- Volume 140, Issue 3, 1994

Glucose-repressed growth of Saccharomyces cerevisiae was analysed in a nitrogen-limited continuous culture at different dilution rates (D). The glucose consumption of the yeast decreased from 3.4 g g-1 h-1 to 3.0 g g-1 h-1 when D was decreased from 0.3 h-1 to 0.15 h-1. No transcripts of the SUC2 and HXK1 genes, encoding, respectively, invertase and hexokinase isoenzyme 1, could be detected. Because both genes are regulated by glucose repression at the transcriptional level, this confirmed that the culture was glucose repressed at every D. During the decrease in D, no change in the activities or mRNA levels of key enzymes in carbon metabolism was observed, except for alcohol dehydrogenases I and II and phosphoglucomutase. These enzymes increased in activity and/or mRNA level when D was decreased, which was also observed in glucose- and galactose-limited continuous cultures. This demonstrates that the expression levels of alcohol dehydrogenases I and II, and also phosphoglucomutase, are coupled to the growth rate of the organism. A comparison between the alcohol dehydrogenase II activity in glucose- and nitrogen-limited continuous cultures demonstrated that the growth rate contributes as much to repression of alcohol dehydrogenase II activity as does glucose. Both the glucose consumption and the activity of the glycolytic enzymes were relatively constant when D was decreased and, as a consequence, the concentrations of intracellular metabolites remained constant. A slight decrease in the glucose 6-phosphate concentration was observed, which could be caused by the slight decrease in glucose consumption at low D values. The 2-oxoglutarate and cAMP concentrations increased twofold when D was decreased. The first probably reflects the increased NH4 consumption at high D values, while the latter is caused by the high amount of extracellular cAMP compared with the amount of intracellular cAMP. The decrease in growth rate raised the amount of biomass, while nitrogen was limiting in all cases. Analysis of the biomass composition at the different D values revealed that the amount of nitrogen per gram dry weight was constant, while the amounts of carbon, hydrogen and oxygen were higher at low D values. In addition, higher concentrations of trehalose and glycogen were found at low D values. This demonstrates that the glucose which is used at high D values for the production of biomass is converted into storage carbohydrates, e.g. trehalose and glycogen, at low D values. A nitrogen pulse did not result in a dramatic response of S. cerevisiae except for a rapid change in the free amino acid pool. Thus S. cerevisiae is not able rapidly to enhance growth upon a nitrogen pulse, which is in contrast with the response to a glucose pulse of a carbon-limited continuous culture of S. cerevisiae.

Saccharomyces cerevisiae T23C (pda1::Tn5ble) is an isogenic gene replacement mutant of the wild-type strain S. cerevisiae T23D. The mutation causes a complete loss of pyruvate dehydrogenase activity. Pyruvate metabolism in this pyruvate-dehydrogenase-negative (Pdh-) strain was investigated in aerobic glucose-limited chemostat cultures, grown at a dilution rate of 0.10 h-, and compared with the metabolism in the isogenic wild-type strain. Under these conditions, growth of the Pdh- strain was fully respiratory. Enzyme activities in cell-free extracts indicated that the enzymes pyruvate decarboxylase, acetaldehyde dehydrogenase and acetyl-coenzyme A (acetyl-CoA) synthetase could provide a functional bypass of the pyruvate dehydrogenase complex. Since this metabolic sequence involves ATP hydrolysis in the acetyl-CoA synthetase reaction, a negative effect of the pda1::Tn5ble mutation on the growth efficiency was anticipated. Indeed, the biomass yield of the Pdh− strain [0.44 g biomass (g glucose)-1] was significantly lower than that of wild-type S. cerevisiae [0.52 g biomass (g glucose)-1]. The effect of the mutation on biomass yield could be quantitatively explained in terms of a lower ATP yield from glucose catabolism and an increased ATP requirement for the synthesis of acetyl-CoA used in anabolism. Control experiments showed that the pda1::Tn5ble mutation did not affect biomass yield in ethanol-limited chemostat cultures. The results support the view that, during aerobic glucose-limited growth of S. cerevisiae at low growth rates, the pyruvate dehydrogenase complex accounts for the major part of the pyruvate flux. Moreover, it is concluded that hydrolysis of pyrophosphate formed in the acetyl-CoA synthetase reaction does not contribute significantly to energy transduction in this yeast. Respiratory-deficient cells did not contribute to glucose metabolism in the chemostat cultures and were probably formed upon plating.

