Streptococcus mutans GS-5 and IB1600 adapted to growth in acidic environments in continuous culture at slow (generation time = 8.3 h) or fast (generation time = 2.4 h) rates of growth in complex medium with a restricted glucose supply. The extent of adaptation was indicated by changes in minimum pH values attained by harvested cells suspended in dense suspensions with excess glucose and by increased levels of ATPase activity assayed in permeabilized cells. Also, adapted cells better withstood potentially lethal acidification. Cells harvested from cultures growing at pH values close to 5 reduced suspension pH to lower values than cells from cultures maintained at pH 7. Cells from pH 6 cultures were intermediate. The IB1600 strain had a higher level of constitutive acid resistance than the GS-5 strain and also was better able to adapt to growth in acidified media. Both had less adaptive capacity than Enterococcus hirae ATCC 9790. Adaptation occurred rapidly, mainly within a single generation in continuous culture, while deadaptation occurred more slowly over multiple generations. The capacity of S. mutans to adapt to acid conditions is likely to be important in the ecology of dental plaque and also for the cariogenicity of the organism.

Fluoride and a variety of other weak acids acted to reduce reversibily the acid tolerance of glycolysis by intact cells of Streptococcus mutans GS-5 as shown by higher final pH values in acid-drop experiments with glucose in excess. The order of effectiveness was fluoride > indomethacin > ibuprofen > ketoprofen > salicylate > sorbate > cinnamate > p-hydroxybenzoate > benzoate > ascorbate. Only fluoride also acted as an inhibitor of the glycolytic enzyme enolase. However, enolase in permeabilized cells was also inhibited by acidification with a sharp drop-off in activity between pH 6 and 5. It was proposed that the weak acids, including fluoride, acted to reduce glycolytic acid tolerance by enhancing cytoplasmic acidification and thereby inhibiting enzymes such as enolase. The potencies of the acids could not be predicted accurately from knowledge of pKa values, octanol-water partition coefficients, and molecular weights. It was concluded that their modes of action in acid sensitization involved perturbations of membrane function in addition to their acting as transmembrane carriers of protons. Methylparaben (methyl ester of p-hydroxybenzoate) was also a sensitizer but was less effective than the parent acid.

AniA (formerly Pan1) is the major anaerobically induced outer membrane protein in Neisseria gonorrhoeae. AniA has been shown to be a major antigen in patients with gonococcal disease, and we have been studying its regulation in order to understand the gonococcal response to anaerobiosis and its potential role in virulence. This study presents a genetic analysis of aniA regulation. Through deletion analysis of the upstream region, we have determined the minimal promoter region necessary for aniA expression. This 130-bp region contains a sigma 70-type promoter and an FNR (fumarate and nitrate reductase regulator protein) binding site, both of which are absolutely required for anaerobic expression. Also located in the minimal promoter region are three T-rich direct repeats and several potential NarP binding sites. This 80-bp region is required for induction by nitrite. By site-directed mutagenesis of promoter sequences, we have determined that the transcription ofaniA is initiated only from the sigma 70-type promoter. The gearbox promoter, previously believed to be the major promoter, does not appear to be active during anaerobiosis. The gonococcal FNR and NarP homologs are involved in the regulation of aniA, and we demonstrate that placing aniA under the control of thetac promoter compensates for the inability of a gonococcalfnr mutant to grow anaerobically.

The acid tolerance of Streptococcus mutans GS-5 in standard pH-drop assays was found to be affected by the sugar used in the assay and also by the sugar used for growth of the organism. For example, acid tolerance was lower when galactose was used as catabolite than when glucose was used, apparently because galactose/proton symport brought protons extruded by the F-ATPase back into the cell and thus reduced delta pH across the cell membrane. The acid tolerance of glycolysis was related directly to the capacities of the cells to produce acid glycolytically, or probably more correctly, to their capacities to produce adenosine triphosphate but not to acid tolerance of phosphotransferase systems for sugar uptake. Thus, glycolytic acid tolerance of S. mutans depends not only on environmental factors such as potassium or magnesium levels but also on the specific catabolites the organism is metabolizing or to which it has become metabolically adapted.

No abstract