- Volume 128, Issue 1, 1982

N2-fixing Azospirillum brasilense possesses an H2-uptake hydrogenase activity capable of supporting H2-dependent acetylene reduction by whole cells starved of carbon metabolites. H2 did not support acetylene reduction in carbon-sufficient bacteria. although carbon substrates did not inhibit H2-dependent respiration. H2-dependent respiration was extremely O2 sensitive and could not usually protect nitrogenase against inhibition by O2. H2 partly inhibited nitrogenase activity at sub-optimal O2 concentrations: this may be because H2-dependent respiration has a greater affinity for O2 but is less efficiently coupled to ATP production than is carbon-dependent respiration.

The concentrations of glycolytic intermediates and ATP and the activities of certain glycolytic and gluconeogenic enzymes were determined in Propionibacterium shermanii cultures grown on a fully defined medium with glucose, glycerol or lactate as energy source. On all three energy sources, enzyme activities were similar and pyruvate kinase was considerably more active than the gluconeogenic enzyme pyruvate, orthophosphate dikinase, indicating the need for regulation of pyruvate kinase activity. The intracellular concentration of glucose 6-phosphate, a specific activator of pyruvate kinase in this organism, changed markedly according to both the nature and the concentration of the growth substrate: the concentration (7–10 mm) during growth with excess glucose or glycerol was higher than that (1–2 mm) during growth with lactate or at growth-limiting concentrations of glycerol or glucose. Other glycolytic intermediates, apart from pyruvate, were present at concentrations below 2 mm. Glucose 6-phosphate overcame inhibition of pyruvate kinase activity by ATP and inorganic phosphate. With 1 mm-ATP and more than 10 mm inorganic phosphate, a change in glucose 6-phosphate concentration from 1–2 mm to 7–10 mm was sufficient to switch pyruvate kinase from a strongly inhibited to a fully active state. The results provide a plausible mechanism for the regulation of glycolysis and gluconeogenesis in P. shermanii.

In medium saturated with oxygen, the cyclopropane synthetase (unsaturated-phospholipid methyltransferase; EC 2.1.1.16) of Proteus vulgaris was generally synthesized after the mid-exponential phase of growth. The enzyme could also be induced by rapidly limiting the oxygen supply, or by initiating respiration on nitrate or thiosulphate following an initial period of growth in a highly aerobic environment. In each of these ‘step-down’ situations the specific activity of cyclopropane synthetase rose to a maximum prior to the stationary phase of growth and subsequently decreased. The cyclopropane fatty acids, methylene hexadecanoic acid and methylene octadecanoic acid accumulated throughout exponential growth following the induction of the enzyme. During a 12 h period in the stationary phase there was little synthesis of either of the fatty acids, despite detectable cyclopropane synthetase activity in the cells, indicating that essentially all the fatty acid synthesis was complete prior to entering the stationary phase. When nitrate was used as a respiratory electron acceptor, a twofold increase in octadecenoic acid was observed, giving rise to an increase in methylene octadecanoic acid. This increase in octadecenoic acid was not apparent in mutants unable to respire on nitrate.

Both early- and late-exponential phase cells of Acanthamoeba castellanii possess a terminal oxidase which by 1 mm-cyanide but not by azide, salicylhydroxamic acid or CO. In early-exponential organisms this oxidase has a low affinity for O2, but in late-exponential it is modified or replaced by an oxidase which can function rapidly at around 1 μm-O2

An antigenic fraction extracted from mycelium of Absidia cylindrospora contained several mannose-containing glycoproteins which differed in molecular weight and in their affinities to concanavalin A (Con A) and DEAE-Sephadex A-50. The fraction of the extract that bound to concanavalin A was separated into high-mannoproteins (minor fraction) and fucomannopeptides (major fraction). The former showed higher antibody-precipitating activity than the latter. Subfractions of the fucomannopeptides obtained by DEAE-Sephadex A-50 chromatography showed increased serological reactivity and an increased mannose to fucose molar ratio with increasing acidity, but neutral and acidic fractions of the mannoproteins showed the same degree of serological reactivity. The results indicate that the mannosecontaining antigenic fractions are highly heterogeneous and can be classified into high-mannose type (mol. wt 15000–35000) and fucomannan type (mol. wt 15000–70000).

