The pool of ADP and ATP regulates anaerobic product formation in resting cells of Lactococcus lactis

Abstract

Lactococcus lactis grows homofermentatively on glucose, while its growth on maltose under anaerobic conditions results in mixed acid product formation in which formate, acetate, and ethanol are formed in addition to lactate. Maltose was used as a carbon source to study mixed acid product formation as a function of the growth rate. In batch and nitrogen-limited chemostat cultures mixed acid product formation was shown to be linked to the growth rate, and homolactic fermentation occurred only in resting cells. Two of the four lactococcal strains investigated with maltose, L. lactis 65.1 and MG1363, showed more pronounced mixed acid product formation during growth than L. lactis ATCC 19435 or IL-1403. In resting cell experiments all four strains exhibited homolactic fermentation. In resting cells the intracellular concentrations of ADP, ATP, and fructose 1,6-bisphosphate were increased and the concentration of P(i) was decreased compared with the concentrations in growing cells. Addition of an ionophore (monensin or valinomycin) to resting cultures of L. lactis 65.1 induced mixed acid product formation concomitant with decreases in the ADP, ATP, and fructose 1,6-bisphosphate concentrations. ADP and ATP were shown to inhibit glyceraldehyde-3-phosphate dehydrogenase, lactate dehydrogenase, and alcohol dehydrogenase in vitro. Alcohol dehydrogenase was the most sensitive enzyme and was totally inhibited at an adenine nucleotide concentration of 16 mM, which is close to the sum of the intracellular concentrations of ADP and ATP of resting cells. This inhibition of alcohol dehydrogenase might be partially responsible for the homolactic behavior of resting cells. A hypothesis regarding the level of the ATP-ADP pool as a regulating mechanism for the glycolytic flux and product formation in L. lactis is discussed.

FIG. 1.

Maltose consumption and product formation in pH-controlled batch fermentation of L. lactis 65.1. Symbols: ▪, optical density at 620 nm (OD₆₂₀); •, maltose concentration; ▴, lactate concentration; □, formate concentration; ○, acetate concentration; ▵, ethanol concentration.

FIG. 2.

Percentage of lactate (A) and concentrations of ADP and ATP (B), FBP (C), and P_i (D) in L. lactis 65.1 cells metabolizing 10 g of maltose per liter. The culture conditions were as follows: mid-exponential growth (n = 6) (bars 1), mid-exponential growth with 30 mM arginine (n = 3) (bars 2), resting cells with 0.5 μM monensin (n = 3) (bars 3), resting cells with 2 μM valinomycin (n = 3) (bars 4), and resting cells (n = 6) (bars 5), where n is the number of cell samples analyzed for intracellular metabolites.

FIG. 3.

Inhibition kinetics for dehydrogenase in vitro activity as a function of ADP and ATP. (A) GAPDH; (B) LDH; (C) ADH. The inhibitors were ATP (○) and ADP (•). ADP_crit and ATP_crit are the inhibitor concentrations at which the enzyme was totally inhibited, and n is the degree of inhibition. The values for these parameters were estimated by fitting equation 2 through the data points.

FIG. 4.

Schematic pathway for metabolism of maltose to anaerobic fermentation end products. A plus sign in a circle and a minus sign in a circle indicate metabolites that activate and inhibit dehydrogenase activities in L. lactis, respectively. MP, maltose phosphorylase; GK, glucokinase; PTA, phosphotransacetylase; ACK, acetate kinase; GAP, glyceraldehyde 3-phosphate; DHAP, dihydroxyacetone phosphate; CoA, coenzyme A.