- Volume 130, Issue 12, 1984

The induction parameters of levansucrase synthesis were the same in Bacillus subtilis strain 168 Marburg and in a derivative, hyperproducing (sacU h) strain. However, only the hyperproducing strain showed an induction lag period. The kinetics of appearance of functional levansucrase mRNA was established. Strain 168 did not release levansucrase, but washing the cells with high ionic strength buffer released different proteins of which levansucrase represented 2%. In contrast, the great majority of levansucrase synthesized by the hyperproducer was released in a homogeneous form into the culture medium. In this case high ionic strength treatment caused the cells to release the remaining levansucrase but not other proteins. A Triton X-100 sensitive form of levansucrase was isolated by phenol treatment of the sacU h strain; this form was absent in strain 168. We suggest that the sacU gene product possibly controls the synthesis of cellular components with which levansucrase is associated and thus its release is normally prevented.

Glycerol : NAD+ 2-oxidoreductase (EC 1.1.1.6) was purified to homogeneity from the non-methylotrophic yeast Candida valida H 122. Results of electrophoresis in polyacrylamide gels, gel filtration and ultracentrifugation were compatible with the enzyme’s consisting of two subunits with a molecular weight 38000. No heterogeneity was observed by isoelectric focusing. The pH optima were10·0 for glycerol oxidation and 7·5 for dihydroxyacetone reduction. The K m values for glycerol, dihydroxyacetone, NAD+ and NADH were 5·8 10−2 m, 7·7 10−4 m, 1·4 10−4 m and 4·8 10−5 m, respectively. 1,2-Propanediol also served as substrate in the forward and dl-glyceraldehyde in the reverse reaction. The oxidation product of glycerol was identified as dihydroxyacetone. An ordered bi-bi mechanism was deduced from product-inhibition studies. Of several anions and cations tested, pyrophosphate and ammonium ions stimulated the dehydrogenating activity the most. 2-Mercaptoethanol, ethylene glycol and Tris inhibited activity; an inhibition was also observed by phosphorylated coenzymes and substrates. The purified enzyme, which is labile at low concentrations, was stabilized by the addition of neutralized filtrate from the culture liquid.

Changes in cell composition of Rhodosporidium toruloides CBS 14 were monitored during growth of batch cultures with NH4Cl and glutamate as nitrogen sources. Carbohydrate was synthesized at the expense of lipid in NH4 +-grown cells, whereas in glutamate-grown cells lipid accumulation was predominant. Total biomass and protein concentration were similar in both cultures. Uptake of [U-14C], [1-14C] and [6-14C]glucose, and evolution of 14CO2 from these sources, by washed suspensions of cells grown on glutamate revealed the flux of carbon during glucose dissimilation was principally via the Embden-Meyerhof pathway (72%), with 28% being metabolized by the pentose phosphate pathway. Both urea and glutamate, when added to the cell suspensions, significantly stimulated glucose catabolism, with the flux of carbon via the former pathway increasing to about 89% of the total. Phosphofructokinase (PFK) was implicated as the likely controlling enzyme to explain these events.

PFK was only detected in extracts prepared from the yeast grown in a carbon-limited (nitrogen-excess) medium; no activity was detected in extracts of cells grown in nitrogen-limited medium. The presence of a protease in these latter extracts was revealed. PFK was purified 92-fold to a final specific activity (in the presence of 10 mm-NH4 +) of 4·2 units (mg protein)−1 and exhibited one broad band on polyacrylamide gel electrophoresis. The apparent mol. wt (mt ) of the enzyme was approx. 700000. The major properties of the enzyme were examined to determine its regulatory role during lipid biosynthesis. Unlike the enzyme from Saccharomyces cerevisiae, no inhibition was found with 10 mm-ATP. ADP was not inhibitory either. NH4 + ions increased activity 11-fold by increasing the affinity of the enzyme for both fructose 6-phosphate and ATP. K+ ions also stimulated activity but to a lesser extent. Activity was severely inhibited by citrate, isocitrate and cis-aconitate but this inhibition was dramatically alleviated by NH4 +. Inhibition by citrate was competitive with fructose 6-phosphate in the absence of NH4 + ions. The K i values for citrate were 1·0 mm (with no NH4 +) and 7·2 mm (with 10 mm-NH4 +). Long-chain fatty acyl-CoA esters had no significant inhibitory effect. It is concluded that the interplay between the prevailing intracellular concentrations of NH4 + and citrate is the major determinant of the activity of PFK in vivo and thus governs the extent to which glucose is converted either to lipid or carbohydrate.

Candida tropicalis Pk 233 produced extracellular glutathione during growth in filamentous form caused by adding ethanol (⩾ 2·5%, v/v). The intracellular concentration of glutathione also was greater in cultures with added ethanol. myo-Inositol added at a physiological concentration (5 µg ml−1) prevented the ethanol-induced production of extracellular glutathione as well as the morphological change. In batch culture, production of extracellular glutathione was optimal at an ethanol concentration of 5%, and reached a maximum concentration of 42 mg ml−1 after 96 h cultivation, before decreasing gradually.