E colicins are those inactive on btuB mutants, which lack the outer membrane protein that adsorbs vitamin B12, E colicins and phage BF23; types E1, E2, E3 and E7 have been defined by the specific immunity of E-colicinogenic strains to the type of E colicin they produce. Further immunity types - E4, E5 and E6 - are now described. Shigella sonnei of colicin type 9 makes colicin E4, and Shigella sonnei of types 9A, 12 and 14 make colicin E6 (not colicin E3, as previously supposed). Many local Shigella sonnei isolates make E colicin of a new type - E5. The action of colicins E1 to E7 was (incompletely) blocked by vitamin B12, which also reduced the effect of A colicins (which are weakly active on Escherichia coli K12 btuB indicators). Escherichia coli K12 given plasmid ColA-23 by transformation was immune to A colicins but sensitive to colicins E1 to E7. A purported ‘colicin E4’ was shown to be of class colicin A. Escherichia coli K12(CloDF13) transformants were immune to colicin E6. A ‘cap’ of colicin-sensitive indicator bacteria developed over, but not around, killed colonies of btuB + E-colicinogenic strains, because of adsorption of colicin by non-induced bacteria of the colony. Killed colonies of btuB + E-colicinogenic strains gave partly discrete lobes of colicin action on indicator lawns, instead of circular zones, apparently as a result of unstable variation in colicin production. The presence of a colicin E4, E5 or E6 plasmid in K12 btuB + strains made them more sensitive to colicins E2 and E7, as shown by zones of partial translucency surrounding the ordinary colicin zones and by increased titre of colicin E2 and E7 preparations on such indicators.

The genetic stability of the capacity of an improved strain of Streptomyces hygroscopicus to produce the macrolide antibiotic turimycin was investigated during long-term continuous culture. Dilution rate, growth-limiting substrate and culture temperature were varied. Certain culture conditions resulted in the stable propagation of the inoculated turimycin-producing population. Other conditions led to segregation of the initial population. Turimycin non-producing phenotypes appeared, and in each case the simultaneous loss of ability to form aerial mycelium was observed. The non-differentiating clones were found to be stable, without any reversion to the parental phenotype, indicating that a loss of genetic information probably took place.

The generalized transducing phage SV1 of Streptomyces venezuelae lysogenizes its host with low efficiency and is therefore temperate. Phage SV2 is also temperate in S. venezuelae, which it lysogenizes more efficiently than SV1. Prophage SV2 is the first example of a u.v.-inducible prophage in Streptomyces. Stable double lysogens able to produce both SV1 and SV2 and insensitive to each phage have been isolated. The relative efficiencies of plating of SV1 and SV2 on four different strains of S. venezuelae indicate that, between them, these strains have four different host specificities. The molecular basis of each host specificity remains to be determined.

Intraspecific protoplast fusion, induced by polyethylene glycol and Ca2+. was carried out in various pairings between auxotrophic nystatin-sensitive, ergosterol-producing strains of Candida albicans and their nystatin-resistant, ergosterol-less mutants of different origins. Nutritionally-complemented stable heterozygous diploid hybrids were obtained, which proved to be sensitive. semi-resistant or resistant to nystatin as a consequence of complementation or non-complementation for ergosterol biosynthesis. Dominant mutation control of the resistance was not found.

Histidine-grown cells of Rhizobium leguminosarum can accumulate more than 100 mm free ammonia in the medium. Growth and respiration are unaffected by these concentrations of ammonia. Measurements of internal and external ammonia concentrations for cells grown on histidine or NH4Cl as nitrogen sources suggest rapid equilibration of ammonia across the cell membrane under conditions where actively accumulated solutes are retained. Implications of these results for movement of ammonia out of N2-fixing bacteroids are discussed.

