- Volume 128, Issue 11, 1982

The efflux of Ca2+ from the yeast Saccharomyces cerevisiae is induced by the influx of mono- or divalent cations. The mechanism of the tight coupling between K+ influx and Ca2+ efflux was investigated. Two possibilities were examined: (1) Ca2+ efflux is induced only by the carriermediated and energy-dependent K+ uptake or (2) Ca2+ efflux can be induced by electric coupling to K+ influx which is driven by its inward electrochemical gradient in the presence of K+ ionophores. It was found that K+ influx in the presence of valinomycin or nigericin and in the absence of glucose did not induce Ca2+ efflux. It is suggested, therefore, that the coupling is mediated by antiport carriers which exchange Ca2+ and K+.

[U-14C]-D-Glucose (1 and 20 mm) was added to glucose-grown suspension cultures of Puccinia graminis, and the sequence of movement of the label into metabolic intermediates was determined under steady-state conditions. Radioactive label moved most rapidly into cellular pools of free glucose, amino acids and phosphate esters of trehalose and glucose, and less rapidly into free fructose. No lag was detected in the movement of label into any of these compounds. Thus fructose was the first free carbohydrate synthesized from glucose. Radioactive label moved more slowly, and with an initial lag phase, into trehalose, glucitol and mannitol; the lag was more pronounced for mannitol than glucitol. Kinetic data and specific activity measurements to identify precursors suggest that trehalose was formed via trehalose phosphate, mannitol via fructose, and glucitol via both glucose and fructose. Steady-state specific activities of all free intracellular carbohydrates were much lower (15-50%) than that of the exogenous glucose, indicating that unlabelled carbon was entering these pools from storage reserves or other components from the culture medium.

Saccharomyces cerevisiae grown on glucose (1 %, w/v) medium exhibited diauxic growth because of the transition from catabolite-repressed fermentative metabolism to derepressed oxidative metabolism. 3-Hydroxy-3-methylglutaryl-CoA reductase (HMG-CoA reductase; EC 1.1.1.34) was object to catabolite repression and required cytoplasmic protein synthesis for derepression. The addition of glucose (2 %, w/v) at up to 4 h prior to glucose exhaustion had no effect on subsequent derepression of HMG-CoA reductase, suggesting that the enzyme was irreversibly committed to derepression at this stage. However, other derepressible enzyme activities such as NAD-dependent glutamate dehydrogenase (EC 1.4.1.2) and malate dehydrogenase (EC 1.1.1.37) did not show a commitment to derepression at this time. In contrast, NADP-dependent glutamate dehydrogenase (EC 1.4.1.4) was induced by the addition of glucose.

An extracellular pectinolytic enzyme produced by Butyrivibrio fibrisolvens isolated from the bovine rumen was studied. The enzyme had a pH optimum of 8·0 to 8·5 and was stimulated by Ca2+ and inhibited by EDTA. The products of pectinolysis had an absorption peak at 235 nm and reacted with thiobarbituric acid, indicating a lyase type of action. The enzyme cleaved the substrates terminally from the reducing end; action on poly- and oligogalacturonates resulted in the formation of an unsaturated trigalacturonate. The enzyme was classified as an exopectate lyase (EC 4.2.2.9). A pectinesterase was also produced by B. fibrisolvens but polygalacturonase was not detected.

Biosynthesis of cell-wall glucans was studied during regeneration of protoplasts from Schizophylhum commune. Double-labelling experiments using [14C]glucose and [3H]glucose indicated a larger pool size of precursors for an alkali-insoluble (1 → 3)-β-glucan than for the other cell-wall components, (1 → 3)-β-glucan and chitin. Pulse-chase experiments established the existence of a water-soluble, partly alkali-soluble (1 → 3)-β-glucan as a precursor for the alkali-insoluble wall glucan, containing only (1 → 3)-β-linkages. Polyoxin D, an inhibitor of chitin synthase, completely arrested accumulation of alkali-insoluble (1 → 3)-β-glucan. This antibiotic did not inhibit the synthesis of the water-soluble glucan, but prevented the incorporation of this material into the alkali-insoluble glucan/chitin complex. Cycloheximide added at the start of regeneration prevented the synthesis of the alkali-insoluble (1 → 3)-β-glucan and of the water-soluble glucan precursor, whereas no effect on the formation of the alkali-insoluble glucan was observed when cycloheximide was added 3 h after the onset of the regeneration.