Glucan synthases of Saprolegnia assayed in the presence of MgCl2 [(1→4)-β-glucan production] were stimulated by ATP or GTP, while the non-phosphorylating nucleotide analogues adenylyl (β,γ-methylene)diphosphonate and guanylyl (·γ-methylene)diphosphonate did not stimulate glucan synthesis. The analogues adenosine 5′-O-(3-thiotriphosphate) and guanosine 5′-O-(3-thiotriphosphate) stimulated the enzyme activity but to a lesser extent than the nucleotides. The activators stimulated membrane-bound enzymes, particularly those equilibrating at the endoplasmic reticulum density in sucrose gradients. Glucan synthases solubilized by digitonin were not stimulated by nucleotides. Nucleotides, whose action seems to be related to the integrity of the phosphate groups, may produce their stimulative effect by increasing the translocation of the substrate through membranes to the enzymes.

Acid-soluble and alkali-insoluble glucan fractions were prepared from yeast, hyphal and germ-tube forming cells of Candida albicans. Alkali-insoluble glucan was also extracted from purified yeast cell walls. Paper chromatography of partial acid hydrolysates confirmed that the glucan preparations contained β(1→3)-and β(1→6)-chains but no mixed intra-chain β(1→3)/β(1→6) linkages. Methylation and 13C-NMR analyses showed that the acid-soluble glucan consisted of a highly branched polymer composed mainly (67·0% to 76·6%) of β(1→6)-linked glucose residues. The alkali-insoluble glucan from yeast and hyphal cells contained from 29·6% to 38·9% β(1→3) and 43·3% to 53·2% (1→6) linkages. Alkali-insoluble glucan from germ-tube forming cells consisted of 67·0% β(1→3) and 14% β(1→6) linkages. Branch points accounted for 6·7%, 12·3% and 17·4% of the residues in the alkali-insoluble glucan of yeast, germ-tube forming and hyphal cells, respectively.

2-Deoxy-d-glucose added to cultures of Candida albicans in the stationary phase of growth inhibited the incorporation of glucose into the (1→3)-β-glucan fraction of the organisms. In the presence of ATP and cell extracts it was converted to 2-deoxy-d-glucose phosphate and when UTP was also present, material with the electrophoretic properties of UDP-2-deoxy-d-glucose was formed. In similar conditions glucose formed glucose phosphates, UDP-glucose and other products. Evidence was obtained that the analogue, after conversion to a phosphate derivative, was an inhibitor of phosphoglucomutase.

When C. albicans was grown in the presence of 2-deoxy-d-glucose for 24 h, analogue residues became incorporated into the (1→3)-β-glucan fraction and the subsequent rate of incorporation of glucose into that fraction was enhanced. The rate of turnover of glucose in this β-glucan fraction was greater than in controls. Pretreatment of cultures with β-glucanase, or incubation under conditions known to stimulate endogenous β-glucanases, increased the subsequent rate of glucose incorporation and this increase was enhanced by growth in the presence of 2-deoxy-d-glucose. The analogue thus had the effect of altering the stability and glucose-acceptor function of (1→3)-β-glucan chains. This could affect the properties of the polymer network leading to the known effect of the analogue in delaying the onset of phenotypic resistance to amphotericin methyl ester in stationary phase cultures of C. albicans.

The soluble and membrane-bound cytochromes c of methylotrophic bacteria were investigated in order to determine their relationships with each other, and their roles in electron transport. The proportions of soluble cytochromes cH and cL in various methylotrophs were not markedly dependent on the type of methylotroph nor on conditions of growth. Between 30 and 50% of the cytochrome c of Methylophilus methqdotrophus was bound tightly to membranes; 8% of this was cytochrome cH, 37% was cytochrome cLand 55% was the cytochrome c component of the oxidase, cytochrome co. These cytochromes were purified and characterized. It was concluded that cytochrome cH has no role in the membrane. By contrast, the membrane cytochrome cL has a separate role from that of soluble cytochrome cL; the membrane-bound cytochrome cL may play a role analogous to that of mitochondria1 cytochrome c1 in mediating between cytochrome b and the terminal oxidase during the oxidation of NADH.

Representative mycelial fungi from the phycomycete, ascomycete and basidiomycete groups (Achlya bisexualis, Neurospora crassa, Aspergillus nidulans, Schizophyllum commune and Coprinus cinereus) all generated steady electrical currents around their hyphal tips; the generation of a transhyphal ion current may therefore be a universal characteristic of hyphal growth. As with all other tip growing organisms, positive current always entered apically and left distally; non-growing hyphae did not drive transcellular currents. The current density, measured approximately 30 m from the membrane surface at the hyphal tips, varied between 0·05 and 0·60 μA cm−2 in different fungi and tended to be larger in wider, rapidly extending hyphae than in thinner, slow growing hyphae. The possibility that these currents serve to localize growth at the apex is discussed.