The obligate chemolithotroph Thiobacillus neapolitanus can grow with NH4 + NO3 − or urea as source of nitrogen. Gradual and rapid mechanisms were detected for regulating both the activity and the rate of synthesis of enzymes required for the metabolism of these nitrogen compounds. Glutamine synthetase (GS) in combination with glutamate synthase (GOGAT) was active under most growth conditions. Alanine dehydrogenase appears to be the major pathway of NH4 + assimilation during energy-limited growth in the presence of excess NH4 +. GS was regulated in this organism by repression/derepression of enzyme synthesis, by inhibition by low molecular weight compounds, and also by adenylylation and deadenylylation. GS was deadenylylated during CO2- and N-limited growth and also during energy-limited growth when NO3 − or urea were supplied as the nitrogen source. GS was adenylylated during energy-limited growth in the presence of 7·7 mm-NH4 +. The activity of GS increased with decreasing dilution rate during NH4 +-limited growth, whereas the activity of GOGAT remained almost constant. The ability of whole cells to reduce NO3 − was derepressed during N-limited growth. During NH4 +-limited growth 24 % of total carbon fixed was excreted as 2-oxoglutarate, pyruvate, succinate, p-hydroxyphenylacetate and ethylmalonate.

The effect of dissolved oxygen concentration on the metabolism of glucose in Pseudomonas aeruginosa was studied with chemostat cultures using both single-step and gradual transitions from either ammonium or glucose limitation to oxygen limitation and studying transient and steady states. The pathway of glucose metabolism was regulated by the availability of oxygen. The organism responded to oxygen limitation by adjusting its metabolism of glucose from the extracellular direct oxidative pathway, which produces gluconate and 2-oxogluconate, to the intracellular phosphorylative route. This change was a consequence of decreased activities of glucose dehydrogenase and gluconate dehydrogenase and of the transport systems for gluconate and 2-oxogluconate, and an increased activity of glucose transport, while relatively high activities of hexokinase and glucose-6-phosphate dehydrogenase were maintained. Citrate synthase, isocitrate dehydrogenase and malate dehydrogenase activities responded to changes in dissolved oxygen concentration rather than to changes in the glucose or ammonium concentrations. The effect of oxygen limitation on the oxo-acid dehydrogenases and aconitase was probably due, wholly or in part, to repression by glucose consequent upon the increase in residual glucose concentration. Succinate dehydrogenase was repressed by an increase in ammonium concentration under an oxygen limitation.

Of the seven antimicrotubular drugs tested, nocodazole, mebendazole and trifluralin at saturable concentrations failed to inhibit cell division in Schizosaccharomyces pombe, while carbendazim, thiabendazole and chloropropham each at 50 μg ml−1 and amiprophos methyl at 200 μg ml−1 completely arrested cell division. This inhibition was associated with striking morphological changes in which carbendazim- and thiabendazole-treated cells became elongated and pseudohyphal, whereas chloropropham- and amiprophos methyl-treated cells appeared small and rounded with occasional V-shaped pairs. Lomofungin staining revealed that nuclear division was also arrested by these drugs. Suspected blockage of defined cell cycle stages was confirmed by pulse-induction experiments which revealed that cells could be synchronized into division using exposure to a drug for one generation. Further experiments with synchronous cultures prepared by size selection showed that different drugs possessed different transition points; for example, carbendazim and thiabendazole were effective in blocking a late stage of the cell cycle just prior to division, whereas amiprophos methyl affected a very early stage. The results suggest that some of the drugs used exert cell cycle specificity in S. pombe either by impairing microtubule assembly mechanisms (as with carbendazim and thiabendazole) or by inhibiting synthesis of tubulin subunits (as with amiprophos methyl). These drugs could prove useful in studies of microtubule biogenesis during the cell cycle in yeast.

The effects of N and P limitation on cell division and on cellular contents of limiting and non-limiting nutrients were examined in cyclostat cultures of the freshwater diatoms Asterionella formosa and Fragilaria crotonensis. Nutrient-limited cell cycles of both species were readily entrained by a 14 h/10 h light/dark cycle. In F. crotonensis, the maxima relative to the onset of the light phase of the photocycle occurred at approximately − 1 to +1 h for cell division, +11 to 13 h for cell N, and +21 to 23 h for cell P. In A. formosa the maxima occurred at approximately +3 to 10 h for cell division and +2 to 4 h for cell N and cell P. Changes in culture dilution rate (integrated growth) and in the type of nutrient limiting growth did not significantly alter these times for either species. The cell-number oscillations can be explained by a simple exponential function for the instantaneous population growth rate, which occurs after a single modal transition time τ, which is characteristic for each species. The consistency of phase relations between N and P limitations and culture dilution rates suggests that the temporal organization for events within the cell cycle of each organism is independent of the nutrient-limited status of the cell populations.