The ability of cell-free extracts of Chromobacterium violaceum to form cyanide has been investigated. For maximal activity, extracts had to be prepared from bacteria harvested at the end of growth and stored under anaerobic conditions in the presence of dithiothreitol. Glycine was the substrate for cyanide formation by extracts and an electron acceptor such as phenazine methosulphate was required. Radiolabelling studies confirmed that cyanide was produced by decarboxylation of glycine. Formaldoxime, glyoxylic acid oxime, oxamic acid and N-hydroxyglycine, potential intermediates in the conversion of glycine to cyanide, were tested as substrates for cyanogenesis, but were inactive. However, N-hydroxyglycine was a noncompetitive inhibitor of cyanogenesis from glycine. Cyanogenic activity was found principally in the particulate fraction, but a significant amount was also present in the supernatant fraction from high-speed centrifugation. The activity in the particulate fraction was solubilized with detergents.

The turimycin-producing Streptomyces hygroscopicus strain JA6599/NG60-93 (Tur+Amy+) and strain CCl, which is unable to produce antibiotic and aerial mycelium (Tur−Amy−), were compared with respect to their mycelial enzyme activities and cellular lipid composition. Changed activities of six enzymes of intermediary metabolism during submerged growth of strain CCl on chemically defined medium attest to alterations of the life cycle. In addition, strain CCl contained decreased amounts of 12-methyltetradecanoic acid in relation to 14-methylpentadecanoic acid (isopalmitic acid) and displayed a quantitatively altered phospholipid composition.

The extracellular killer toxin of Saccharomyces cerevisiae strain 28 was concentrated by ultrafiltration of culture supernatants and purified by ion-exchange chromatography. Polyacrylamide gradient gel electrophoresis in SDS indicated that the toxin is a glycoprotein with a molecular weight of about 16000. Amino acid analysis revealed that the killer toxin contains 111 amino acid residues, equivalent to a molecular weight of 14045; the ratio of protein to carbohydrate in the molecule is therefore about 9 to 1. The isoelectric point of the killer toxin was pH 4·4 to 4·5. The toxin was unaffected by heating at 40 °C for 1 h and its maximum activity against sensitive yeast cells was observed at pH 5v0. Cell-free extracts prepared from well-washed cells of S. cerevisiae strain 28 were toxic for sensitive yeasts. The toxin present in these extracts (intracellular toxin) was partially purified by ultrafiltration and ion-exchange chromatography. The isoelectric points of the extracellular and intracellular killer toxin were similar.

The inhibitory action of adenosine on the initiation of aggregation centre formation in starving Dictyostelium discoideum was examined and used to test assumptions of theoretical models of cyclic AMP signalling. Adenosine at 5 mm strongly inhibited aggregation centre initiation with-out preventing signal relay. Inosine or AMP at 10 mm had no effect and did not prevent the effect of adenosine. Adenosine had no effect on cellular phosphodiesterase activity, contact site A formation, or chemotaxis to small drops of cyclic AMP. The chemotactically-related responses of rapid cyclic GMP formation and changes in light scattering after pulses of cyclic AMP did not show any inhibition or decrease in sensitivity to the signal in the presence of adenosine. However, binding of 3H-labelled cyclic AMP to cell surface receptors was strongly inhibited by adenosine. The maximum inhibition was approximately 90% and was non-competitive with respect to cyclic AMP. The role of cell surface cyclic AMP receptors in the mechanism of initiation and the implications of the effects of adenosine for the role of cyclic GMP as a secondary messenger are discussed.

Tyrosine aminotransferase, induced in Trichoderma viride by l-tyrosine, was isolated from the culture medium, partially purified and characterized. The enzyme was inducible by both l- and dl-tyrosine, although l-tyrosine was the better inducer. The enzyme required l-tyrosine and a-ketoglutarate as amino donor and amino acceptor, respectively. Its pH optimum in 50 mm-potassium phosphate buffer was 7·6, the apparent Michaelis constants were 0·5 mm for l-tyrosine and 0·25 mm for a-ketoglutarate; it had a molecular weight of 110000.

