Streptococci and lactococci synthesize large amounts of HPr(Ser-P)(His~P) Denis J. Roy, Israe ¨ l Casabon, Katy Vaillancourt, Jonathan L. Huot, and Christian Vadeboncoeur Abstract: HPr is a protein of the phosphoenolpyruvate:sugar phosphotransferase transport system (PTS). In gram-positive bacteria, HPr can be phosphorylated on Ser-46 by the kinase/phosphorylase HprK/P and on His-15 by phospho-enzyme I (EI~P) of the PTS. In vitro studies with purified HPrs from Bacillus subtilis, Enterococcus faecalis, and Streptococcus sal- ivarius have indicated that the phosphorylation of one residue impedes the phosphorylation of the other. However, a recent study showed that while the rate of Streptococcus salivarius HPr phosphorylation by EI~P is reduced at acidic pH, the phosphorylation of HPr(Ser-P) by EI~P, generating HPr(Ser-P)(His~P), is stimulated. This suggests that HPr(Ser-P)(His~P) synthesis may occur in acidogenic bacteria unable to maintain their intracellular pH near neutrality. Consistent with this hypothesis, significant amounts of HPr(Ser-P)(His~P) have been detected in some streptococci. The present study was aimed at determining whether the capacity to synthesize HPr(Ser-P)(His~P) is common to streptococcal species, as well as to lactococci, which are also unable to maintain their intracellular pH near neutrality in response to a decrease in extra cellular pH. Our results indicated that unlike Staphylococcus aureus, B. subtilis, and E. faecalis, all the streptococcal and lactococcal species tested were able to synthesize large amounts of HPr(Ser-P)(His~P) during growth. We also showed that Streptococcus salivarius IIAB L Man , a protein involved in sugar transport by the PTS, could be efficiently phosphory- lated by HPr(Ser-P)(His~P). Key words: doubly phosphorylated HPr, HPr(Ser-P) and HPr(His~P), EI and HprK/P, phosphoenolpyruvate:sugar phosphotransferase system, streptococci, lactococci. Re ´sume ´: La prote ´ine HPr appartient au syste `me de transport phosphoe ´nolpyruvate : phosphotransfe ´rase des sucres (PTS). Chez les bacte ´ries a ` gram positives, la HPr peut e ˆtre phosphoryle ´e sur la se ´rine 46 par la kinase/phosphorylase HprK/P et sur l’histidine 15 par la phosphoenzyme I (EI~P) du PTS. Des e ´tudes in vitro re ´alise ´es sur la HPr purifie ´e de Bacillus subtilis, En- terococcus faecalis et Streptococcus salivarus ont indique ´ que la phosphorylation d’un re ´sidu empe ˆche la phosphorylation de l’autre. Cependant, une e ´tude re ´cente a de ´montre ´ que alors que le taux de phosphorylation de la HPr de S. salivarus par la EI~P est re ´duit a ` pH acide, la phosphorylation de la HPr(Ser-P) par la EI~P, ge ´ne ´rant la HPr(Ser-P)(His~P), est stimule ´e. Ceci sugge `re que la synthe `se de HPr(Ser-P)(His~P) puisse se produire chez les bacte ´ries acidoge `nes incapables de maintenir leur pH intracellulaire pre `s de la neutralite ´. En appui a ` cette hypothe `se, des quantite ´s significatives de HPr(Ser-P)(His~P) ont e ´te ´ de ´tecte ´es chez quelques streptocoques. La pre ´sente e ´tude avait pour but de de ´terminer si la capacite ´ de synthe ´tiser la HPr(Ser-P)(His~P) e ´tait commune aux streptocoques et lactocoques, qui sont aussi incapables de maintenir leur pH in- tracellulaire pre `s de la neutralite ´ en re ´ponse a ` une diminution du pH extracellulaire. Nos re ´sultats indiquent que, contrairement a ` Staphylococcus aureus, B. subtilis et E. faecalis, toutes les espe `ces de streptocoques et de lactocoques teste ´es pouvaient synthe ´tiser de grandes quantite ´s de HPr(Ser-P)(His~P) durant leur croissance. Nous avons aussi de ´- montre ´ que la prote ´ine IIAB L Man de S. salivarus, une prote ´ine implique ´e dans le transport des sucres par le PTS, pou- vait e ˆtre efficacement phosphoryle ´e par la HPr(Ser-P)(His~P). Mots-cle ´s : HPr doublement phosphoryle ´e, HPr(SerP) et HPr(His~P), syste `me phosphoe ´nolpyruvate : phosphotransfe ´rase des sucres, EI et HprK/P, streptocoques, lactocoques. [Traduit par la Re ´daction] Introduction Several bacterial species of the Firmicutes subdivision of gram-positive bacteria, which possess a guanosine (G) + cy- tosine (C) content of less than 50%, transport a number of sugars via the phosphoenolpyruvate: sugar phosphotransfer- ase system (PTS) (Postma et al. 1993; Vadeboncoeur and Pelletier 1997; Titgemeyer and Hillen 2002; Kok et al. 2005; Deutscher et al. 2006; Ajdic ´ and Pham 2007). The HPr protein is a key PTS component that serves as a phos- Received 14 February 2008. Revision received 26 May 2008. Accepted 21 August 2008. Published on the NRC Research Press Web site at cjm.nrc.ca on 31 October 2008. D.J. Roy, I. Casabon, K. Vaillancourt, J.L. Huot, and C. Vadeboncoeur. 1 Groupe de Recherche en E ´ cologie buccale, Faculte ´ de me ´decine dentaire and De ´partement de biochimie et de microbiologie, Faculte ´ des sciences et de ge ´nie, Universite ´ Laval, 2420 rue de la Terrasse, Quebec City, QC G1V 0A6, Canada. 1 Corresponding author (e-mail: Christian.Vadeboncoeur@bcm.ulaval.ca). 941 Can. J. Microbiol. 54: 941–949 (2008) doi:10.1139/W08-085 # 2008 NRC Canada