Low-Affinity Sucrose Phosphotransferase Activity

Abstract

Sucrose metabolism by the oral pathogen Streptococcus mutans plays at least two roles in the ability of this organism to initiate carious lesions in teeth. (i) Sucrose is a substrate for glucosyltransferases that synthesize extracellular glucans which are important in the colonization of S. mutans in the oral cavity. (ii) Sucrose is an efficient fermentation substrate for S. mutans, leading to excretion of lactic acid, which is responsible in part for the initiation of dental caries (2, 6). The mechanism and regulation of sucrose uptake in S. mutans has therefore been studied extensively (2a, 4, 5, 8-12). Sucrose is taken up in S. mutans by as many as three different uptake systems (2a, 11). The best-characterized of these is a high-affinity (Ki,m ca. 70 ,uM) phosphoenolpyruvate (PEP)-dependent phosphotransferase system (PTS) which depends on the protein products of the genes scrA, which encodes a sucrose-specific enzyme II of the PTS (8), and scrB, which encodes a sucrose 6-phosphate hydrolase (7). A second sucrose PTS with a Km of ca. 250 ,uM was discovered by Slee and Tanzer (11) in a mutant of S. mutans that lacked the high-affinity sucrose uptake system. A third, low-affinity, sucrose uptake system (Ki,, ca. 3 mM) which is independent of the PTS has also been described by a number of workers (4, 10, 11). As a first step in the characterization of these latter two uptake systems, we have investigated the identity of the lower-affinity sucrose PTS in S. mutans by using a mutant carrying a Mu dE insertion in the scrA gene (8). The GS-5 strain of S. mutans used in this study and a derivative of this strain containing a Mu dE insertion in scrA (insertion 188) (8) were provided by H. Kuramitsu. Cells were grown at 37°C to the midexponential phase on TYEMOPS medium (5) containing the appropriate sugar at 0.4% and were harvested and washed as previously described (5). PEP-dependent sucrose phosphorylation was assayed in toluene-treated cells (5) by using [14C]sucrose (5 ,uCi/,mol; Dupont, NEN Research Products) at the concentrations indicated below. We have reported that Mu dE insertions into scrA nearly completely abolished sucrose uptake in S. mutans GS-5 when measured at 25 ,uM sucrose (8). However, some residual sucrose phosphorylation activity dependent on PEP was detected at a sucrose concentration of 0.1 mM in these mutants (8). Since Slee and Tanzer (11) had reported a second sucrose uptake activity dependent on the PTS, it seemed reasonable that this residual activity in the insertion

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Cite this paper

@inproceedings{PoyLowAffinitySP, title={Low-Affinity Sucrose Phosphotransferase Activity}, author={Florence Poy and Gary R Jacobson} }