- Volume 128, Issue 10, 1982

The inducible systems of uptake of β-d-galactopyranosides into Kluyveromyces fragilis, Kluyveromyces marxianus and Debaryomyces polymorphus were studied under aerobic and anaerobic conditions, using both lactose and the non-metabolized analogue, methyl 1-thio-β-d-galactopyranoside (TMG). A common carrier served both substrates, and aerobic entry was by active transport, involving proton symport. The rate of uptake and the final equilibrium concentration were decreased on adding N,N′-dicyclohexylcarbodiimide, diethylstilbestrol or antimycin A, and active transport was completely abolished by carbonyl cyanide m-chlorophenylhydrazone. Uptake under anaerobic conditions differed markedly from that occurring aerobically: TMG was not concentrated anaerobically, even by strongly fermenting yeasts. Instead, it was transported into the cells by facilitated diffusion, which could sustain a rate of entry of over half the maximum rate observed aerobically. That this difference between aerobic and anaerobic transport into yeasts might apply to glycosides in general, was suggested by the finding that a strain of Saccharomyces cerevisiae also took up maltose by active transport under aerobic conditions, but by facilitated diffusion anaerobically. By contrast, the amino acid, 2-aminoisobutyric acid, was concentrated by Kluyveromyces fragilis, even under anaerobic conditions. The significance of these findings in relation to the Kluyver effect is discussed.

Neurospora crassa produces a phase-specific cationic mucopolysaccharide composed primarily of galactosamine (galactosaminoglycan mucopolysaccharide; GAG-MP), which becomes part of the cell wall and is later secreted into the medium. Its appearance coincides with the onset of the restricted phase of growth, and causes efflux of small molecular weight metabolites when incubated with conidial cells. This activity may be a product of electrostatic interactions of the GAG-MP with the conidial plasma membrane. The activity is blocked by acetylation of the primary amines on the molecule, digestion by enzymes that hydrolyse carbohydrate linkages and the inclusion of NaCl (1 m) in the GAG-MP/conidia reaction mixture. The physiological activity of the GAG-MP mimics that of known depolarizing agents. A model is proposed in which GAG-MP depolarizes the plasma membrane at the onset of the restricted phase of growth, stimulating a cell surface enzyme to produce endogenous cyclic AMP which in turn switches on the enzymic and genetic machinery necessary for that phase of growth.

A number of organic compounds were tested to determine their ability to induce encystment or germination of zoospores of the fungus, Phytophthora cinnamomi. The basic amino acids, lysine and arginine, induced encystment at concentrations in the millimolar range. Pectin (500 μg ml−1) also induced encystment and in addition stimulated the encysted cells to germinate. A wide range of carbohydrates and the amino acids, glutamate and aspartate, stimulated cysts to germinate although they did not themselves induce encystment.

The polypeptide, poly-l-lysine and the basic proteins, histone 1, histone 4 and lysozyme together with the lectin, concanavalin A, immobilized swimming zoospores at concentrations between 0.3 and 10 μg ml−1. At concentrations below 1 μg ml−1 cysts were formed but at higher concentrations cells lysed and the viability of the population was reduced. These compounds did not induce germination of the encysted zoospores. Ethanol, isovaleraldehyde and hexanal also induced encystment but only at concentrations in the millimolar range.

These data, together with previous work on cations show that the triggering of motile cells to encyst does not in this species invariably result in the cells being committed to germination.

The dominant cytochrome in thiosulphate-grown Thiobacillus A2 was found to be of the c-type with a reduced α-band at 548 nm (c 548). This c 548 component did not constitute an integral part of the membrane carrier system. It did, however, appear to be part of a large complex not tightly bound to membranes. Reconstitution experiments showed that cytochromes of the membrane ‘abc’ system could be reduced by the c 548 component and vice versa. The reduction of membrane cytochromes of either lithotrophic or organotrophic origin by thiosulphate electrons was achieved, but it required the presence of a soluble fraction containing cytochrome c 548. Evidence tending to rule out a reductive cleavage as the first step of the thiosulphate oxidation pathway in Thiobacillus A2 was obtained by following the reduction of partially purified c 548 particles under various conditions.

The growth rate of Bacillus stearothermophilus cells in a chemically defined medium was inversely proportional to the concentration of Mn2+ between 15 and 300 mu;m. In the presence of >100 mu;m-Mn2+, cells grew with doubling times of >60 min. Cessation of exponential growth due to a high concentration of Mn2+ was most pronounced in cultures low in sulphur or carbon. This was due to the toxic effect of high Mn2+ concentrations since dilution of cultures to a final Mn2+ concentration of 15 mu;m or less restored the growth rate to maximum. Sporulation depended upon the nature of the growth-limiting nutrient. The manganese effect on sporulation of sulphur-limited cells depended upon the concentration of glucose and the aeration rate in a qualitatively similar manner. The highest spore yield at optimal aeration rates was obtained when the initial Mn2+ concentration in the medium was 10–30 μm. Although the growth rate of bacteria in low-sulphate media was determined by the manganese concentration, its effect on sporulation frequency was independent of the growth rate.

