- Volume 129, Issue 8, 1983

Transient exposure of mycelia from Aspergillus niger to the cytidine analogue 5-azacytidine, at concentrations which do not affect the growth rate of the fungus on nearly minimal media, result in a dose-dependent, heritable change in the timing of the conidiation programme as well as heritable over-production of adaptive enzymes (glycosidases and phosphatases) and modification in the control properties of acid phosphatase. These heritable changes are induced by 5-azacytidine in a non-random way since the new phenotypes are exhibited not only by isolated clones but also by mixed populations of mycelia several life cycles (thousands of mitoses) after exposure to the drug.

Entomocidal proteins produced by certain subspecies of Bacillus thuringiensis were isolated from purified crystals (parasporal bodies) by column chromatography using Sephacryl S-300. The crystals of most subspecies contained only one entomocidal protein (P-1), but some strains of B. thuringiensis which had been classified as subsp. kurstaki, subsp. thuringiensis, subsp. kenyae and subsp. tolworthi appeared to produce an additional protein (P-2) as a minor component of the crystal. The protein (P-2) was serologically similar to the mosquito factor previously discovered in the HD-1 strain of subsp. kurstaki. These entomocidal proteins (P-1 and P-2) were isolated and digested by trypsin. The peptides resulting from the trypsin digestion were mapped by high performance liquid chromatography (HPLC). HPLC patterns of the toxins, in particular P-1, were reliably reproducible and revealed differences in P-1 toxins between strains even in the same serotype. Analysis of P-2 by HPLC indicated that it is different from P-1 in protein structure.

Chromosomal genes of the facultative methylotroph, Pseudomonas AM1, have been mobilized using the broad host-range plasmid, R68.45. Genes coding for C1 metabolism were transferred at a frequency of 10−5 to 10−4 per donor cell. The genes coding for resistance to cycloserine, phosphonomycin and streptomycin are linked to the methanol dehydrogenase gene, while the marker for thiamin auxotrophy is not.

A mixture of two phages, B44/1 and B44/2, protected calves against a potentially lethal oral infection with an O9: K30,99 enteropathogenic strain of Escherichia coli, called B44, when given before, but not after, the onset of diarrhoea; a mixture in which phage B44/3 was replaced by phage B44/3 was effective after the onset of diarrhoea. Calves that responded to phage treatment had much lower numbers of E. coli B44 in their alimentary tract than untreated calves. Usually, high numbers of phage B44/1 and rather lower numbers of phage B44/2 or B44/3 were present in the alimentary tract of these animals. At death, most calves that had not responded to treatment with phages B44/1 and B44/2 had high numbers of mutants of E. coli B44 resistant to phage B44/1 in their small intestine. Phage-treated calves that survived E. coli infection continued to excrete phage in their faeces, at least until the numbers of E. coli B44 also excreted were low. The phages survived longer than E. coli B44 in faecal samples taken from phage-treated calves and exposed to the atmosphere in an unheated animal house. Calves inoculated orally with faecal samples from phage-treated calves that contained sufficient E. coli B44 to cause a lethal infection remained healthy.

A mixture of two phages, P433/1 and P433/2, and phage P433/1 alone cured diarrhoea in piglets caused by an O20:K101,987P strain of E. coli called P433. The numbers of the infecting bacteria and phages in the alimentary tract of the piglets resembled those in the calves. Another phage given to lambs 8 h after they were infected with an O8:K85,99 enteropathogenic strain of E. coli, called S13, reduced the numbers of these organisms in the alimentary tract and had an ameliorating effect on the course of the disease. No phage-resistant mutants of E. coli S13 were isolated from the lambs. The only mutants of E. coli B44 and P433 that emerged in the calves and piglets were K30- or K101- and resistant to phage B44/1 or P433/1 respectively; those tested were much less virulent than their parent strains.

