Molecular Physiology of Sugar Catabolism in Lactococcus lactis IL1403

Abstract

The metabolic characteristics of Lactococcus lactis IL1403 were examined on two different growth media with respect to the physiological response to two sugars, glucose and galactose. Analysis of specific metabolic rates indicated that despite significant variations in the rates of both growth and sugar consumption, homolactic fermentation was maintained for all cultures due to the low concentration of either pyruvate-formate lyase or alcohol dehydrogenase. When the ionophore monensin was added to the medium, flux through glycolysis was not increased, suggesting a catabolic flux limitation, which, with the low intracellular concentrations of glycolytic intermediates and high in vivo glycolytic enzyme capacities, may be at the level of sugar transport. To assess transcription, a novel DNA macroarray technology employed RNA labeled in vitro with digoxigenin and detection of hybrids with an alkaline phosphatase-antidigoxigenin conjugate. This method showed that several genes of glycolysis were expressed to higher levels on glucose and that the genes of the mixed-acid pathway were expressed to higher levels on galactose. When rates of enzyme synthesis are compared to transcript concentrations, it can be deduced that some translational regulation occurs with threefold-higher translational efficiency in cells grown on glucose.Lactococcus lactis is generally recognized as the model organism for the study of lactic acid bacteria, and the complete genome sequence of the IL1403 strain (3, 4) will undoubtedly consolidate this position. When growing on rapidly metabolized sugars, this species shows homolactic metabolism in which more than 90% of metabolized sugar is converted to lactic acid. However, under certain conditions, this homolactic metabolism is replaced by a shift in pyruvate metabolism towards alternative fermentation pathways. Under anaerobic conditions, this shift involves an increased flux through the pyruvate-formate lyase reaction and leads to mixed acid fermentation and the accumulation of formate, acetate, and ethanol. These products accumulate in only low quantities during homolactic metabolism but have been shown to account for the majority of carbon flux under circumstances in which the rate of glycolysis of sugars is significantly diminished.This shift in pyruvate metabolism has been correlated with the NADH/NAD ratio (11). When flux through glycolysis is high, the high NADH/NAD ratio favors lactate dehydrogenase activity and provokes the inhibition of glyceraldehyde-3P dehydrogenase activity upstream of pyruvate. Under such conditions, metabolite pools upstream of glyceraldehyde-3P dehydrogenase (notably triose-Ps) increase within the cell, due to the controlling influence of this enzyme on glycolytic flux (10). Triose-Ps have a negative allosteric effect on pyruvate-formate lyase activity, thereby greatly diminishing the in vivo activity of the enzyme. Sugars metabolized at diminished rates lead to the relaxation of this coordinated control and facilitate the shift towards the more energetically fav...

Transcriptional, translational and metabolic regulation of glycolysis in Lactococcus lactis subsp. cremoris MG 1363 grown in continuous acidic cultures

“…the cells were in the same physiological state -were analysed in parallel. The standard deviation value was found to be approximately constant for the various genes of glycolysis and the mean standard deviation value for the mRNA cellular concentrations was found to be ±36 % (Fontaine et al, 2001;Even et al, 2001). As done previously (Even et al, , 2002, expression ratios were considered to be significantly different when differing by at least two standard deviations (i.e.…”

“…Since the total RNA concentration was normalized in the assay (2 mg), the alkaline phosphatase activity revealed the abundance of a specific messenger in the total RNA population. This value was corrected using the cellular RNA concentration, which is known to vary with the environmental conditions, in order to obtain the cellular concentration of a specific messenger as described previously (Fontaine et al, 2001;Even et al, 2001Even et al, , 2002. Results were standardized using the values obtained from the steady-state culture at pH 6?6 and expressed as expression ratios.…”

“…Membranes were incubated in a solution containing anti-digoxigenin conjugate coupled to alkaline phosphatase (75 mU ml 21 ) (Roche). The alkaline phosphatase substrate AttoPhos (Amersham Pharmacia Biotech) was then added and the membranes were scanned with a phosphofluoroimager (STORM 860, Molecular Dynamics) as described previously (Fontaine et al, 2001;Even et al, 2001). Quantification was performed at different times after the addition of AttoPhos, leading to the calculation of the alkaline phosphatase activity for each slot.…”

“…PCR-based macro-arrays that included all the glycolytic genes were made on nylon membranes as described previously (Fontaine et al, 2001;Even et al, 2001). Pre-hybridization, hybridization and washing steps were performed as described by Fontaine et al (2001).…”

“…As described previously (Fontaine et al, 2001;Even et al, 2001Even et al, , 2002, total RNA was extracted using a variant of the acid/phenol method and quantified spectrophotometrically; 2 mg of this RNA was then chemically labelled with digoxigenin and stored at 220 uC until hybridization.…”