- Volume 121, Issue 1, 1980

Genetic analyses of mutations for increased penicillin titre were carried out on two strains of Aspergillus nidulans isolated after independent programmes of recurrent mutation and selection, similar to those used industrially for strain improvement. Both selected strains were stable in terms of colony morphology and penicillin titre. Backcrosses to the un-selected ancestor indicated that the increased yield of each strain was due to an induced polygenic system with both additive and non-additive gene action. The titre-increasing mutations in one strain (A6-9) were recessive to their wild-type alleles, while those in the other (B6-27) were either semi-dominant or showed ambidirectional dominance. The diploid between A6-9 and B6-27 had a titre less than either component haploid, suggesting that the mutations in the two strains involved different genes. Strain B6-27 carried a translocation between chromosomes III and VII which had been induced during the selection programme. This investigation complements previous work on the genetics of penicillin production in A. nidulans and Penicillium chrysogenum and the results are directly relevant to industrial strain improvement by hybridization of divergent strains.

Mutations in the B incompatibility factor of Coprinus cinereus were obtained using two techniques. Both selected for the expression of B-controlled functions (associated with nuclear migration during dikaryon morphogenesis) in heterokaryotic mycelia where these were blocked by having identical B alleles present. The first technique selected for clamp cell fusion in a common B heterokaryon, the second selected for septum dissolution in a heterokaryon following a common B mating. Two mutants, one derived by each of the selection techniques, were studied in detail. In both, mutation was inseparable by recombination from the B locus and resulted in self-compatibility. Mutant monokaryons had uninucleate cells and intact septa. Thus, full regulatory control of morphogenesis in the mutant monokaryon was maintained. However, both mutations promoted clamp cell fusion in dikaryons formed between the mutants and monokaryons having the progenitor B allele or the mutant self allele. Therefore, mutation leads to the loss of regulation of clamp cell fusion. A combination of a B mutation and an A mutation (Amut Bmut) in the same nucleus gave a haploid mycelium which resembled a true dikaryon; thus, both the A and B morphogenetic sequences which are expressed in the dikaryon were operating. However, these dikaryons were not fertile.

Activities of five enzymes of the pyrimidine biosynthetic pathway and one enzyme involved in arginine synthesis were measured during batch culture of Salmonella typhimurium. Aspartate carbamoyltransferase, dihydroorotase, and the arginine pathway enzyme, ornithine carbamoyltransferase, remained constant during the growth cycle but showed a sharp decrease in activity after entering the stationary phase. Dihydroorotate dehydrogenase, orotate phosphoribosyltransferase and orotidine-5′-monophosphate (OMP) decarboxylase showed peaks of activity corresponding to the mid-point of the exponential phase of growth while remaining comparatively stable in the stationary phase. Derepression studies carried out by starving individual pyrimidine (Pyr−) deletion mutants for uracil showed that the extent of derepression obtained for aspartate carbamoyltransferase, dihydroorotase and dihydroorotate dehydrogenase depended on the location of the pyr gene mutation. Orotate phosphoribosyltransferase and OMP decarboxylase derepression levels were independent of the location of the pyr mutation. Aspartate carbamoyltransferase showed the greatest degree of derepression of the six enzymes studied, with pyrA strains (blocked in the first step of the pathway) showing about twice as much derepression as pyrF strains (blocked in the sixth step of the pathway). A study of the kinetics of repression on derepressed levels of the pyrimidine enzymes produced data that were compatible with dilution of specific activity by cell division when repressive amounts of uracil were added to the derepression medium.

Chlorella fusca var. vacuolata has high affinity active transport systems for the amino acids l-leucine and l-tyrosine. Leucine at 0·2 μ m was concentrated at least 222-fold and tyrosine 920-fold. Neither uptake process was inhibited by high concentrations of serine, threonine or phenylalanine. The concentration giving half-maximum rate of uptake was 2·5 μ m for leucine with a smaller value for tyrosine. The maximum uptake rate for leucine was 10 amol cell−1 min−1. Uptake of leucine and tyrosine was not inhibited by 0·2 mm-2,4-dinitrophenol, which completely inhibited the incorporation of radioactive leucine and tyrosine into protein. Intracellular radioactive leucine did not exchange with an excess of unlabelled leucine added to the medium, except during nitrogen starvation, and only partial exchange occurred with tyrosine. The apparent overall pool size for these amino acids was dependent upon the growth state of the algal cells. The kinetics of incorporation of radioactive amino acids from intracellular pools into protein suggested that both leucine and tyrosine occupied at least two distinct pools within the cells. Tyrosine and methionine were present in smallest amounts (38 and 37 amol cell−1, respectively). Leucine was present at 90 amol cell−1. At least 11 amino acids had higher pool concentrations than leucine, whilst only alanine and glycine were more abundant than leucine in C. fusca protein.

