Growth profiles of <i>L. lactis</i> at different pH values.

<p>Growth was performed in CDM with 1% glucose (w/v) at 30 ^°C, with pH controlled at 6.5, 5.1 and 4.8. pH was controlled by addition of 10 M NaOH. Growth rate (μ): pH 6.5, 0.97±0.08 h^-1; pH 5.1, 0.63±0.04 h^-1; pH 4.8, 0.53±0.01 h^-1. Symbols: diamond, pH 6.5; triangle, pH 5.1; square, pH 4.8. Data shown are representative from at least two identical experiments.</p

Sequences of ¹³C-NMR spectra of <i>L. lactis</i> cell suspensions (pH 4.8 and 5.5), showing separate resonances due to intra- and extracellular lactic acid.

<p>Due to the fast rate of proton exchange each resonance represents the total contribution of the two forms of lactic acid (dissociated and non-dissociated forms). The extracellular pH varies by about 0.2 units because of a lag in the addition of base in the circulating system used for the NMR experiment.</p

Effect of various conditions on the maximal glucose uptake rate in <i>L. lactis</i>.

<p>Effect of pH alone (white bars), extracellular lactic acid (grey bars) and intra and extracellular lactic acid (black bars) on <i>V</i>_max (nmol min^-1 mg protein^-1) for glucose uptake assessed by [¹⁴C] radio-labelled assays with <i>L. lactis</i> cells. Glucose was added to a final concentration of 0.1 mM.</p

Time courses obtained by <i>in vivo</i> NMR during glucose metabolism in <i>L. lactis</i>.

<p>A, B, C and D: time courses obtained during the metabolism of [1-¹³C] glucose in non-growing cells of <i>L. lactis</i> as monitored online by <i>in vivo </i>¹³C-NMR. E and F: biochemical parameters determined during the metabolism of 40 mM glucose in <i>L. lactis</i> MG1363 as monitored online by <i>in vivo </i>³¹P-NMR. The experiments were carried out at 30 ^°C, under anaerobic conditions and pH controlled at 6.5 (orange diamonds), 5.5 (black diamonds), 5.1 (blue diamonds) and 4.8 (green diamonds). (A) Kinetics of [1-¹³C] glucose (40 mM) consumption, (B) extracellular pool of lactic acid, (C) pools of FBP (fructose 1,6-bisphosphate), (D) profiles of intracellular lactic acid, (E) profiles of intracellular P_i, and (F) intracellular pH. The time points for glucose exhaustion are indicated by vertical dashed lines (graph A, B, C, D and E). The horizontal lines in graph F represent the constant values at which the extracellular pH was controlled. The lack of information on intra and extracellular concentrations of lactic acid at pH 6.5 is due to severe overlap of the lactic acid resonances at this pH and consequent large uncertainty in the measurements of individual areas. Each experiment was performed at least twice with good reproducibility.</p

Determination of <i>n</i>, the number of protons extruded from the cell concomitantly with each lactate.

<p>Profiles of total amounts of internal (open diamonds) and external (filled diamonds) lactate (mmol) in <i>L. lactis</i> cell suspensions metabolizing glucose at pH 5.5 (A), 5.1 (B) and 4.8 (C) by cells grown at pH 6.5 or cells at pH 5.1 and previously grown at pH 5.1 (D). The experimental points represented were obtained while glucose was available. The black lines indicate the value of <i>n</i>, that was obtained numerically from exponential fits to the intra- and extracellular lactic acid curves.</p

A qualitative model for pH effects and acid stress adaptation of <i>L. lactis</i> cells.

<p>(A) Non-adapted cells (grown at pH 6.5 and suspended in buffer at pH 5.1); and (B) adapted (grown at pH 5.1 and suspended in buffer at pH 5.1). Dotted arrows indicate metabolic effects with the sign indicated; signs without arrows indicate transcriptional effects. Abbrev: HLac, non-dissociated form of lactic acid; Lac-, lactate anion. The adapted and non-adapted energized cells both had the same internal pH of 6.3.</p