When Neurospora crassa was grown in the presence of chloramphenicol, the oxidation of NAD+-linked substrates by isolated mitochondria became largely insensitive to rotenone. It appears that chloramphenicol hindered the biogenesis of a functional rotenone-sensitive NADH-ubiquinone oxidoreductase. The rotenone-resistant pathway was able to donate electrons to both cyanide-sensitive and -resistant oxidases, indicating the operation of a branched system rather than of parallel respiratory chains. Growth of N. crassa in the presence of chloramphenicol also strongly enhanced the rates of oxidation of exogenous NADH and NADPH. This increased oxidation by mitochondria was largely insensitive to cyanide. The addition of exogenous AMP to isolated mitochondria did not specifically stimulate the electron flux through the alternative oxidase.

Batch cultures of Hyphomicrobium X mut 1 lacking methanol dehydrogenase activity were grown in media containing both methylamine and ethanol as carbon source. The pattern of growth did not show the characteristics of diauxic growth but methylamine was the preferred substrate. When the concentration of the methylamine in the medium fell to 9 mm (±0·8 mm), ethanol was utilized and the two substrates were then utilized simultaneously until the methylamine was exhausted from the medium. Growth then continued using ethanol. The levels of key enzymes and the Q O2 values for the two substrates supported this growth pattern and accounted for the measured uptake of the two substrates from the medium. When the wild-type Hyphomicrobium X was used in similar experiments, methylamine and ethanol appeared to be used simultaneously. However, the Q O2 values for methanol and ethanol were the same until ethanol dehydrogenase activity was induced. This suggested that the ethanol utilization observed before the induction of ethanol dehydrogenase was due to the activity of methanol dehydrogenase induced by the presence of methylamine. When this was taken into account the growth pattern obtained with the wild-type organism was the same as that seen with the mutant.

The synthesis, activity and breakdown of the denitrifying enzymes of Rhizobium japonicum, R. lupini and R. meliloti were found to be regulated by O2. Nitrogen oxide reductases were present in anaerobically grown and symbiotic R. japonicum, but in the case of organisms that had been grown aerobically the enzymes were induced only after a period of incubation under anaerobic conditions. Activity of the denitrification system that had been induced in aerobically grown cells was inhibited by O2. Denitrification by anaerobically grown cells and bacteroids was stimulated by 5% O2. Air inhibited denitrification completely. Little loss of denitrifying activity was shown by cells incubated in 5% O2, but cells incubated at ⩾10% O2 showed a rapid loss of denitrification activity.

Streptococcus BL 78/7 was isolated from alkaline potato-processing effluent, and resembled S. casseliflavus in morphology, motility and pigment. In aerobic conditions, at controlled pH, growth occurred between pH 5 and 11 with a maximum growth rate between pH 8 and 9 and a maximum yield at pH 8. The major metabolic products formed from glucose were lactate and acetate. The molar proportion of lactate to acetate decreased with increasing pH, from 4:1 at pH 5 to approximately 1:1 above pH 9. Growth occurred under nitrogen over the pH range from below 6 to 11, with a maximum growth rate and yield at pH 7 to 8. Lactate was the major metabolic product, with formate, acetate and ethanol also present in the molar ratio of 2:1:1. The molar proportion of lactate to formate decreased from 5:1 at pH 6 to 1·3:1 at pH 11.

A method for the axenic growth of Dictyostelium discoideum wild-type strain NC-4 is described. There were some differences between the nutritional requirements of strain NC-4 and an axenic strain Ax-2 derived from NC-4. Of the numerous components in growth media, the nature and concentration of the peptone was especially critical for axenic growth of NC-4 cells; 3% Bacteriological-peptone (Oxoid) giving the best growth rate and yield. NC-4 cells seemed to require more vitamin B12 than Ax-2 cells. Other vitamins like folic acid, biotin, and riboflavin also stimulated axenic growth of NC-4 cells. Pinocytosis was examined by the use of FITC-dextran and the results showed that the above peptone considerably enhanced the pinocytotic activity of both NC-4 and Ax-2 cells. Bacto-peptone (Difco) also enhanced pinocytosis to a similar extent, though it never supported axenic growth of NC-4 cells. Therefore, stimulation of pinocytosis may be necessary for nutrient uptake, but is not sufficient for axenic growth. The pinocytotic ability of cells decreased conspicuously during the course of development, particularly just before aggregation. The biological significance of this is discussed in relation to cell differentiation.

