Our previous continuous culture studies with strains of Streptococcus mutans have indicated that the organism has the capacity of adapt to growth in acidic environments. This study was undertaken to examine this question in more detail. S. mutans Ingbritt and the phosphotransferase system (PTS)-defective mutant, S. mutans DR0001/6, were grown in continuous culture at pH 7.5 and 5.5 or 5.1, and the pH optimum for glucose uptake and glycolysis and the capacity of the cells to generate pH gradients were determined over the pH range 4.5 to 8.0 with steady state, washed cells. In addition, the proton permeability of the cells was measured over the pH range by an acid pulse technique. The results indicate that the pH optimum for glucose uptake by S. mutans Ingbritt grown at pH 7.5 was 7.5 and this optimum shifted to 7.0 and 6.0 for cells grown at pH 5.5 and 5.1, whereas with the S. mutans DR0001/6, the optimum shifted from 7.5 to 7.0 for the pH 5.5 cells. A similar shift in the pH optimum for glycolysis was observed for the 2 strains, and this was particularly pronounced for cells incubated with glucose in the presence of gramicidin to dissipate proton gradients. The capacity of the cells to generate pH gradients was related to their metabolic activity, and although larger gradients were not formed by the pH 5.5 cells, these cells were nevertheless capable of maintaining gradients at a lower pH; S. mutans DR0001/6 generated 2-fold larger pH gradients at pH 5.5 than S. mutans Ingbritt.(ABSTRACT TRUNCATED AT 250 WORDS)

The global distribution of individual species of oral bacteria demonstrates their ability to survive among their human hosts. Such an ubiquitous existence is the result of efficient transmission of strains and their persistence in the oral environment. Genetic analysis has identified specific clones of pathogenic bacteria causing infection. Presumably, these express virulence-associated characteristics enhancing colonization and survival in their hosts. A similar situation may occur with the oral resident flora, where genetic variants may express specific phenotypic characteristics related to survival. Survival in the mouth is enhanced by dental plaque formation, where persistence is associated with the bacteria's capacity not only to adhere and grow, but also to withstand oxygen, wide fluctuations in pH and carbohydrate concentration, and a diverse array of microbial interactions. Streptococcus mutans has been discussed as a 'model' organism possessing the biochemical flexibility that permits it to persist and dominate the indigenous microflora under conditions of stress.

Using 21 species of oral bacteria, representing six acidogenic genera, we undertook to determine whether the pH-limiting exponential growth is related to the ability of the organisms to generate an acid-tolerance response that results in enhanced survival at low pH. The lower pH limit of exponential growth varied by more than two units with that of Neisseria A182 at pH 6.34; growth of Lactobacillus casei RB1014 stopped at pH 3.81, with species of Actinomyces, Enterococcus, Prevotella and Streptococcus falling between these limits. The working hypothesis was that the organisms with the higher pH limits for growth are unable to respond to acidic environments in order to survive, whereas the more aciduric organisms would possess or acquire acid tolerance. Adaptation to acid tolerance was tested by determining whether the prior exposure of exponential-phase cells to a low, sub-lethal pH would trigger the induction of a mechanism that would enhance survival at a pH killing pH 7.5 control cells. The killing pH varied from pH 4.5 for Prevotella intermedia ATCC 25611 to pH 2.3 for the three Lactobacillus casei strains in the study, with the three Streptococcus mutans strains killed at pH 3.0 for 3 h. The adaptation experiments revealed three groups of organisms: non-acid-responders, generally representing strains with the highest terminal pH values; weak acid-responders in the middle of the pH list, generating low numbers of survivors at one or two pH values, and the aciduric, strong responders generating a high number of survivors at pH values in the range 6.0 to 3.5, but not at pH 7.5. Predominant among the latter group were the S. mutans and Lactobacilli casei strains, with the most significant adaptive response exhibited by S. mutans LT11 and S. mutans Ingbritt, involving a process that required protein synthesis. Time course experiments with the latter organisms indicated that 90-120 min was required after exposure to the triggering pH before the acid response was fully functional. These results indicate that the sudden exposure of strains of oral streptococci and lactobacilli, as well as Enterococcus faecalis, to pH values between 6.0 and 3.5 results in the induction of an acid tolerance response that enhances the survival of these strains at or below pH 3.5.