Measurements were made of the rate of intracellular protein turnover in Chlorella fusca var. vacuolata, using two methods. Incorporation of 18O into protein from H2 18O-enriched medium occurred at linear rates in both autotrophically growing and nitrogen-starved cells. On the assumption that the only port of 18O incorporation is via the hydrolytic cleavage of peptide bonds followed by resynthesis of the labelled amino acids into new protein, apparent turnover rates were between 1.2 and 2·13% h−1 in growing cells, falling to 0·5% h−1 at the commencement of starvation. After 17·5 h starvation, the rate rose again to the growing cell value. However, analysis of the exchanges between the acid and amides of aspartate and glutamate in the free pool showed that 18O can also be incorporated via side-chain exchange, thus causing overestimation of turnover. Labelling of protein by growth in 3H2O followed by selective measurement of the α-carbon tritium has also been used to avoid the problem of isotope reincorporation. However, in growing cells, incorporation continued after washing the cells free of unincorporated tritium. In nitrogen-starved cells, two stability classes were seen, 85% of protein being degraded at 3·47% h−1, and 15% at 1·11% h−1. It is suggested that this high rate of turnover results from the exposure of the culture to high levels of radioactivity for long periods, and concluded that neither method alone will yield an absolute value for the rate of protein turnover, but differences between classes of protein with different decay rates will be apparent.

Chlorella fusca var. vacuolata protein was labelled by incorporation of 14C-labelled leucine or tyrosine. On subsequent incubation with unlabelled amino acid in otherwise nitrogen-free medium, radioactive amino acid accumulated in the medium as a consequence of intracellular protein degradation (turnover). The kinetics of loss of label from protein was first order with two major components. By varying the period of incorporation of 14C-labelled amino acid into protein of growing cells, it was deduced that about 8% of protein was degraded at about 20% h−1 in growing cells. The relative size of the ‘unstable’ fraction decreased at the onset of nitrogen starvation. The remaining cell protein was degraded much more slowly during nitrogen starvation and may have been essentially stable in growing cells.

From a strain carrying spoIIA69, a mutation giving rise to asporogeny, a revertant was isolated which sporulated at about 10-2 of the wild-type frequency but in which the time-course of sporulation was much protracted. Genetic analysis of this revertant showed that it retained spoIIA69 but had acquired a secondary mutation sas. sas failed to suppress mutations in spoIID, spoIIE or spoIIG; it also failed to suppress another mutation in the spoIIA locus. sas is extremely closely linked (recombination frequency ⩽ 1%) with the mutation spoIIA69 that it specifically suppresses. Strains carrying sas alone sporulated at a frequency at least two orders of magnitude below that in the spoIIA69 sas double mutant. It is suggested that spoIIA69 and sas lead to compensating amino acid changes in the protein specified by the spoIIA locus.

Rapid development of competence can be induced in cultures of Bacillus subtilis by incubation at 37 °C after previous growth at 42 °C. This temperature-induced competence was accompanied by an increase in DNA binding capacity and breakdown of donor DNA. Inhibition of protein synthesis prevented the rapid increase of competence. This is probably due to the inhibition of de novo synthesis of a constituent that enables the bacteria to bind DNA.

A strain of Neisseria gonorrhoeae requiring arginine, proline, glutamate and cystine as nutritional supplements was transformed, in several steps, to grow in a simple mineral medium containing cystine as the only growth factor with DNA from several clinically isolated strains of this organism. Using DNA from naturally occurring auxotrophs (auxo-types) known to require arginine, hypoxanthine and uracil (Arg− Hyx− Ura−), as well as other factors, it was possible to transfer nutritional markers, one at a time, into such prototrophs to obtain seven single marker auxotrophic strains. Three different uracil markers, two different hypoxanthine markers, an arginine marker, and an isoleucine-valine marker were each introduced into separate strains. Of 114 DNA samples from independently isolated strains of N. gonorrhoeae, 54 were able to transform all seven single marker strains to prototrophy. Six of the single marker strains failed to be transformed to prototrophy by DNA samples from 43 strains, thus demonstrating that all these strains possess at least six nutritional lesions in common. Two strains were shown to contain all seven nutritional lesions, whereas several strains contained some but not all of the seven lesions. Six of the seven single marker strains have been shown to revert spontaneously to prototrophy at low frequencies. During construction of prototrophic strains it was observed that genes conferring sensitivity to growth inhibition by nutrients in complex media were occasionally transferred along with prototrophy.

