Mechanism of inducer expulsion in Streptococcus pyogenes: a two-step process activated by ATP

Abstract

The mechanism of methyl-beta-D-thiogalactoside-phosphate (TMG-P) expulsion from Streptococcus pyogenes was studied. The expulsion elicited by glucose was not due to exchange vectorial transphosphorylation between the expelled TMG and the incoming glucose since more beta-galactoside was displaced than glucose taken up, and the stoichiometry between TMG and glucose transport was inconstant. Instead, two distinct and sequential reactions, intracellular dephosphorylation of TMG-P followed by efflux of free TMG, mediated the expulsion. This was shown by temporary accumulation of free TMG effected by competitive inhibition of its efflux and by the aid of arsenate, which arrested dephosphorylation of TMG-P but did not affect efflux of free TMG formed intracellularly before arsenate addition. The competitive inhibition of TMG efflux by its structural analogs suggests that a transport protein facilitates the expulsion. Iodoacetate or fluoride prevented TMG-P dephosphorylation and its expulsion. However, provision of ATP via the arginine deiminase pathway restored these activities in the presence of the glycolytic inhibitors and stimulated expulsion in their absence. Other amino acids tested did not promote this restoration, and canavanine or norvaline severely inhibited it. Arginine without glucose neither elicited the dephosphorylation nor evoked the expulsion of TMG-P. Ionophores or ATPase inhibitors did not prevent the expulsion as elicited by glucose or its restoration by arginine. The results suggest that activation of the dephosphorylation-expulsion mechanism occurs independently of a functional glycolytic pathway, requires ATP provision, and is possibly due to protein phosphorylation controlled by a yet unknown metabolite. The in vivo phosphorylation of a protein (approximate molecular weight - 10,000) under the conditions of expulsion was demonstrated.

Expulsion mechanism of xylitol 5‐phosphate in Streptococcus mutans

“…Our experiments do not allow us to conclude which one of tbe two expulsion steps needed energy. In S. pyogenes the energy may be needed for the activation of the dephosphorylation mechanism (29). The results of the present experiments involving L-arginine, D-glucose,, and glycolysis inhibitors resenabled those reported for the expulsion of TMG-P by S. pyogenes: the results indicate that both ATP and a gSycolytic intermediate are required for optimal dephosphorylation (29).…”

“…4A shows the efflux of xylitol 5-P as triggered by 10 mM D-glucose (control). In theory, the addition of unlabeled xylitol to this reaction tnixture caused an uptake of the unlabeled xylitol and resulted in competition for efflux (29). In fact, for a high concentration (100 tnM) of external xylitol, the amount of ' ""Cxylitol within the cells was clearly higher than in the controls ( Fig.…”

“…The results speak for a two-step process in which xylitol 5-P is first dephosphorylated with subsequent expulsion of free xylitol. Such a naechanism has been demonstrated to be responsible for the expulsion of methyl-p-Dthiogalactoside phosphate (TMG-P) by S. pyogmes and xylitol 5-P by L. casei (20,29,33). Expulsion of sugar phosphates appears to be one of the diverse mechanisms grampositive bacteria use to regulate sugar accumulation (14,34).…”

“…The results of the present experiments involving L-arginine, D-glucose,, and glycolysis inhibitors resenabled those reported for the expulsion of TMG-P by S. pyogenes: the results indicate that both ATP and a gSycolytic intermediate are required for optimal dephosphorylation (29). In S. pyogenes phosphorylation of a protein is also involved in the expulsion process (29).…”

“…The second set of experiments was performed with cells preincubated with 10 mM NaF and 5 mM L-arginine essentially according to REIZER et at. (29). Fig.…”