Fluoride inhibition of carbohydrate metabolism by the acidogenic plaque microflora is well-established, although it has not always been appreciated that oral bacteria vary considerably in their susceptibility to fluoride. Early studies demonstrated that the F-induced reduction in acid production was due, in part, to the inhibition of the glycolytic enzyme, enolase, which converts 2-P-glycerate to P-enolpyruvate. The decreased output of PEP in the presence of F, in turn, results in the inhibition of sugar transport via the PEP phosphotransferase system (PTS). Bacterial accumulation of fluoride involves the transport of HF, a process requiring a transmembrane pH difference or pH gradient, which is generated only by metabolically active cells. The uptake of HF into the more alkaline cytoplasm results in the dissociation of HF to H+ and F- and, if allowed to continue, the accumulation of protons acidifies the cytoplasm, causing a reduction in both the proton gradient and enzyme activity. Current information indicates that in addition to enolase, F- also inhibits the membrane-bound, proton-pumping H+/ATPase, which is involved in the generation of proton gradients through the efflux of protons from the cell at the expense of ATP. Thus, fluoride has the dual action of dissipating proton gradients and preventing their generation through its action on H+/ATPase. The collapse of transmembrane proton gradient, in turn, reduces the ability of cells to transport solutes via mechanisms involving proton motive force. In spite of these known effects on the bacterial cell, there is no general agreement that the anti-microbial effects of F contribute to the anti-caries effect of fluoride.(ABSTRACT TRUNCATED AT 250 WORDS)

The authors have previously demonstrated that Streptococcus mutans shows an exponential-phase acid-tolerance response following an acid shock from pH 75 to 55 that enhances survival at pH 30. In this study the response of S. mutans H7 to acid shock was compared with the responses generated by salt, heat, oxidation and starvation. Prior induction of the acid-tolerance response did not cross-protect the cells from a subsequent challenge by the other stresses ; however, prior adaptation to the other stresses, except heat (42 mC), protected the cells during a subsequent acid challenge at pH 35. Starvation by fivefold dilution of the basal medium (BM) plus fivefold reduction of its glucose content increased the numbers of survivors 12-fold, whereas elimination of glucose from fivefold-diluted BM led to a sevenfold enhancement compared to the control cells ; this indicated a relationship between the acid and starvation responses. The stress responses were further characterized by comparing the 2D electrophoretic protein profiles of exponential-phase cells subjected to the various stress conditions. Cells were grown to exponential phase at pH 75 (37 mC) and then incubated for 30 min under the various stress conditions in the presence of 14 C-labelled amino acids followed by cell extraction, protein separation by 2D gel electrophoresis and image analysis of the resulting autoradiograms. Using consistent twofold or greater changes in IOD % as a measure, oxidative stress resulted in the upregulation of 69 proteins, 15 of which were oxidation-specific, and in the downregulation of 24 proteins, when compared to the control cells. An acid shock from pH 75 to 55 enhanced synthesis of 64 proteins, 25 of them acidspecific, while 49 proteins exhibited diminished synthesis. The dilution of BM resulted in the increased formation of 58 proteins, with 11 starvation-specific proteins and 20 showing decreased synthesis. Some 52 and 40 proteins were enhanced by salt and heat stress, with 10 and 6 of these proteins, respectively, specific to the stress. The synthesis of a significant number of proteins was increased by more than one, but not all stress conditions ; six proteins were enhanced by all five stress conditions and could be classified as general stress proteins. Clearly, the response of S. mutans to adverse environmental conditons results in complex and diverse alterations in protein synthesis to further cell survival.