A convenient, sensitive, and reliable assay for the conversion of radiolabeled sucrose to alcohol-insoluble dextran by the Streptococcus mutans dextransucrase has been developed.

The dextransucrase (EC 2.4.1.5) activity from cell-free culture supernatants of Streptococcus mutans strain 6715 has been purified approximately 1,500-fold by ammonium sulfate precipitation, hydroxylapatite chromatography, and isoelectric focusing. The enzyme was eluted as a single peak of activity from hydroxylapatite, and isoelectric focusing of the resulting preparation gave a single band of dextransucrase activity which focused at a pH of 4.0. The final enzyme preparation contained two distinct, enzymatically active proteins as judged by assay in situ after polyacrylamide gel electrophoresis. One of the proteins represented 90% of the total dextransucrase activity and 53% of the total protein. The molecular weight of the enzyme was estimated by gel filtration to be 94,000. The temperature optimum of the enzyme was broad (34 to 42 C) and its pH range was rather narrow, with optimal activity at pH 5.5. The Km for sucrose was 3 mM, and fructose competitively inhibited the enzyme reaction with a Ki of 27 mM. Streptococcus mutans has been associated with dental caries in experimental animals (7, 14) and man (3, 6, 15). The cariogenic potential of this bacterium is thought to reside in its ability to produce water-soluble and waterinsoluble extracellular dextrans from sucrose (8, 9, 13, 27). The S. mutans dextrans are made up of a-(1-6)-linked glucose molecules with a high and variable proportion of a-(1-3)-linked branch points (11, 17, 18). The heterogeneity of the S. mutans dextrans has been proposed (12) to be due to the production of multiple forms of the enzyme dextransucrase (EC 2.4.1.5). This enzyme occurs in a cell-associated (insoluble) state and in a cell-free (soluble) form (10, 20, 22). In one study (12), the soluble enzyme was fractionated into at least seven activities which demonstrated distinct physical properties. This observation and the importance of elucidating the exact mechanism of dextran synthesis by S. mutans seemed to warrant further investigation of the dextransucrase produced by this organism. In this communication, we report on the purification and properties of dextransucrase activity from S. mutans strain 6715. MATERIALS AND METHODS Bacteria and growth conditions. S. mutans strain 6715 was obtained from the Forsyth Dental Center, Boston, and was used throughout this investigation. Cells were grown anaerobically (Gas Pak system,

Dextran stimulation (priming) of the dextransucrase (EC 2.4.1.5) from Streptococcus mutans strain 6715 was studied. The dextransucrase activity in supernatant fluids from glucose-grown cultures was shown to be partially primer dependent. During extended storage at 4 C the enzyme retained its activity. However, the ability to make dextran became increasingly primer dependent. Hydroxylapatite-chromatographed enzyme preparations were completely dependent upon added dextran for rapid synthesis of methanol-insoluble glucan from sucrose. Half-maximal stimulation of new dextran synthesis occurred with dextran at a concentration of 2 to 3 uM and with a molecular weight of about 2,600. Neither glycogen, amnylose, inulin, nor isomaltose functioned as primer. Studies with the dextransucrase activities detectable by in situ assay in polyacrylamide gels subjected to electrophoresis under nondenaturing conditions revealed that the major activity was detectable in the presence of sucrose alone and was stimulated by addition of primer dextran. The minor activity was only detected when primer dextran was present. Homogeneous preparations of both enzymes contained 30 to 40% carbohydrate.

We attempted to purify dextransucrase from S mutans strain 6715 to investigate its properties and determine if multiple species of the enzyme existed. It was concluded that the properties of this enzyme such as the pH (5.5), temperature (37 C) optimum, and Km for sucrose (3 mM) are very similar to those reported for S sanguis, S bovis, S mutans strain OMZ-176 isozymes, S mutans strain GS-5, and the single dextransucrase purified from S mutans strain HS-6. The IEF enzyme preparation consisted of two enzyme species, possibly differing in their ability to synthesize different dextran linkages. The minor enzyme activity demonstrated a strict primer dependency. Similarly, primer dependency has been reported for dextransucrases from S mutans, S sanguis, and L mesenteroides. S mutans strain 6715 dextransucrase also showed both the insertion and stepwise mechanisms for dextran synthesis. Sucrose was the sole glucose donor, whereas dextran was a specific, highly efficient glucose acceptor. The complex primer kinetics are not fully understood at this time and require further investigation. Without linkage analysis of the products of our enzymes, we can only postulate that each enzyme has a different function in the synthesis of interresidue and interchain alpha1-3 and alpha1-6 bonds. Insoluble dextran synthesis may involve a special enzyme mechanism characteristic of S mutans. This synthesis would require both enzymes, possibly in some aggregated form, with one enzyme synthesizing endogenous primer dextran. This endogeneous primer or some cell wall polysaccharide could stimulate both enzymes to rapidly synthesize heterogeneously linked insoluble dextran.

A marine bacterium, Vibrio MB22, has been studied to determine the pattern of feedback regulation of the first enzyme unique to the biosynthesis of the aromatic amino acids, 3-deoxy-D-arabino-heptulosonate 7-phosphate (DAHP) synthetase. The crude extract was used to study response of the enzyme to various salts as well as possible feedback inhibitors. Ethylenediaminetetraacetic acid was found to be inhibitory to enzyme activity, and only CoCl2, of the salts tested, allowed full recovery as well as apparent stimulation of the DAHP synthetase activity. The DAHP synthetase activity was inhibited solely by the aromatic amino acids, tyrosine, tryptophan, and phenylalanine, of the possible effectors tested. Further work demonstrated the existence of three isozymes of DAHP synthetase, each primarily inhibited by one of the aromatic amino acids. 'Journal Series no. 4063, Agriculture Experiment Station.