Glycogen and glycogen phosphorylase content of microplasmodia from Physarum polycephalum were studied during carbohydrate-limited growth and spherulation, induced by starvation. The results indicate that glycogen metabolism in this organism responds most strongly to the availability of external glucose. Glycogen phosphorylase is a constitutive enzyme, present with the same specific molecular activity at all phases of growth and during transition to spherules, supporting the observation that the enzyme cannot be regulated covalently by phosphorylation-dephosphorylation. The enzyme is regulated only by metabolites, in a rather inefficient way: even during net synthesis of glycogen its degradation is not entirely stopped, resulting in a futile cycle. Its activity is merely slowed down to approximately 30% of its rate during glycogen breakdown after consumption of glucose. At this time, part of the glycogen, broken down, is apparently used for the synthesis of slime.

The effect of three β-galactosides on the components membrane potential (Δψ) and pH gradient (ΔpH) of protonmotive force and growth of Escherichia coli has been examined. A good correlation between the reduction of the protonmotive force and growth inhibition was observed. Thus some galactosides had little effect on either the protonmotive force or growth while lactose diminished the protonmotive force and caused growth inhibition. This effect of lactose was dependent on the ionic composition of the growth media. In Medium A (77 mm- Na+, 85 mm- K+) lactose diminished Δψ but had no effect on ΔPH. Growth inhibition was transient at an external pH 6·0 but complete at pH 7·5. In medium KA (approximately 1 mm-Na+, 162 mm-K+) ΔpH was diminished and Δψ was not affected and consequently growth inhibition was complete at pH 6·0. In medium NA (163 mm-Na+, 20 mm-K+) lactose had little effect on Δψ,ΔpH or growth. These data support Skulachev’s hypothesis of buffering of the protonmotive force by K+ and Na+ gradients.

The general control of amino acid biosynthesis was shown to play an important role in the coordination between cell growth and cell division under amino acid limitations. Mutant strains defective in this regulatory system, as studied here mainly with mutant strain RH375 (ndr1-1), showed excessive and aberrant cell growth under mild limitation, and rapid loss of cell viability under severe amino acid limitation. Furthermore, wild-type (NDR1) cells were able to derepress, or at least maintain, levels of enzymes subject to the general control under amino acid limitations. The ndr1-1 mutant cells showed significantly decreased enzyme levels under these conditions. The loss of viability of ndr1-1 mutant cells was not due to inability to accumulate at ‘Start’ under amino acid limitation. In conclusion, we postulate that the aberrant behaviour of ndr-mutant cells is due to an inability to maintain adequate levels of amino acid biosynthetic enzymes throughout the mitotic cell cycle.

The cyanobacterium Anabaena variabilis CCAP 1403/13a (UTEX 1444, ATCC 29413) was grown diazotrophically in the dark in the presence of fructose with a doubling time of 25 h at 35 °C. This was 40% of the maximum rate observed in the light. Growth rates in the dark were similar with nitrate or ammonium as nitrogen sources or under diazotrophic conditions. Dark-grown cyanobacteria reduced acetylene in the dark at 25% of the rate observed for photoautotrophic cultures in the light. Heterotrophic growth yields for dark growth with different nitrogen sources were estimated from the extent of fructose-dependent growth in batch cultures. The ratio between cell carbon and fructose carbon was 0.38 in diazotrophically grown cells, 0.38 with nitrate and 0.52 with ammonia. Thus the energy requirement for growth on molecular nitrogen is similar to the energy requirement for growth on nitrate, but greater than that for growth on ammonia. Since the diazotrophic culture always had a dissolved O2 concentration of at least 80% of air saturation, this demonstrates that there are no detectable extra energy requirements for aerobic nitrogen fixation compared to nitrate reduction, such as would result from a need for respiratory protection of nitrogenase.

Chloramphenicol inhibited growth of asynchronous cells of Alcaligenes eutrophus. In synchronous cultures, different effects on cell division were observed, depending on the time of addition of chloramphenicol. The earlier the time of addition, the greater the inhibition of cell division, which indicates that protein necessary for cell division is synthesized at the beginning of the cell cycle.