Criterion for Rapid Classification

In recent years much interest has focused upon Streptococcus mutans as the etiological agent of smooth-surface caries in both rodents (7, 8, 10) and humans (5, 11, 12). Although similar in many respects to other homolactic streptococci found in the oral environment, a nutritional characteristic which has been used to distinguish S. mutans is its ability to utilize either mannitol or sorbitol as a primary energy source (6, 8, 9). Our recent studies with S. mutans 10449 (Brown and Wittenberger, submitted for publication) have shown that both mannitol and sorbitol are ultimately converted to the glycolytic intermediate fructose-6-phosphate by the action of an inducible mannitol-l-phosphate dehydrogenase or an inducible sorbitol-6-phosphate dehydrogenase after initial phosphorylation of the hexitols to mannitol-l-phosphate or sorbitol-6-phosphate. The purpose of this communication is to report on comparative studies performed on hexitol phosphate dehydrogenases isolated from a variety ofS. mutans strains. All organisms were originally obtained from H. V. Jordan and were grown anaerobically in the complex medium described previously (2) with either mannitol, sorbitol, or glucose as the primary energy source. Cell-free extracts were also prepared, as described previously (2), by sonification of the cells obtained from a 150-ml culture which had been washed in 0.05 M potassium phosphate buffer, pH 7.0, before being suspended in 12 ml of 0.05 M tris(hydroxymethyl)aminomethane (Tris) hydrochloride buffer, pH 7.5, which contained 0.005 M ,B-mercaptoethanol. The hexitol phosphate dehydrogenases were assayed at 25 C by monitoring the hexitol phosphate-dependent reduction of nicotinamide adenine dinucleotide (NAD) at 340 nm with a Gilford model 240 spectrophotometer equipped with a Gilford model 6040 recorder. The assay system contained the following components in a final volume of 1.0 ml: mannitol-I phosphate or sorbitol-6-phosphate, 2.0 ,umoles; NAD, 1.0 ,umoles; Tris-HCl, pH 8.5, 100 ,umoles; and distilled water to volume. The reactions were always initiated by the addition of enzyme and one unit is the amount of enzyme required to catalyze the reduction of 1 j,mole of NAD per minute. All strains of S. mutans examined had very high levels of hexitol phosphate dehydrogenase activity in extracts obtained from hexitol-adapted cells, whereas extracts obtained from glucosegrown cells had only low levels of enzyme activity (Table 1). Consequently, the induction of mannitol-l-phosphate dehydrogenase and sorbitol-6-phosphate dehydrogenase occurred in all S. mutans strains studied and was not a property unique to S. mutans strain 10449. It is of interest to note in Table 1 that the specific activity of mannitol-l-phosphate dehydrogenase and sorbitol-6-phosphate dehydrogenase was approximately the same in all S. mutans strains examined, with the exception of S. mutans strain FA-1 where the specific activity of both hexitol phosphate dehydrogenases was two to three times higher than in extracts obtained from other S. mutans strains. The physiological significance of relatively higher levels of hexitol phosphate debydrogenases in S. mutans strain FA-1 is not clear at this time. Polyacrylamide, disc gel electrophoresis of cell-free extracts from a number of S. mutans strains yielded the results shown in Fig. 1 when the gels were stained for hexitol phosphate dehydrogenase activity. Four classes of mannitol-l-