Twofold Reduction of Phosphofructokinase Activity in Lactococcus lactis Results in Strong Decreases in Growth Rate and in Glycolytic Flux

Abstract

Two mutant strains of Lactococcus lactis in which the promoter of the las operon, harboring pfk, pyk, and ldh, were replaced by synthetic promoters were constructed. These las mutants had an approximately twofold decrease in the activity of phosphofructokinase, whereas the activities of pyruvate kinase and lactate dehydrogenase remained closer to the wild-type level. In defined medium supplemented with glucose, the growth rate of the mutants was reduced to 57 to 70% of wild-type levels and the glycolytic flux was reduced to 62 to 76% of wild-type levels. In complex medium growth was even further reduced. Surprisingly, the mutants still showed homolactic fermentation, which indicated that the limitation was different from standard glucose-limited conditions. One explanation could be that the reduced activity of phosphofructokinase resulted in the accumulation of sugar-phosphates. Indeed, when one of the mutants was starved for glucose in glucose-limited chemostat, the growth rate could gradually be increased to 195% of the growth rate observed in glucosesaturated batch culture, suggesting that phosphofructokinase does affect the concentration of upstream metabolites. The pools of glucose-6-phosphate and fructose-6-phosphate were subsequently found to be increased two-to fourfold in the las mutants, which indicates that phosphofructokinase exerts strong control over the concentration of these metabolites.Lactococcus lactis plays an important role in dairy fermentations, mainly in the production of cheeses. During such fermentation processes, lactose is present at very high concentrations (50 g/liter) and is converted through glycolysis to primarily form lactic acid as well as minor amounts of other compounds (homolactic fermentation). The resulting low pH contributes to the texture and flavor of cheeses and inhibits the growth of other bacterial species. During homolactic fermentation more than 90% of the lactose consumed is recovered as by-products (42), which shows that the glycolytic pathway functions almost exclusively as a catabolic pathway to supply ATP to the cells. When sugar becomes limiting for growth, or in the presence of a less readily metabolized carbon source, the pattern of product formation switches from homolactic to mixedacid fermentation, i.e., to the formation of formate, ethanol, and acetate with smaller amounts of lactate (43).The mechanisms responsible for regulation of glycolytic flux and the shift between different fermentation modes in L. lactis have been studied intensively. The concentrations of intermediary metabolites and cofactors are affected by the external sugar concentration (8,10,37,46). In the presence of excess sugar, the concentrations of fructose-1,6-bisphosphate, the triose-phosphates, and pyruvate and the NADH/NAD ϩ ratio are high, whereas the concentrations of phosphoenolpyruvate and inorganic phosphate are relatively low. The glycolytic flux was proposed to be regulated through the level of fructose-1,6-bisphosphate (43), which is known to activate both pyruvate kinase ...

Loss of Antibiotic Tolerance in Sod-Deficient Mutants Is Dependent on the Energy Source and Arginine Catabolism in Enterococci

“…However, all attempts to obtain this mutant failed, suggesting that the upper part of the glycolysis pathway is essential in E. faecalis. The finding that a 2-fold reduction of phosphofructokinase activity strongly decreased the growth rate of the lactic acid bacterium Lactococcus lactis is in agreement with this conclusion (33).…”

CTP Limitation Increases Expression of CTP Synthase in Lactococcus lactis

“…Growth of the pyrG::lacLM fusion strain in defined medium with cytidine and galactose or maltose as a carbon source increased the doubling time to 157 and 111 min, respectively, compared to 56 min when grown on glucose (Table 3). These doubling times are similar to previous observations of growth of MG1363 on defined medium (1,24). The nucleoside triphosphate pools, and thus the CTP pool, are lower when the strain is grown on these carbon sources than when it is grown on glucose.…”

The Glycolytic Flux in Escherichia coli Is Controlled by the Demand for ATP

“…The HPLC equipment used was from Shimadza Corporation and was controlled by the system program Class VP 5.0. The separation was performed as described earlier (1).…”

“…In order to obtain a quantitative answer as to how strong the control is, we split the energy metabolism into a block of enzymatic reactions that synthesize ATP (i.e., e 1 Under the assumption that anabolism and catabolism is only linked via ATP, this block elasticity approach is independent of the complexity of the components in the blocks (11,29). The control of flux in this simple system can be calculated from the "elasticities" (the metabolic control analysis word for sensitivities …”