. However, rates of O2 uptake, in the absence or presence of carbonyl cyanide-m-chlorophenylhydrazone, increased smoothly throughout this period. The results suggest that, in 2?-deoxyadenosine-induced synchronous growth, respiration rates may be controlled by the intracellular ATP/ADP ratio, and demonstrate that division synchrony is not induced by depletion of ATP pools.

The activities of nitrogenase, glutamine synthetase and other ammonia-assimilating enzymes were studied in bacteroids of Rhizobium leguminosarum isolated 9 to 21 d after infection. Total bacteroid numbers (per plant) increased proportionally with nitrogenase, but glutamine synthetase activity decreased with increasing age of the root nodule. Glutamine synthetase was adenylylated throughout the period of increasing nitrogenase activity and during the period of constant nitrogenase activity.