Control of glycolysis by glyceraldehyde-3-phosphate dehydrogenase in Streptococcus cremoris and Streptococcus lactis

Abstract

The decreased response of the energy metabolism of lactose-starved Streptococcus cremoris upon readdition of lactose is caused by a decrease of the glycolytic activity (B. Poolman, E. J. Smid, and W. N. Konings, J. Bacteriol. 169:1460-1468. The decrease in glycolysis is accompanied by a decrease in the activities of glyceraldehyde-3-phosphate dehydrogenase and phosphoglycerate mutase. The steady-state levels of pathway intermediates upon refeeding with lactose after various periods of starvation indicate that the decreased glycolysis is primarily due to diminished glyceraldehyde-3-phosphate dehydrogenase activity. Furthermore, quantification of the control strength exerted by glyceraldehyde-3-phosphate dehydrogenase on the overall activity of the glycolytic pathway shows that this enzyme can be significantly rate limiting in nongrowing cells.In a previous communication, we have presented evidence that the survival of Streptococcus cremoris upon lactose starvation in chemostat cultures is related to the glycolytic capacity maintained (12). A rapid decrease of glycolytic activity was observed for lactose-starved cells in growth medium (anaerobically) and in phosphate-lactate buffer (aerobically) (12). Initially, the decrease of glycolysis did not result in a lower steady-state level of the proton motive force (Ap), but the rate of proton motive force generation was severely affected. However, when glycolysis decreased further over time, the high proton motive force could not be maintained and the activity of proton motive force-driven processes, like amino acid transport, decreased (12). In this paper, the cause of the drop in glycolytic activity upon (lactose) starvation has been analyzed.Enzymatic activities. The decrease in glycolysis during starvation as described previously (12) was also observed for S. cremoris Wg2 (and S. lactis ML3) cells grown on complex broth (4) in batch cultures (Fig. 1). Cells were grown to an optical density at 660 nm of about 0.8, washed twice, and suspended in sterile phosphate-lactate buffer (pH 6.4) as described elsewhere (12). At the times indicated, samples were taken and the acidification of the medium was monitored (11). To study the effect of starvation on the activities of glycolytic enzymes, cell-free extracts were prepared (10) after three different periods of starvation, as indicated by the arrows (Fig. 1). Enzymatic activities were determined by standard procedures involving NAD, NADH-, or NADPcoupled assays (3). The effects of starvation on the activites of glyceraldehyde-3-phosphate (G3P) dehydrogenase (EC 1.2.1.12) and phosphoglycerate mutase (EC 5.4.2.1) are shown in Table 1. Both activities decreased concomitant with the decrease in glycolysis. The activities of all other glycolytic enzymes were not significantly affected by the starvation regime (data not shown). The activities of these enzymes varied between 1 and 6 U/mg of protein, except for phosphoglycerate kinase (EC 2.7.2.3), which had an activity of about 0.4 U/mg of protein, independent of the starvation ...

Physiological responses of Lactococcus lactis ML3 to alternating conditions of growth and starvation

“…Although the loss of glycolytic activity could be correlated with specific loss of glyceraldehyde 3-phosphate dehydrogenase, phosphoglycerate mutase and pyruvate kinase activity, two-dimensional gel analyses indicated that overall protein degradation is non-specific during starvation. In a previous study (Poolman et al 1987b) it has been shown that during lactose starvation of L. lactis subsp, cremoris Wg2 glyceraldehyde 3-phosphate dehydrogenase is inactivated but not (specifically) degraded. A similar situation may hold for L. lactis The analysis of arginine/ornithine exchange activities in membrane vesicles isolated from growing and starving cells indicates that the transport system is not inactivated during starvation; rather a slight increase in exchange activity is observed (Fig.…”

“…Cells were broken by cycle sonication (8 x 15 s with 45-s intervals; amplitude intensity of 8 gm) under mtrogen at 4 ~ To remove the cell debris the extracts were centrifuged for 60 rain at 48000 • g at 4 ~ The enzymatic activities in the cell lysates were determined by standard procedures involving NAD(H)-or NADPcoupled assays (Bergmeier 1974), essentially as described by Poolman et al (1987b).…”

“…In the first hour(s) of lactose starvation ofL. lactis, when culture is still fully viable, ATP levels fall to concentrations below 0.1 mM, and the membrane potential (A ~) and pH gradient (ApH) collapse (Otto et al 1985;Poolman et al 1987b). In the initial phase of starvation a transient increase in "PEP-pool" intermediates (3-phosphoglycerate, 2-phosphoglycerate and phosphoenolpyruvate) has been observed (Mason et al i981;Otto et al 1985;Poolman et al 1987a; Thompson and Thomas 1977).…”

“…The loss of glycolytic activity in Lactococcus lactis subsp. cremoris Wg2 has been associated with specific loss of glyceraldehyde 3-phosphate dehydrogenase and phosphoglycerate mutase activities (Poolman et al 1987b). It has also been shown that under certain conditions glyceraldehyde 3-phosphate dehydrogenase can be significantly rate limiting for glycolysis in L. lactis (Poolman et al 1987b), emphasizing the importance of the enzyme in starvation survival.…”

Lactococcus lactis glyceraldehyde-3-phosphate dehydrogenase gene, gap: further evidence for strongly biased codon usage in glycolytic pathway genes

“…therefore has a key role in glycolysis. This has been underscored by metabolic studies which showed that the decrease in glycolytic activity observed after starved L. lactis cells were provided with an energy source was primarily due to diminished glyceraldehyde-3-phosphate dehydrogenase activity (Poolman et a/., 1987). Since the publication of the amino acid sequence of lobster glyceraldehyde-3-phosphate dehydrogenase (Davidson e t al., 1967), the primary structures and predicted sequences of glyceraldehyde-3-phosphate dehydrogenases from many organisms have been reported, and all of the sequences share considerable similarity (Fothergill-Gilmore & Michels, 1993).…”