mutans UA159 genome (Fig. 1). We also identified a cysteine aminopeptidase C gene (SMU.466) downstream of the

tcyABC locus. In Lactococcus lactis, the PepC aminopeptidase has broad substrate specificity and hydrolyzes napthylamide-substituted amino acids and di- and tri-peptides; its activity can be inhibited by thiol-group-blocking compounds, but not by serine or metalloproteinase inhibitors (Kredich, 1992; Lau et al., this website 2002). The presence of cysteine aminopeptidases suggests that S. mutans can access smaller peptides to free up amino acids such as cysteine. To confirm the role of TcyABC in cystine uptake, we tested the abilities of S. mutans UA159 wild-type strain and its ΔtcyABC mutant to transport l-[14C]cystine. As shown in Fig. 2, a significant (55%) decrease

in l-[14C]cystine uptake was observed in the ΔtcyABC mutant SmTcyABC compared with wild-type UA159 cells (0.48 ± 0.13 vs. 1.06 ± 0.49 nmol mg−1 dry cell per min, respectively) over 8 min, consistent with TcyABC being involved in l-cystine transport. In both the mutant and Volasertib order wild-type UA159 strains, l-cystine uptake was highest in the first 2 min and then continued linearly at a decreased rate, tapering off in both strains after 10 min. Not surprisingly, transport of l-cystine was not completely abolished in the mutant, given that there are other transporters involved in the uptake of l-cystine in S. mutans. Studies of cystine uptake in B. subtilis have also shown the highest uptake in the first 2 min at a rate of 1.9 nmol min−1 mg−1 of protein, which then continued linearly up to 6 min and began to plateau, while a ΔtcyJKLMN mutant showed a decreased cystine transport rate of 1.4 nmol min−1 mg−1 of protein relative to the parent strain (Burguiere et al., 2005). It is likely that the drastic impairment of cysteine uptake in S. mutans was because of the presence of only two cysteine transport systems in this bacterium, which is in contrast to that of B. subtilis equipped with three cystine transport systems. To determine the substrate specificity

of the S. mutans TcyABC transport system, we measured the level of inhibition of l-[14C]cystine uptake in the presence of a 100-fold excess of different unlabeled amino acids and sulfur-containing compounds in the wild-type strain and its ΔtcyABC mutant. When unlabeled l-cystine Phosphatidylinositol diacylglycerol-lyase was added in excess, a 94% decrease in l-[14C]cystine uptake was observed in the wild-type strain confirming the transporter specificity for l-cystine. In the wild-type strain, unlabeled cystathionine, djenkolic acid, S-methyl-l-cysteine, and cysteine competitively inhibited cystine uptake by > 50% (Fig. 3) while arginine, glutamine, glutamate, leucine, and methionine did not effectively inhibit l-cystine uptake. In contrast, the SmCysBPA mutant showed very little l-cystine uptake and no inhibition by unlabeled l-cystine, djenkolic acid, and S-methyl-l-cysteine.