Toluene permeabilization differentially affects F- and P-type ATPase activities present in the plasma membrane of Streptococcus mutans

Abstract

Streptococcus mutans membrane-bound P-and F-type ATPases are responsible for H + extrusion from the cytoplasm thus keeping intracellular pH appropriate for cell metabolism. Toluene-permeabilized bacterial cells have long been used to study total membrane-bound ATPase activity, and to compare the properties of ATPase in situ with those in membrane-rich fractions. The aim of the present research was to determine if toluene permeabilization can significantly modify the activity of membranebound ATPase of both F-type and P-type. ATPase activity was assayed discontinuously by measuring phosphate release from ATP as substrate. Treatment of S. mutans membrane fractions with toluene reduced total ATPase activity by approximately 80% and did not allow differentiation between F-and P-type ATPase activities by use of the standard inhibitors vanadate (3 µM) and oligomycin (4 µg/mL). Transmission electron microscopy shows that, after S. mutans cells permeabilization with toluene, bacterial cell wall and plasma membrane are severely injured, causing cytoplasmic leakage. As a consequence, loss of cell viability and disruption of H + extrusion were observed. These data suggest that treatment of S. mutans with toluene is an efficient method for cell disruption, but care should be taken in the interpretation of ATPase activity when toluene-permeabilized cells are used, because results may not reflect the real P-and F-type ATPase activities present in intact cell membranes. The mild conditions used for the preparation of membrane fractions may be more suitable to study specific ATPase activity in the presence of biological agents, since this method preserves ATPase selectivity for standard inhibitors.

Genotypic and phenotypic analysis of S. mutans isolated from dental biofilms formed in vivo under high cariogenic conditions

“…Although this was not the aim of the current investigation, it is an interesting approach for evaluation in future studies. Furthermore, a previous study suggested that toluene, which is used for cell permeabilization, may inhibit the activity of F-ATPase (20). This study also suggests that toluene may induce rupture of the cell membrane, causing an outflow of ATPases found in the cytoplasm.…”

Acid‐adaptive mechanisms of Streptococcus mutans–the more we know, the more we don't

“…The regulator CcpA has been implicated in the regulation of the F‐type ATPase (Abranches et al ., ); however, regulation of the ATPase enzymes at the transcriptional level is not well understood. Although it has been hypothesized that the shifts in membrane unsaturation may affect activity of the S. mutans F‐ATPase (Fozo & Quivey, ; MacGilvray et al ., ), measuring ATPase activity in live, intact cells presents a technical challenge (Thedei et al ., ); and, so a link between ATPase activity and the UFA : saturated fatty acids ratio in the S. mutans membrane has yet to be directly confirmed. In E. faecalis , F‐type ATPase activity is regulated at the level of assembly, where a reduction in intracellular pH halts degradation of the complex subunits and allows association of the active enzyme in the membrane.…”

Photodynamic Therapy with Rose Bengal Induces GroEL Expression inStreptococcus mutans

“…30,31,38 Figure 1 shows that PDT abolishes the acidogenic capacity of a S. mutans cell suspension, sustaining the effect of this therapy on bacterial metabolism. 25,38 In addition, when the per se toxicity of RB or of the light applied to a nondye-treated suspension was evaluated, results were similar to those of the controls. That indicates that the decrease of the acidogenic capacity of S. mutans is due to the combination of the dye treatment followed by irradiation (which generates reactive oxygen species) and not to dye or light applied alone.…”

“…Then glucose was added to an initial concentration of 50 mmol/L, and the resulting decrease in pH was monitored with a thin-bulb glass pH electrode, reflecting metabolic activities of both samples. 38 …”