Supply-demand analysis of energy metabolism in Lactococcus lactis under anaerobic conditions

[摘要] ENGLISH ABSTRACT: The interests in understanding the metabolic processes of microbial systems are numerous. The interest in the species Lactococcus lactis (L. lactis) lies in applications to the food industry and in studies comparing the metabolism of related organisms.The aim of this study was to perform in vivo supply-demand analysis on anaerobically fermenting L. lactis. This was done by perturbing both the supply and demand pathways, then measuring glycolytic flux (by means of 13C NMR spectroscopy) and intracellular ATP/ADP (by means of 31P NMR spectroscopy) at steady state – where the central metabolite, ATP, is produced at the same rate as it is consumed and its concentration thereby remains constant. The L. lactis reference strain MG1363 was supplemented with glucose and analysed 'online by 13C-NMR under anaerobically-fermentative conditions. The rates of glucose consumption and lactate production were determined from this 13C flux. Due to experimental difficulties with the online detection of 31P (possibly due to the low biomass yield and the choice of growth medium), ATP/ADP levels had to be determined offline: from the same batch cultures as the 13C samples, fermentations were performed and halted at time points when the cells had attained a steady state. These fermentation cultures were then subjected to cell lysis and centrifugation in order to extract intracellular metabolites. These cell extracts were analysed offline by 31P NMR in order to determined levels of phosphate metabolites, specifically ATP and ADP.Perturbation of the supply pathway was achieved by utilising a genetically modified strain (the CS8 strain with over-expressed las operon) and comparing it to the reference strain MG1363. This resulted in a slight increase in ATP/ADP, but also yielded a slightly reduced flux, which is contrary to expectations from a mutant with over expressed glycolytic enzymes.The demand pathway was perturbed by two methods: 1) utilisating a genetically modified strain (the BK1506 strain with over-expressed F1-ATPase) and comparing it to the reference strain MG1363, and 2) by treating wild-type MG1363 with sodium acetate and comparing flux and ATP/ADP values to the untreated wild-type. Sodium acetate dissociates in the cytoplasm and causes dissipation of the transmembrane proton motive force, which is re-established by upregulation of membrane-bound H+-translocating ATPases. While the use of genetically modified strains provided only one flux-vs-ATP/ADP data point to compare to the wild-type (not sufficient for complete supply-demand analysis), the treatment of the wild type with uncoupler yielded several data points where flux and ATP/ADP values differed according to the concentration of uncoupler added.The CS8 strain demonstrated a 19 % reduced glucose flux (24 % reduced lactate flux) with respect to the wild type MG1363. The BK1506 strain demonstrated a 72 % increase in glucose flux (33 % increase in lactate flux) with respect to the wild type. The treatment with 2 mM acetate resulted in a 72 % increase in glucose flux (123 % increase in lactate flux), whereas treatment with 4 mM acetate resulted in a 107 % and a 126 % increase in glucose and lactate fluxes, respectively. The treatment with different concentrations of acetate provided several data points with corresponding flux and ATP/ADP, enabling the calculation of the elasticity coefficient of the supply pathway to changes in ATP/ADP (εsupply ) which was found to be -5.6 and -6.3 for glucose and lactate, respectively.ATP/ADPThe elasticity coefficient was high compared with values obtained in similar studies on other organisms. Considering that at steady state the supply and demand fluxes are equal, the high supply elasticity (which is easier to measure), when incorporated into control coefficient summation theorems, gives the indication that: 1) a greater amount of control may reside in the ATP demand pathway (the elasticity of which is more difficult to determine experimentally, but which may well be lower than the supply elasticity), and 2) ATP/ADP homeostasis is good, as indicated by a high elasticity of the supply pathway to ATP/ADP. This study represents a basis for further supply-demand analysis with non-growing batch cultures of L. lactis.

[发布机构] Stellenbosch University