Phosphoenolpyruvate and 2-phosphoglycerate: endogenous energy source(s) for sugar accumulation by starved cells of Streptococcus lactis

Abstract

In the absence of an exogenous energy source, galactose-grown cells of Streptococcus lactis Mb3 rapidly accumulated thiomethyl-,8-galactopyranoside (TMG) and 2-deoxyglucose to intracellular concentrations of 40 to 50 mM. Starved cells maintained the capacity for TMG uptake for many hours, and accumulation of the f8-galactoside was insensitive to proton-conducting ionophores (tetrachlorosalicylanilide and carbonylcyanide-m-chlorophenyl hydrazone) and sulfydryl group reagents including iodoacetate and N-ethylmaleimide. Fluorimetric analysis of glycolytic intermediates in extracts prepared from starved cells revealed (a) high intracellular levels of phosphoenolpyruvate (13 mM; PEP) and 2-phosphoglycerate (-39 mM; 2-PG), but (b) an absence of other metabolites including glucose 6-phosphate, fructose 6-phosphate, fructose 1,6diphosphate, and triosephosphates. The following criteria showed PEP (and 2-PG) to be the endogenous energy source for TMG accumulation by the phosphotransferase system: (i) the intracellular concentrations of PEP and 2-PG decreased with concomitant uptake of TMG, and a close correlation was observed between maximum accumulation of the B-galactoside and the total available concentration of the two intermediates; (ii) TMG accumulated as an anionic derivative, which after extraction and incubation with alkaline phosphatase (EC 3.1.3.1) formed the original analogue; (iii) fluoride inhibition of 2-phospho-Dglycerate hydrolyase (EC 4.2.1.11) prevented the conversion of 2-PG to PEP, and uptake of TMG by the starved cells was reduced by 80%; and (iv) the stoichiometric ratio [TMG] accumulated/[PEP] consumed was almost unity (0.93). In cells metabolizing glucose, all intermediates listed in (a) and (b) were found. Upon exhaustion of glucose from the medium, the metabolites in (b) were not longer detectable, while the intracellular concentrations of PEP and 2-PG increased to the levels previously observed in starved cells. The glycolytic intermediates in (b) are all in vitro heterotropic effectors of pyruvate kinase (adenosine 5'triphosphate:pyruvate 2-O-phosphotransferase, EC 2.7.1.40) from S. lactis ML3. It is suggested that the capacity of starved cells to maintain high intracellular concentrations of PEP and 2-PG is a consequence of decreased in vivo activity of this key regulatory enzyme of glycolysis. Streptococcus lactis and S. faecalis possess certain physiological characteristics (10, 11, 14) that have proved extremely useful in the study of energy coupling to solute accumulation by microorganisms (9, 10, 34). For example, under normal growth conditions these organisms lack a functional cytochrome system and so are unable to produce adenosine 5'-triphosphate (ATP) via oxidative phosphorylation. S. lactis and S. faecalis usually rely upon glycolysis or arginine catabolism for the generation of the compounds that serve directly (phosphoenolpyruvate, PEP) or indirectly (ATP) as energy

Quantitative analysis of cellulose degradation and growth of cellulolytic bacteria in the rumen

“…Growth and maintenance can be described by À dS/ dt  Y = mx1ax, but this equation does not address 'starvation' (Dawes, 1985), when transmembrane gradients and ATP pools dissipate unless endogenous metabolism prevents this de-energetization. Bacteria with phosphotransferase systems (PTS) reserve a pool of phosphoenolpyruvate and can use it to reinitiate transport (Thompson & Thomas, 1977). However, cellulolytic ruminal bacteria lack a PTS (Maas & Glass, 1991;Chow & Russell, 1992) and use stored glycogen to prevent cellular death (Wells & Russell, 1994).…”

Regulation of sugar transport and metabolism in lactic acid bacteria

“…In 1969-1970 McKay and coworkers [18,19] first described the lac-PTS in the lactic strepto- cocci by in vitro complementation of soluble components (EI, HPr, EIII l"~) with a sugar-specific membrane fraction (Eli la~) prepared from S. lactis. However, it was not until 1977 following the discovery of the endogenous PEP-potential in starved cells of S. lactis [20] that the importance of PEP, and the highly efficient coupling of sugar transport to phosphorylation via the lac-PTS, were demonstrated in vivo. The PEP-potential, which was comprised of three glycolytic intermediates (3-PGA, 2-PGA and PEP, approx.…”

“…The PEP-potential, which was comprised of three glycolytic intermediates (3-PGA, 2-PGA and PEP, approx. 40 mM total, Table 1), was formed at the onset of sugar exhaustion and was maintained by some species for many hours e.g., S. lactis [20,21], S. pyogenes [22], L. casei [23], S. faecalis [24,25] or for a matter of minutes as for some strains of S. cremoris [26]. The biochemical basis for the formation of the endogenous PEP-potential has been the subject of controversy [20,21,[26][27][28].…”

“…40 mM total, Table 1), was formed at the onset of sugar exhaustion and was maintained by some species for many hours e.g., S. lactis [20,21], S. pyogenes [22], L. casei [23], S. faecalis [24,25] or for a matter of minutes as for some strains of S. cremoris [26]. The biochemical basis for the formation of the endogenous PEP-potential has been the subject of controversy [20,21,[26][27][28]. However, data obtained by enzymatic analysis [20,21], [14C]fluorography [27] and in vivo [31p]NMR spectroscopy [27,28] suggest that the establishment of the PEPpotential (Fig.…”

“…The biochemical basis for the formation of the endogenous PEP-potential has been the subject of controversy [20,21,[26][27][28]. However, data obtained by enzymatic analysis [20,21], [14C]fluorography [27] and in vivo [31p]NMR spectroscopy [27,28] suggest that the establishment of the PEPpotential (Fig. 3,A and B) results from the inactivation of pyruvate kinase by: (a) depletion of positive effectors of the allosteric enzyme (e.g., FDP, [29][30][31][32]), and (b) marked increase in intracellular Pi concentration, a potent inhibitor of PK in vitro [29,31].…”