Spores of Bacillus stearothermophilus NCTC 10003 germinated without delay in glucose/glutamate chemically defined medium at 60 °C. The nature of the nutrient limitation inducing sporulation affected the ability of spores to germinate under the conditions studied. Spores produced after glucose depletion of the medium (carbon-depleted spores) germinated faster and to a greater extent than did spores produced after sulphate depletion of the medium (sulphur-depleted spores). Sub-lethal heat treatment (80 °C, 10 min) prior to germination enhanced dormancy. l-Alanine (0·2 mg ml−1) did not reverse this effect, nor did it enhance the germination of sulphur-depleted spores. The rate and extent of germination for both kinds of spores was proportional to the intrasporal manganese concentration. In contrast, extrasporal manganese exerted an inhibitory effect and it was not required for germination. We conclude that the nature of the nutrient depletion and the level of intrasporal manganese distinctly affect the extent and rate of spore germination.

A newly devised method to obtain diffuse growth of Streptomyces coelicolor A3(2) in liquid minimal medium was used to study glucose repression. Although diauxic growth was not obtained, glucose repression of uptake of 14C-labelled carbon sources was demonstrated. Active, arabinose-induced, arabinose transport was repressed at the level of transcription by glucose. Of two glycerol-inducible glycerol transport systems, one was glucose-inhibited but not repressed (and operated by facilitated diffusion), whilst the other (an active transport system) was glucose-repressed. Active transport systems for galactose and fructose which did not require induction by their respective sugars were both inhibited by glucose. Galactose- and fructose-metabolizing enzymes were inducible by the respective sugars, but only in the absence of glucose. This was because glucose both inhibited galactose and fructose transport and repressed the metabolic enzymes concerned. Constitutive active glucose uptake was also demonstrated in arabinose-grown cells. Mutants that grew on arabinose or glycerol in the presence of 2-deoxy-glucose were glucose-derepressed for both soluble carbon source utilization and extracellular agarose. Three glucose-derepressed mutants were studied in detail. One of these could not utilize glucose (and probably lacks glucose kinase), whilst the other two could utilize glucose to differing degrees.

Synchronous cultures of the cellular slime mould, Dictyostelium discoideum, were prepared by a selection method which involved low-speed centrifugation of exponential cultures. The method gave a relatively high degree of synchronous growth and cell doubling and a discrete phase of DNA synthesis was discernible. Amoebae which were induced to sporulation at different times during synchronous growth differed in their differentiation responses. Those which were about to divide produced more fruiting bodies and had higher levels of α-mannosidase and N-acetylglucosaminidase than cells removed to sporulation conditions at other times during synchronous growth.

Growth of six pure cultures of methanogens was inhibited by low concentrations of dissolved acetylene (C2H2); other archaebacteria (three Halobacterium species) and several eubacteria were not similarly affected. The minimum concentration of dissolved C2H2 required to inhibit growth of Methanospirillum hungatei completely was about 8 mu;m; dissolved ethylene at 20 μm had little effect on growth. Dissolved acetylene (33 mu;m) did not alter the E h of the medium, or result in a loss in viability of M. hungatei after 16 h exposures. In anaerobic cell extracts of M. hungatei, activities of hydrogenase, NADP reductase, methyl-coenzyme M reductase and ATP hydrolase were not inhibited by C2H2 concentrations several times higher than those required for growth inhibition. The intracellular ATP content of all of the methanogens dropped dramatically on exposure to C2H2. Moreover, cells of M. hungatei and Methanobacterium bryantii on exposure to C2H2 lost their ability to maintain a transmembrane pH gradient. We suggest that exposure to C2H2 results in a decline in methanogen functions which require a H+-flux, including ATP synthesis, Ni2+ uptake and methanogenesis.

Evidence is presented for the first time for the presence of a b-type cytochrome in extracts of Corynebacterium renale, C. bovis, C. kutscheri and C. pseudotuberculosis, and b- and c-type cytochromes in extracts of Rhodococcus equi (syn. C. equi). Previous observations on the presence of a b-type cytochrome in C. pyogenes were extended.

Susceptibility to bleomycin proved to be a useful criterion for differentiating Rhodococcus from Nocardia, the former being susceptible and the latter resistant to this agent. The test was also useful for discriminating between various species of the genus Mycobacterium.