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, ⌬ppc. 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.

The relevance of carbon dioxide metabolism in Streptococcus thermophilus

“…Wild-type PpC + CpS + S. thermophilus DSM 20617 T , its PpC 2 derivative A18(Dppc) (Arioli et al, 2007), and the PpC 2 CpS 2 double mutant A19(DppcDcarB), were maintained in M17 broth (Difco Laboratories) at 37 uC. Plasmid-containing S. thermophilus strains were maintained in M17 broth (10 g lactose l 21 ) supplemented with 5 mg erythromycin ml 21 , at 28 uC, while strains containing the pG + host9-derived vector integrated into the chromosome were maintained in the M17 broth supplemented with 2 mg erythromycin ml 21 , at 42 uC.…”

“…Escherichia coli strains were routinely maintained in Luria-Bertani broth at 37 uC with aeration; the medium was supplemented with 10 mg kanamycin ml 21 and, when necessary, with 200 mg erythromycin ml 21 . The auxotrophy for aspartic acid, arginine and uracil of S. thermophilus wild-type, A18(Dppc) and A19(DppcDcarB) was evaluated in a chemically defined medium (CDM) containing lactose (10 g l 21 ); this medium was derived from that described by Reiter & Oram (1962), and modified as described by Arioli et al (2007). A mixture containing lactose, salts and vitamins was prepared at a concentration that was 26 the concentration in CDM.…”

“…The DcarB gene was obtained by PCR, as previously described (Arioli et al, 2007;Mora et al, 2004). Briefly, DNA fragments located upstream and downstream of the 1687 bp deletion were independently amplified using CarB1-CarB2 and CarB3-CarB4 primer pairs (CarB1, 59-CTATTATCATTGGTCAGGCT-39; CarB2, 59-GTTACATACCACA-AGAGCAT-39; CarB3, 59-GCGTGATGCCGAAGACAATGCTCTTG-TGGTATGTAACTCCAAGAGACTATCGCTGA-39; CarB4, 59-TAT-CAAGGGCTGTGAAGAGT-39).…”

“…1). Recently, the CO 2 metabolism of S. thermophilus has been investigated by evaluating the phenotype of a PpC 2 mutant (Arioli et al, 2007). That work demonstrated that the fixation of CO 2 by PpC activity is fundamental for the biosynthesis of aspartic acid, and for the growth of S. thermophilus in milk (Arioli et al, 2007).…”

“…Recently, the CO 2 metabolism of S. thermophilus has been investigated by evaluating the phenotype of a PpC 2 mutant (Arioli et al, 2007). That work demonstrated that the fixation of CO 2 by PpC activity is fundamental for the biosynthesis of aspartic acid, and for the growth of S. thermophilus in milk (Arioli et al, 2007). That study also reported that the availability of aspartic acid modulates the level of urea amidohydrolase activity, underlining the existence of a link between CO 2 and N 2 metabolism in this species.…”