The accumulation of compatible solutes, such as glycine betaine, is known to stimulate growth under conditions of osmotic stress. In Salmonella typhimurium the accumulation of glycine betaine is mediated by two osmotically activated transport systems, ProP and ProU. This study was undertaken to determine the quantitative relationship between glycine betaine accumulation from the environment and growth stimulation, and also the relative roles of the high affinity (ProU) and low affinity (ProP) transport systems. Our data show that relatively low concentrations of glycine betaine (= 10μM) are sufficient to stimulate growth and that under these conditions ProP and ProU transport systems are equivalent. At external concentrations of glycine betaine below 1 μM, cells able to express the ProU transport system possess a significant advantage over cells that only possess ProP. At high osmolarity the correlation between growth stimulation and cytoplasmic glycine betaine concentration is limited. At low glycine betaine concentrations further accumulation of the compatible solute stimulated growth. However, once the cells had accumulated 100 nmol glycine betaine per OD650 unit biomass no greater growth stimulation was observed in cells with higher levels of the compatible solute. The implications of these data for growth and pathogenicity of bacteria in natural ecosystems, such as foods, are discussed.

Different classes of cell wall degrading enzymes produced by the biocontrol fungi Trichoderma harzianum and Gliocladium virens inhibited spore germination of Botrytis cinerea in a bioassay in vitro. The addition of any chitinolytic or glucanolytic enzyme to the reaction mixture synergistically enhanced the antifungal properties of five different fungitoxic compounds against B. cinerea. The chemicals tested were gliotoxin, flusilazole, miconazole, captan and benomyl. Dose response curves were determined for each combination of toxin and enzyme, and in all cases the ED50 values of the mixtures were substantially lower than ED50 values of the two compounds used alone. For instance, the addition of endochitinase from T. harzianum at a concentration of 10λg ml-1 reduced the ED50 values of toxins up to 86-fold. The level of synergism appeared to be higher when enzymes were combined with toxins having primary sites of action associated with membrane structure, compared with pesticides having multiple or cytoplasmic sites of action. Among enzymes tested, the highest levels of synergism with synthetic fungicides were detected for the endochitinase from T. harzianum strain P1, which, when used alone, was the most effective chitinolytic enzyme against phytopathogenic fungi of those tested. The use of hydrolytic enzymes to synergistically enhance the antifungal ability of fungitoxic compounds may reduce the impact of some chemical pesticides on plants and animals.

The dsRNA viruses of the corn pathogen Ustilago maydis encode toxins that affect sensitive strains of the same species and related species. The KP6 toxin encoded by the P6 virus subtype is a binary toxin, consisting of two polypeptides, α (8-6 kDa) and β (9-1 kDa) with no covalent or hydrogen bonds between them. In this study the effect of each polypeptide was tested on sensitive and resistant cells and spheroplasts of U. maydis. The results indicate that both polypeptides bind to intact cells from sensitive and resistant strains; however, only spheroplasts of sensitive strains are affected by the toxin and both α and β are necessary to affect the spheroplasts.

A series of Tn5-induced mutants which showed enhanced symbiotic effectiveness (Eff++) was isolated from Rhizobium meliloti strains CXM1-105 and CXM1-188. Alfalfa plants inoculated by the Eff++ mutants had significantly higher shoot dry mass than plants inoculated by the parental strains. In the greenhouse, the most effective mutants increased the shoot dry mass of the host plants by 23-26% and plant total nitrogen by 23-27%. Interestingly, the frequency of the Eff++ mutants in strain CXM1-188 was higher than in the strain CXM1-105 (1.1% versus 0.4%); this was also the case for the auxotrophic mutants (1.2% in CXM1-188 versus 0.3% in CXM1-105). Genetic analysis of the mutants showed that the enhanced symbiotic effectiveness was cotransducible with Tn5. By the use of Southern hybridization and plasmid transfer, it was found that ten Tn5 insertions were located in the chromosome, five in megaplasmid 1, and six in megaplasmid 2.

Erwinia amylovora CFBP1430 was mutagenized by phage MudllPR13 insertion Thirty prototrophic pathogenicity mutants with a single insertion were isolated. Among these, 17 non-pathogenic (Path-) mutants were obtained; 11 of them were unable to induce a hypersensitive response (HR) on tobacco (Hrp- mutants), whereas the remaining six were still able to do so (Dsp- mutants). Thirteen other mutants showed reduced virulence (Rvi-) and were still able to induce an HR. One of them appeared to be Path- on apple seedlings and Rvi- pear seedlings. All the Hrp- mutants and all but three of the Dsp- mutants mapped in the hrp-dsp gene cluster previously reported. Some Rvi- mutants also proved to map in this hrp-dsp region; most of them, as well as two Dsp- mutants map in an unknown genomic region. Cell-surface components thought to play a role in bacterial pathogenicity were examined, including exopolysaccharides (EPS), lipopolysaccharides (LPS), and outer-membrane proteins. One mutant only was found to be non-capsulated and unable to produce EPS. The insertion in this mutant mapped in a genomic cluster involved in amylovoran synthesis. Unlike the parental strain, some mutants exhibited sensitivity to the phage Ffm and this phenotype was associated with a modified LPS electrophoretic profile. Rvi- mutants and some Path- mutants were able to multiply in planta to some extent; other Path- mutants reached only a low population level, except the non-capsulated one, which rapidly decreased to an undetectable level.