Cells of Bacillus subtilis RodB changed from rods to cocci when shifted from 20 to 42 °C in media containing no additional anions. Quantitative studies of surface growth, including cross-wall formation and pole construction, have been made from reconstructions obtained from central, longitudinal sections of cells. Measurements of surface area and volume were obtained by mathematical rotation of axial sections about their longitudinal axis. Surface markers, perhaps analogous to the wall bands of streptococci, have been used to distinguish septal from cylindrical wall. During the shape change, wall volume increased most rapidly, in relation to cell volume, at the division site. The average volume of wall distal to the septum also increased but the slopes of the lines relating distal wall volume to cell volume were the same at all stages of the shape change. The quantity of wall per distal pole gradually declined with increase of cell volume and as the cells became more coccus-like. Collectively, these features suggest that wall continues to be produced at sites of cylindrical extension but fails to become incorporated into the existing cylinder to maintain a constant diameter. Instead, wall material may be used to thicken the surface of distal poles, but the rate of addition may decline as the cells assume a coccal morphology. The change from rods to cocci may involve a progressive dependence on septal growth for surface expansion and a modification of the time at which the cross-wall is closed. Septal closure is progressively delayed as the organisms change into cocci, so that cross-wall separation precedes septal closure, as in streptococci.

When cells from cultures of Streptococcus mutans strain FA-1 grown at 37 °C were exposed to incubation temperatures of 26 C or less for 5 min or more, an extensive aggregation of particles was observed on the convex fracture faces of their freeze-cleaved membranes. Aggregation of particles was accompanied by a parallel increase in the activation energy for growth. By shifting the growth temperature from 37 to 24 °C for one doubling of culture mass, the transition temperature for membrane particle aggregation could be lowered from about 26 to 0 °C. Although membrane lipids became enriched with unsaturated fatty acids during this period of growth at 24 °C, this enrichment was not accompanied by an increased growth rate of the culture. However, the period of growth at 24 °C did result in bacteria that could grow more rapidly at 10 °C than could bacteria directly transferred from cultures grown at 37 °C. These observations suggest that the increase in membrane fluidity that occurs when bacteria are grown at 24 °C does not allow bacteria to grow faster at 24 °C, but rather allows them to adapt more readily to further decreases in growth temperature.

A description of the patterns of sporulation in chains of Bacillus subtilis cells has been obtained by fitting a statistical model to data obtained from a resuspended culture. The underlying assumptions of this model are: (a) resuspension causes an immediate response in the growing bacteria which is necessary for subsequent sporulation; (b) the probability of this response occurring is the same for all the bacteria present at the time of resuspension; (c) after resuspension some of the bacteria pass through a single round of cell division to produce a pair of sporulating bacteria, while the rest divide twice to produce four; and (d) the two sub-populations described in (c) differ in their ability to complete sporulation.

Respiration of early-exponential phase cultures of the ciliate protozoon Tetrahymena pyriformis is inhibited in two stages with increasing concentrations of cyanide. Up to 40 to 50% inhibition occurs at low concentrations (< 15 μ m). Maximal inhibition was obtained with 300 μ m-cyanide; at this concentration 20% of the respiration was still unaffected. Competitive inhibition of respiration by CO occurs (K 1 = 4·25 μ m); azide inhibition of oxygen consumption is uncompetitive (K 1 = 4 to 9 mm). A salicylhydroxamic acid-sensitive oxidase is present which is not inhibited by cyanide, azide, CO or H2S. Three other pathways of terminal oxidation are present which are insensitive to azide, CO and salicylhydroxamic acid. One of these is cyanide- and sulphide-sensitive, a second is cyanide-insensitive but sulphide-sensitive, and a third is cyanide-sensitive but sulphide-insensitive; these pathways may not possess unique terminal oxidases. No oxidase with a very low affinity for oxygen was detected, but the overall affinity for oxygen of T. pyriformis in the absence of inhibitors (K m = 2·9 μ m) is lower than that of some other protozoa.

Thiobacillus A2 was grown in chemostat culture under four distinct types of substrate limitation: chemolithoautotrophically with limitation by thiosulphate or CO2; heterotrophically with limitation by glucose; and mixotrophically with dual limitation by both thiosulphate and glucose. Under mixotrophic conditions energy was obtained from the oxidation of both thiosulphate and glucose, and carbon was derived both from CO2 fixation by the Calvin cycle and from glucose. Ribulosebisphosphate carboxylase (RuBP carboxylase) activity was negligible and chemolithotrophic thiosulphate oxidation and autotrophic CO2 fixation were apparently repressed in bacteria which had been grown heterotrophically. Conversely, under autotrophic conditions the ability to oxidize glucose was repressed. Growth yields from mixotrophic cultures were the sum of those obtained under single substrate limitation. Intermediate activities of RuBP carboxylase were detected in mixotrophic cultures, but more glucose was assimilated mixotrophically than heterotrophically. Glucose was metabolized by the Entner-Doudoroff (85 to 90%) and pentose phosphate (10 to 15%) pathways under both heterotrophic and mixotrophic conditions, with slight involvement also of the Embden-Meyerhof pathway (< 9%) heterotrophically. RuBP carboxylase activity in autotrophic cultures was enhanced four- or fivefold by CO2 limitation. Repression of RuBP carboxylase activity and thiosulphate-oxidizing ability during the transition from autotrophy to heterotrophy and the activities of carbohydrate-metabolizing enzymes in autotrophic, heterotrophic and mixotrophic cultures are described.

