Mechanism of inducer expulsion in Streptococcus pyogenes: a two-step process activated by ATP.
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.
D000251:Adenosine Triphosphatases / Q000037:antagonists & inhibitors; D000255:Adenosine Triphosphate / Q000378:metabolism*; D001149:Arsenates / Q000494:pharmacology; D001426:Bacterial Proteins / Q000378:metabolism; D005947:Glucose / Q000378:metabolism / Q000494:pharmacology; D006019:Glycolysis; D007476:Ionophores / Q000494:pharmacology; D008756:Methylgalactosides / Q000378:metabolism*; D008759:Methylglycosides / Q000378:metabolism*; D010766:Phosphorylation; D012969:Sodium Fluoride / Q000494:pharmacology; D013297:Streptococcus pyogenes / Q000378:metabolism*; D013862:Thiogalactosides / Q000378:metabolism*; D013865:Thioglycosides / Q000378:metabolism*
D001149:Arsenates; D001426:Bacterial Proteins; D007476:Ionophores; D008756:Methylgalactosides; D008759:Methylglycosides; D013862:Thiogalactosides; D013865:Thioglycosides; C009468:thiomethylgalactoside; C027510:methyl beta-D-thiogalactopyranoside phosphate; D000255:Adenosine Triphosphate; D012969:Sodium Fluoride; D000251:Adenosine Triphosphatases; D005947:Glucose; C025657:arsenic acid