A plasmid, pCL34, capable of autonomous replication in Escherichia coli and Pseudomonas aeruginosa has been constructed which carries the promoter and structural gene (amiE) for P. aeruginosa amidase, but not the regulator gene (amiR). Plasmid pCL34 has been mobilized from E. coli to P. aeruginosa using the broad host range plasmid RP4. Complementation studies were performed in P. aeruginosa strains carrying various amidase mutations. Measurements of amidase activity in the recipients under inducing, non-inducing and repressing conditions showed trans-complementation by the chromosomally located regulator gene product. These results confirmed the positive control model for amidase gene expression. Levels of amidase expression seen during these studies were approximately threefold higher than in the parental, amidase-positive strains.

The polypeptides present in 35S-labelled chromatin prepared from Escherichia coli cells, and polypeptides present in the DNA and RNA complexes obtained by micrococcal nuclease digestion of the chromatin, were analysed by two-dimensional non-equilibrium polyacrylamide gel electrophoresis. Three hundred and thirty-five 35S-labelled polypeptides were detected in the chromatin whereas the DNA- and RNA-containing fractions of the micrococcal nuclease digest contained 126 and 183 polypeptides respectively. The major basic low-molecular-weight polypeptides were found in the DNA-containing fractions.

Elementary bodies of lymphogranuloma venereum (LGV) strains of Chlamydia trachomatis contain, in addition to the genomic DNA, a 6·7 kb plasmid. The plasmid from serovar L2 (434-B) was cloned at the BamHI site of pBR327 into Escherichia coli and a restriction cleavage map of this pLGV125 recombinant plasmid determined. All 15 C. trachomatis serovars contained DNA sequences that hybridized with pLGV125. When total DNA from L2 elementary bodies was used as a probe in Southern blotting and spot hybridization, serovars L1, L2 and L3 exhibited significant homology. The detection level of homologous DNA was 100 pg and LGV DNA was detectable in infected cells when total L2 probe was used in the nucleic acid hybridization test. These DNA probes may be useful as investigative and diagnostic reagents for C. trachomatis.

Transformation of competent transformable Neisseria gonorrhoeae F62 to streptomycin resistance was unaffected by antibodies directed against the pilus protein (pilin) of this organism. The pilin component of either crude or purified pilus preparations, separated by SDS gel electrophoresis and transferred to nitrocellulose, failed to bind detectable amounts of DNA; DNA binding to other gonococcal polypeptides was observed under these conditions. These results suggest that gonococcal pilin does not play a direct role in gonococcal transformation.

The mechanism of resistance to the arginine analogue l-canavanine, and of arginine uptake, were examined in the fission yeast Schizosaccharomyces pombe. Two mutants with increased resistance to canavanine were analysed genetically: both were double mutants, and in each case one mutation conferred resistance to canavanine, while the other enhanced this resistance. Evidence is presented that can1.1 strains are defective in one system for arginine uptake, which presumably prevents entry of canavanine into the cell. This system operates in the wild-type whether the nitrogen source supplied is ammonium or glutamate. Double mutants carrying can1.1 and an arginine requirement are unable to grow on ammonium medium even when supplied with exogenous argine, while growth can occur on glutamate plus arginine. This suggested the existence of a second uptake system for arginine which is absent during growth on ammonium, and direct measurements of the rates of arginine uptake under various conditions confirmed this. Our observations closely parallel those made on the budding yeast Saccharomyces cerevisiae. The ability to select for or against function of the can1 gene should facilitate certain types of genetical analysis in S. pombe.

The effect of photon fluence rate on Anacystis nidulans grown in Mg2+- and non-Mg2+-limited continuous cultures was studied. A decline in photon fluence rate from 445 to 30 μE m−2 s−1 resulted in a decrease of the mean cell volume from 2 to 0·8 μm3 in the Mg2+-limited culture. Compared with non-Mg2+-limited cultures, this was the only obvious difference found when decreasing the photon fluence rate. Altering the photon fluence rate for a Mg2+-limited chemostat therefore seems to be a suitable system for studies of the Mg2+-controlled events in cell division of A. nidulans. At constant dilution rate, the DNA content per unit cell volume was independent of photon fluence rate, while the RNA content decreased with this factor. The RNA/protein ratio decreased by a factor of 3 in both Mg2+- and non-Mg2+-limited cultures, when decreasing the photon fluence rate from 445 to 100 μE m−2 s−1. The RNA efficiency therefore varied with photon fluence rate and the organism seemed to have surplus RNA at high light intensities.