Aspartate biosynthesis is essential for the growth of Streptococcus thermophilus in milk, and aspartate availability modulates the level of urease activity

Abstract

We investigated the carbon dioxide metabolism of Streptococcus thermophilus, evaluating the phenotype of a phosphoenolpyruvate carboxylase-negative mutant obtained by replacement of a functional ppc gene with a deleted and inactive version, Deltappc. The growth of the mutant was compared to that of the parent strain in a chemically defined medium and in milk, supplemented or not with L-aspartic acid, the final product of the metabolic pathway governed by phosphoenolpyruvate carboxylase. It was concluded that aspartate present in milk is not sufficient for the growth of S. thermophilus. As a consequence, phosphoenolpyruvate carboxylase activity was considered fundamental for the biosynthesis of L-aspartic acid in S. thermophilus metabolism. This enzymatic activity is therefore essential for growth of S. thermophilus in milk even if S. thermophilus was cultured in association with proteinase-positive Lactobacillus delbrueckii subsp. bulgaricus. It was furthermore observed that the supplementation of milk with aspartate significantly affected the level of urease activity. Further experiments, carried out with a p(ureI)-gusA recombinant strain, revealed that expression of the urease operon was sensitive to the aspartate concentration in milk and to the cell availability of glutamate, glutamine, and ammonium ions.

FIG. 1.

Schematic representation of the enzymatic activities involved in aspartate biosynthesis and urea metabolism in Streptococcus thermophilus. The molecules underlined act on transcription of ure operon. ppc, gene coding for phosphoenolpyruvate carboxylase; aspA, gene coding for aspartate aminotransferase; glnA, gene coding for glutamine synthetase, ureIABCDEFG, operon coding for urease; GluT, hypothetical glutamate membrane transporter. The wide arrow crossing the GluT represents the competitive effect of aspartic acid on the uptake of glutamate.

FIG. 2.

Growth of S. thermophilus wild type (A) and PEP carboxylase-negative mutant A18(Δppc) (B) in CDM (□) or CDM supplemented with l-Asp at a 0.075 mM (⧫), 0.23 mM (▴), 0.75 mM (▪), or 2.25 mM (•) concentration. All CDM cultures were repeated three times, and all standard deviations were <0.05 OD₆₀₀ units.

FIG. 3.

Growth of S. thermophilus wild type (A) and PEP carboxylase negative mutant A18(Δppc) (B) in reconstituted SM (□) or SM supplemented with l-Asp at 0.075 mM (⧫), 0.23 mM (▴), or 0.75 mM (▪). All milk cultures were repeated three times, and standard deviations were always <0.08 OD₆₀₀ units.

FIG. 4.

Acidification rate of S. thermophilus wild type (A) and PEP carboxylase-negative mutant A18(Δppc) (B) in reconstituted SM (□), 0.75 mM l-Asp (▪), or 0.75 mM l-Asp and flurofamide (10 μM) (▵). The decrease in the acidification rate due to urease activity is indicated by arrows. All milk cultures were repeated three times, and the standard deviations were always <0.08 pH units.

FIG. 5.

Growth of mixed cultures of S. thermophilus mutant A18(Δppc) and DSM 20617 wild type with Lactobacillus delbrueckii subsp. bulgaricus ATCC 11842, CNRZ 397, or CNRZ 1159 (CNRZ 397 PrtB negative) in reconstituted SM. (A) Population of A18(Δppc) cultured alone (⧫) or A18(Δppc) in mixed culture of A18(Δppc)/ATCC 11842 (▴), A18(Δppc)/CNRZ 397 (▪), or A18(Δppc)/CNRZ 1159 (□). (B) Population of DSM 20617 cultured alone (⧫) or DSM 20617 in mixed culture of DSM 20617/ATCC 11842 (▴), DSM 20617/CNRZ 397 (▪), or DSM 20617/CNRZ 1159 (□). All milk cultures were repeated three times, and standard deviations were always <0.2 log₁₀ CFU ml⁻¹.