No abstract

The cation-bridged fluoride binding model proposed previously was tested by measuring fluoride binding to Streptococcus mutans R9 in the presence and absence of calcium, magnesium or zinc ions. The dissociation constant for fluoride binding to washed cells was 8.4 ± 7.9 mmol/l and the binding capacity was 4.3 ± 1.7 µmol/g wet weight. Binding was largely accounted for by residual bound divalent magnesium, with a small contribution from calcium. In the presence of 5 mmol/l divalent cation, dissociation constants for fluoride (mmol/l) were: 12.2 ± 3.8 (calcium), 9.9 ± 0.4 (magnesium) and 14.4 ± 0.5 (zinc). Binding capacities (µmol/g wet weight) were: 122 ± 26 (calcium), 130 ± 90 (magnesium) and 142 ± 56 (zinc). Fluoride produced a marked reduction in calcium binding affinity and approximately doubled the calcium binding capacity. In the absence of fluoride, divalent cation binding to plaque is bidentate. It is suggested that fluoride, by competing with macromolecular anionic groups, causes binding to become monodentate. This allows the binding of double the quantity of cations (and of further fluoride). Release of fluoride, bound by calcium bridging, into plaque fluid, as a result of fluoride clearance into saliva, or of a fall in pH, will always be accompanied by a release of calcium which will potentiate the cariostatic effect of fluoride.

Casein phosphopeptides (CPP) stabilize amorphous calcium phosphate (ACP) and may be used to localize ACP in dental plaque, maintaining a state of supersaturation with respect to tooth enamel, reducing demineralization and enhancing remineralization [Reynolds, J Dent Res 1997;76:1587–1595]. The aim of this paper is to investigate these effects by measuring the affinity and capacity of Streptococcus mutans for CPP–ACP. Using the equlibrium dialysis system described by Rose and Hogg [Biochim Biophys Acta 1995;1245:94–98], assessment of calcium binding by a plaque streptococcus at a fixed CPP–ACP concentration gives a series of CPP–ACP–influenced dissociation constants for calcium. These data can then be used to derive a true dissociation constant for CPP–ACP itself. The results demonstrate that CPP–ACP binds with about twice the affinity of the bacterial cells for calcium up to a value of 0.16 g/g wet weight cells. Application of CPP–ACP to plaque may cause a transient rise in plaque fluid free calcium which may assist remineralization. Subsequently, CPP–ACP will form a source of readily available calcium to inhibit demineralization. Hence, CPP–ACP binds well to plaque, providing a large calcium reservoir, which is likely to restrict mineral loss during a cariogenic episode and provide a potential source of calcium for subsequent remineralization. Overall, once in place, CPP–ACP will restrict the caries process.

By means of micro-equilibrium dialysis, calcium binding capacities and affinities were measured in three different oral bacteria, and the effects of extracellular polysaccharide, pH, and aggregation were investigated. Binding capacities of 31.0 +/- 2.1 (C. matruchotii), 34.7 +/- 3.7 (S. sanguis), and 41.5 +/- 5.4 (S. downei) mumol calcium/g wet weight of cells were found at pH 7.0, falling to 21.4 +/- 0.8 mumol calcium/g wet wt. cells at pH 5.0 for S. downei. Dissociation constants were found to vary between 0.78 +/- 0.24 and 1.77 +/- 0.30 mmol/L (at pH 7.0, depending on species), and between 0.62 +/- 0.04 and 1.77 +/- 0.30 (in the pH range 5.0 to 7.0, for S. downei only). Examination suggested that at pH 7.0 calcium-facilitated bacterial association occurs in the streptococci with calcium uptake curves analogous with those of positively cooperative systems. Desorption of calcium from aggregated S. downei suggested that the mechanism of desorption differed from that of uptake. This may be an important factor in plaque formation and in the binding of cells to the surface of formed plaque. Plaque calcium forms a reservoir, readily released by a pH drop, which may increase plaque fluid saturation and reduce demineralization.

Aims:  To compare the properties of the spores of Bacillus subtilis prepared in liquid and on plates. Methods and Results:  The spores of B. subtilis were prepared at 37°C using a nutrient exhaustion medium either in liquid or on agar plates. The levels of core water, dipicolinic acid (DPA) and small, acid‐soluble spore proteins (SASP) were essentially identical in spores made in liquid or on plates. Spores prepared in liquid were killed ∼threefold more rapidly at 90°C in water than the spores prepared on plates, and the spores prepared in liquid were more sensitive to nitrous acid and a diluted stable superoxidized water. Spores prepared in liquid also germinated more rapidly with several agents than those prepared on plates. Pellets of spores prepared on plates were darker than spores prepared in liquid, and spores prepared in liquid had more readily extracted coat protein. However, there were no major differences in the relative levels of individual coat proteins or the cross‐linking of the coat protein GerQ in the two types of spores, although the inner membrane of spores prepared on plates had a higher ratio of anteiso‐ to iso‐fatty acids. Conclusions:  The preparation in liquid yielded spores with some different properties than those made on agar plates. Spores made in liquid had lower resistance to heat and several chemicals, and germinated more readily with several agents. There were also differences in the composition of the inner membrane of spores prepared under these two conditions. However, there were no major differences in the levels of DPA, core water, SASP and individual coat proteins or the cross‐linking of a coat protein in spores made in liquid and on plates. Significance and Impact of the Study:  This work demonstrates that the preparation method can affect the resistance and germination properties of bacterial spores, even if an identical medium and temperature are used. Evidence was also obtained consistent with the role of the inner membrane in spore resistance and germination, and that some factor in addition to core water, DPA and SASP content plays a role in spore resistance to wet heat.