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Inducer expulsion in Streptococcus pyogenes: properties and mechanism of the efflux reaction
S L Sutrina1, J Reizer, M H Saier
1Department of Biology, University of California, San Diego, La Jolla 92093.
Abstract:
Expulsion of preaccumulated methyl-beta-D-thiogalactoside-phosphate (TMG-P) from Streptococcus pyogenes is a two-step process comprising intracellular dephosphorylation of TMG-P followed by rapid efflux of the intracellularly formed free galactoside (J. Reizer, M.J. Novotny, C. Panos, and M.H. Saier, Jr., J. Bacteriol. 156:354-361, 1983). The present study identifies the mechanism and the order and characterizes the temperature dependency of the efflux step. Unidirectional efflux of the intracellularly formed [14C]TMG was only slightly affected when measured in the presence of unlabeled TMG (25 to 400 mM) in the extracellular medium. In contrast, pronounced inhibition of net efflux was observed in the presence of relatively low concentrations (1 to 16 mM) of extracellular [14C]TMG. Since net efflux was nearly arrested when the external concentration of [14C]TMG approached the intracellular concentration of this sugar, we propose that a facilitated diffusion mechanism is responsible for efflux and equilibration of TMG between the intracellular and extracellular milieus. The exit reaction was markedly dependent upon temperature, exhibited a high energy of activation (23 kcal [ca. 96 kJ] per mol), and followed first-order kinetics, indicating that the permease mediating this efflux was not saturated under the conditions of expulsion employed.
Insights
Streptococcus pyogenes expels methyl-beta-D-thiogalactoside-phosphate (TMG-P) via dephosphorylation and efflux. This study reveals TMG efflux occurs through facilitated diffusion, influenced by temperature and concentration gradients.
Area of Science:
- Microbiology
- Molecular Biology
- Biochemistry
Background:
- Streptococcus pyogenes expels methyl-beta-D-thiogalactoside-phosphate (TMG-P) through a two-step process.
- This process involves intracellular dephosphorylation of TMG-P and subsequent efflux of the free galactoside.
Purpose of the Study:
- To elucidate the mechanism of the efflux step in TMG-P expulsion.
- To determine the order of events and characterize the temperature dependency of the efflux process.
Main Methods:
- Investigated unidirectional efflux of intracellular [14C]TMG under varying extracellular TMG concentrations.
- Analyzed the effect of temperature on the efflux reaction rate and kinetics.
Main Results:
- Efflux was minimally affected by unlabeled TMG but significantly inhibited by extracellular [14C]TMG.
- Net efflux ceased when extracellular [14C]TMG approached intracellular concentrations, suggesting facilitated diffusion.
- The efflux reaction showed strong temperature dependence, high activation energy (23 kcal/mol), and first-order kinetics.
Conclusions:
- The efflux of intracellularly formed TMG from Streptococcus pyogenes is mediated by a facilitated diffusion mechanism.
- The permease involved in efflux was not saturated under the experimental conditions.
- Temperature significantly impacts the rate of TMG efflux, with a high energy of activation.