In Bacillus subtilis, the fatty acid moiety of the phospholipids was affected differently during growth in the presence of 1·1 m-methanol or 0·7 m-ethanol, though at these concentrations methanol and ethanol had the same effects on growth rate and completely inhibited sporulation. Synthesis of phosphatidylglycerol was also strongly inhibited and the amount of total cell phospholipids was reduced by 50% by both alcohols. The composition of fatty acids, especially the relative concentration of 12-methyltetradecanoic acid, was modified only by ethanol; in bacteria grown in the presence of methanol, changes in fatty acid composition were negligible. In non-sporulating mutants, synthesis of phosphatidylglycerol was much less affected than in the wild-type and synthesis of phosphatidylethanolamine was increased. In these strains, fatty acid composition was also modified by ethanol but unaffected by methanol.

The composition of neutral, polar and sphingolipids of Entomophthora obscura Hall & Dunn during resting spore formation has been investigated. Sporogenesis was accompanied by an increase in the total lipid concentration and a decrease in the polar/neutral lipid ratio. The most prevalent neutral lipid fatty acids were C10:0, C12:0, C16:0 and C18:0; the concentration of C18:0 increased greatly during sporogenesis. Polar lipids were characterized by high concentrations of C16:0, C20:4 and C20:5; the concentration of C20:5 decreased and those of C18:1 and C20:4 increased during resting spore formation. The fatty acid composition of neutral and polar lipids was similar in several strains of E. obscura and proved their conspecificity irrespective of the facility with which each strain produced resting spores.

Germinating spores of Micromonospora chalcea pass through three morphological stages: darkening, swelling and germ tube emergence. The process of germination has pH and temperature optima of 8·0 and 40 °C, respectively, and is not affected by activation treatments. Darkening, accompanied by a loss of heat resistance and refractility and a decrease in absorbance of the dormant spores, needs only energy, which can be obtained from endogenous sources, and exogenous cations. Agents that inhibit ATP formation block darkening, but inhibitors of macromolecular synthesis do not affect it. Swelling requires exogenous carbon but not nitrogen sources and is characterized by a 30 to 40% increase in spore diameter. RNA synthesis is necessary for swelling and inhibitors of protein synthesis delay this process. During this stage, maximum respiratory, cytochrome oxidase and catalase activities are reached. DNA synthesis starts at the beginning of germ tube emergence. This final stage requires both exogenous carbon and nitrogen sources and the sequence of macromolecular synthesis is RNA, protein and, finally, DNA. Rifampicin, streptomycin and mitomycin C prevent protein and DNA synthesis regardless of when added during germination. Rifampicin inhibits [3H]uridine incorporation immediately but there is a delay of about 160 min in the case of streptomycin or mitomycin C.

The time course of germination of conidia of Colletotrichum musae in the presence of different germination stimulants is consistent with the postulate that the stimulation of germination by anthranilic acid can be attributed to its previously established conversion to 2,3-dihydroxybenzoic acid in conidia. The activity of a number of synthetic chelating agents, such as EDTA and 1,10-phenanthroline, in promoting germination of the same order as 2,3-dihydroxybenzoic acid at about the same molarity provides support for the hypothesis that the chelating properties of the latter compound are important in its biological action.

Experiments with conidia produced on media of different iron concentrations indicate that in the absence of a germination stimulant there is a high inverse correlation between the percentage germination of conidia and conidial iron content. However, in the presence of anthranilic acid there is no significant correlation between these parameters, percentage germination remaining uniformly high regardless of the conidial iron concentration. It is concluded that conidial iron is involved in the inhibition of germination and that this inhibition can be relieved non-specifically by a variety of chelating agents.

The intracellular ATP concentration in plasmodia of the myxomycete Physarum polycephalum was determined by the luciferin-luciferase method after stimulation with various chemotactic chemicals. Repellent salts induced an ATP increase. The change in ATP concentration paralleled changes in contraction and in electrical potential with respect to both time course and concentration dependence. Other repellents (certain organic chemicals and high osmolarity) did not induce appreciable change in ATP concentration within 15 min, but prolonged treatment with hydrophobic repellents and D2O led to diminished ATP. None of the attractants examined (e.g. glucose, alanine, KH2PO4 and 2-deoxyglucose) changed the ATP concentration. A possible role of ATP as a mediator in response to salts is discussed.