The properties of non-differentiating derivatives of Streptomyces hygroscopicus, which appeared spontaneously in continuous cultures of their parental strain, were investigated. These derivatives differ from the original strain by production of only trace amounts of the antibiotic turimycin, inability to form aerial mycelium and spores, and altered morphology of colonies and submerged mycelium. It is proposed that as a consequence of a genetic alteration the derivatives are impaired in a central regulatory function influencing differentiation processes by pleiotropic effects.

Equations were derived expressing the expected frequencies of the parental ditype (PD), non-parental ditype (NPD) and tetratype (T) as a function of the frequencies of 10 diad types with two spores from double heterozygous diploids. Using these equations, a novel procedure, ‘diad analysis’ was proposed that permits the estimation of centromere linkage from the frequencies of 10 diad types with two spores. To test the applicability of the diad analysis, the expected numbers of PD, NPD and T asci were calculated using these equations with simulated data for the 10 diad types, taken from tetrad data, and the results were compared, by x 2 statistics, with the observed data from the same tetrads. A good fit was found between the two. A comparison of diad analysis with tetrad analysis and random spore analysis is also discussed.

The ribosomal RNAs were isolated from cyanobacteria (Anacystis nidulans, Anabaena cylindrica, Anabaena CA and Nostoc MAC) and labelled in vitro with [γ-32P]ATP and polynucleotide kinase. DNA from the corresponding species was isolated, digested with EcoRI or HindIII, and resolved on agarose gels. In situ hybridization was used to measure the number and location of the 16S and 23S ribosomal RNA genes. The comparative results are discussed in relation to the known genome size of these species and to their RNA content per cell.

Wild and laboratory strains of Coprinus cinereus show varying levels of sensitivity to cycloheximide (actidione). Mutants resistant to 1.0 g cycloheximide ml−1 were isolated from UV light-irradiated oidia of two strains unable to grow on more than 0·25 μg cycloheximide ml−1. Of 13 induced mutants, 12 fell into one complementation group, cy-2; mutants at this locus were usually recessive, but one of those isolated carried a dominance modifier gene, modcy +, which led to partial resistance in dikaryons heterozygous for cy-2 r. Modcy+ was dominant in dikaryons to its non-mutant allele modcy −, but it had no effect in diploids. However, it affected all cy-2 r alleles tested in dikaryons. Linkage data are presented for cy-2, modcy and another cycloheximide resistance gene, cy-1, which is found in wild strains.

Plasmid pHH502, of molecular weight 70 × 106, determined resistance to tetracycline, chloramphenicol, trimethoprim, sulphonamides and mercuric chloride and was incompatible with members of IncP and IncIα. It resembled other plasmids of IncIα in the following properties: it determined pili that were morphologically and serologically la pili, whose production was repressed in established plasmid-carrying (R+) cultures; its transfer was equally efficient in liquid or on solid medium; it exerted surface exclusion against other IncIα plasmids; it was non-transferable to Proteus. In a reproducible, recA-independent event, pHH502 gave rise to pHH502-1, a plasmid of molecular weight 40 × 106, lacking determinants for resistance to tetracycline and chloramphenicol and all detectable IncIα characteristics. pHH502-1 was incompatible only with IncP plasmids and resembled other IncP plasmids in determining constitutive production of rigid pili, in its surface exclusion, in transferring at greater frequency on solid than in liquid medium and in being transmissible to Proteus mirabilis. It differed from other IncP plasmids in the morphology and serological type of its pili and in failing to transfer to Pseudomonas aeruginosa or Acinetobacter calcoaceticus. Small numbers of pHH502-1 rigid pili were present on bacteria carrying pHH502. Possible mechanisms for the generation of pHH502 and pHH502-1 are discussed.

Plasmids R394a and R394b which cointegrate to form R394 are described. They have molecular masses of 102 ± 4 and 11·0 ± 0·4 MDal, belong to the T and N incompatibility groups and confer resistance to kanamycin and ampicillin, respectively. R394a is self transmissible and mobilizes R394b, which is non-self transmissible. These findings clarify anomalies in the behaviour of R394 and support suggestions based on the properties of variant phages derived from R394.