The tolerance to acid and the acid-neutralizing activity of three important periodontopathic bacteria, Porphyromonas gingivalis, Prevotella intermedia and Fusobacterium nucleatum were studied. P. gingivalis strains grew only at neutral pH and did not utilize glucose, whereas strains of P. intermedia and F. nucleatum could grow under acidic conditions and increased their growth by utilizing glucose. P. gingivalis tended to raise the culture pH during growth. P. intermedia and F. nucleatum raised the culture pH during growth in the absence of glucose, while in the presence of glucose they decreased the pH. Resting cell suspensions of all the bacteria raised the pH in the presence of tryptone and casamino acids. Acid-neutralizing activity was confirmed by measuring base production at a fixed pH with a pH-stat. During neutralization, the cells produced cytotoxic substrates, ammonia and organic acids (butyric, isobutyric and isovaleric acids by P. gingivalis; isovaleric and succinic acids by P. intermedia; propionic and butyric acids by F. nucleatum). These findings suggest that deamination of amino acids into ammonia and organic acids occurs simultaneously with base production, resulting in acid neutralization. These results could partially explain the survival of P. intermedia and F. nucleatum in both supragingival and subgingival plaque and the apparent restriction of P. gingivalis to subgingival plaque. The former bacteria may aid in creation of an environment fostering colonization of subgingival plaque by P. gingivalis.
The effect of pH on the growth and proteolytic activity of the type strain and fresh isolates of Porphyromonas gingivalis and Bacteroides intermedius was investigated. B. intermedius strains grew with and without glucose at a pH as low as 5.0. These bacteria grew almost as well as Streptococcus mutans at pH 5.0 and better than Actinomyces viscosus at pH 5.5 and 5.0. Some B. intermedius strains raised the culture pH when grown at a low pH without glucose. In contrast, P. gingivalis strains grew only at pH 6.5 to 7.0. The P. gingivalis strains had proteolytic activities against azocoll, azocasein, and azoalbumin, while the B. intermedius strains degraded azocasein and azoalbumin, but not azocoll. B. intermedius showed maximum proteolytic activity at pH 7.0, and high activity over a wide pH range. In contrast, the optimum pH of proteolytic activity in P. gingivalis was pH 7.5 to 8.0. The P. gingivalis activities were more sensitive than those of B. intermedius to low pH. The capacity of B. intermedius to degrade proteins to more readily metabolizable substrates at low pH might explain the growth of this bacterium in an acidic environment. These differences between B. intermedius and P. gingivalis could explain their capacity to survive at different sites in the oral cavity and indicate how B. intermedius might positively influence the growth of P. gingivalis in subgingival plaque.
Changes in human dental plaque pH can be used to obtain estimates of the acidogenic potential of ingested foods. The presence of acid in plaque is influenced by a large number of host, microbial, and substrate factors. Several useful methods have been developed for monitoring changes in plaque pH. Plaque sampling involves repeated removal of small samples of plaque from a number of teeth at intervals after food ingestion, dispersion of the sample, and in vitro measurement of pH. Touch electrode methods utilize glass or antimony microelectrodes, which are placed onto plaque in situ where direct readings can be obtained. Telemetry methods involve placement of glass microelectrodes or ion-sensitive field effect transistors within the dentition. Plaque is allowed to accumulate, and pH changes can subsequently be transmitted with radio or wire. Each of the methods has clear advantages and limitations. The methods have been simultaneously compared in human volunteers using solutions of fermentable carbohydrate. Inter-method differences in response were observed depending upon the site of measurement. Data obtained from caries-prone surfaces via telemetry showed lower pH minima and retarded returns to resting pH levels. The technology is available for controlled comparative plaque pH studies, with the method of choice depending upon the goals of the investigation. It is essential that the results be compared to data obtained with other models designed to evaluate the cariogenic potential of foods.
Streptococcus mutans, S sanguis, and S salivarius use a phosphoenolpyruvate (PEP)-dependent phosphotransferase system that results in phosphorylation of glucose at carbon 6. This enzyme system is not sensitive to fluoride. Glucose uptake into resting cell suspensions is sensitive to fluoride because of inhibition of intracellular PEP production. The glucose phosphotransferase system is constitutive in oral streptococci.
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,
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