The presence of two forms of the phosphocarrier protein HPr of the phosphoenolpyruvate: sugar phosphotransferase system in streptococci

Robitaille, David; Gauthier, Lucie; Vadeboncoeur, Christian

doi:10.1016/0300-9084(91)90025-v

Cited by 17 publications

(19 citation statements)

References 25 publications

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“…Wild-type free HPr migrated as a doublet since a portion of the HPr is not processed by the methionine aminopeptidase. This phenomenon results in a population of HPr consisting of two forms; HPr-1 (without Met) and HPr-2 (with Met) (29,41). Because the cellular extract was heated before electrophoresis, HPr(HisϳP) and the doubly phosphorylated product could not be observed.…”

Section: Resultsmentioning

confidence: 99%

“…During the sonication treatment, the recipient containing the cell suspension was kept on ice. The extract was incubated at 100°C for 5 min, and the proteins were separated by polyacrylamide gel electrophoresis under native conditions with separating gels containing 12.5% acrylamide as described by Robitaille et al (29). The position of HPr in the gel was determined by Western blotting as described by Robitaille et al (29) using specific anti-HPr rabbit polyclonal antibodies.…”

Section: Methodsmentioning

confidence: 99%

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Diversity of Streptococcus salivarius ptsH Mutants That Can Be Isolated in the Presence of 2-Deoxyglucose and Galactose and Characterization of Two Mutants Synthesizing Reduced Levels of HPr, a Phosphocarrier of the Phosphoenolpyruvate:Sugar Phosphotransferase System

2001

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In streptococci, HPr, a phosphocarrier of the phosphoenolpyruvate:sugar phosphotransferase transport system (PTS), undergoes multiple posttranslational chemical modifications resulting in the formation of HPr(HisϳP), HPr(Ser-P), and HPr(Ser-P)(HisϳP), whose cellular concentrations vary with growth conditions. Distinct physiological functions are associated with specific forms of HPr. We do not know, however, the cellular thresholds below which these forms become unable to fulfill their functions and to what extent modifications in the cellular concentrations of the different forms of HPr modify cellular physiology. In this study, we present a glimpse of the diversity of Streptococcus salivarius ptsH mutants that can be isolated by positive selection on a solid medium containing 2-deoxyglucose and galactose and identify 13 amino acids that are essential for HPr to properly accomplish its physiological functions. We also report the characterization of two S. salivarius mutants that produced approximately two-and threefoldless HPr and enzyme I (EI) respectively. The data indicated that (i) a reduction in the synthesis of HPr due to a mutation in the Shine-Dalgarno sequence of ptsH reduced ptsI expression; (ii) a threefold reduction in EI and HPr cellular levels did not affect PTS transport capacity; (iii) a twofold reduction in HPr synthesis was sufficient to reduce the rate at which cells metabolized PTS sugars, increase generation times on PTS sugars and to a lesser extent on non-PTS sugars, and impede the exclusion of non-PTS sugars by PTS sugars; (iv) a threefold reduction in HPr synthesis caused a strong derepression of the genes coding for ␣-galactosidase, ␤-galactosidase, and galactokinase when the cells were grown at the expense of a PTS sugar but did not affect the synthesis of ␣-galactosidase when cells were grown at the expense of lactose, a noninducing non-PTS sugar; and (v) no correlation was found between the magnitude of enzyme derepression and the cellular levels of HPr(Ser-P).Histidine-containing protein, heat-stable protein, and heteromorphous protein are all epithets that have been used to designate HPr, the bacterial phosphocarrier of the phosphoenolpyruvate:sugar phosphotransferase transport system (PTS) (19,41). Results obtained over the past decade unequivocally indicate that the qualifier "heterofunctional protein" also applies to this small, approximately 9-kDa protein (27,31,35).The PTS is a multienzyme complex that sequentially catalyzes the transport and phosphorylation of sugars (27, 32) and plays a cardinal role in regulatory processes that allow bacteria to metabolize sugars differently depending on environmental conditions (31,33,35,45). The HPr of gram-positive bacteria can be phosphorylated on His15 at the expense of phosphoenolpyruvate by enzyme I (EI) of the PTS and on Ser46 by the ATP-dependent HPr(Ser) kinase-phosphatase (HPrK) (27,31,33,35,45). HPr(HisϳP) not only is involved in sugar transport but also controls transcription of several genes by transferring its phosphate grou...

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Diversity of Streptococcus salivarius ptsH Mutants That Can Be Isolated in the Presence of 2-Deoxyglucose and Galactose and Characterization of Two Mutants Synthesizing Reduced Levels of HPr, a Phosphocarrier of the Phosphoenolpyruvate:Sugar Phosphotransferase System

2001

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“…After electrophoresis, the proteins were transferred electrophoretically to a nitrocellulose membrane (pore size, 0.45 m) according to the method of Towbin et al (25). The immunodetection of proteins cross-reacting with anti-III L Man or anti-III H Man antibodies was carried out as described previously (19), except that 5% skim milk and anti-rabbit alkaline phosphatase antibodies were used instead of 1% gelatin and anti-rabbit gold conjugate, respectively.…”

Section: Methodsmentioning

confidence: 99%

Distribution of proteins similar to IIIManH and IIIManL of the Streptococcus salivarius phosphoenolpyruvate:mannose-glucose phosphotransferase system among oral and nonoral bacteria

1995

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“…The reaction product [His 6 -HPr(Ser-P)] was purified on an Ni-NTA column and by size exclusion chromatography on a Superdex 75HR 10/30 column (Pharmacia) equilibrated with 10 mM potassium phosphate (pH 7.5) containing 100 mM NaCl. The purity of the His 6 -HPr(Ser-P) was verified by PAGE under native conditions (28).…”

Section: Methodsmentioning

confidence: 99%

“…Samples were withdrawn at intervals, and the reaction was stopped by adding an equal volume of a solution containing 180 mM Tris-HCl (pH 6.8), 200 mM SDS, 30% glycerol, 2 M ␤-mercaptoethanol, and 0.003% bromophenol blue (stop solution). The proteins were separated by SDS-PAGE and revealed by autoradiography as described previously (28). His 6 -HPr(Ser-P)(Hisϳ 32 P) was synthesized in 50 mM Tris-acetate (pH 7.5) containing 4 mM DTT, 4 mM MgCl 2 , 1.5 M His 6 -EI, and 40 M His 6 -HPr(Ser-P).…”

Section: Methodsmentioning

confidence: 99%

Phosphorylation of Streptococcus salivarius Lactose Permease (LacS) by HPr(His∼P) and HPr(Ser-P)(His∼P) and Effects on Growth

et al. 2003

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The oral bacterium Streptococcus salivarius takes up lactose via a transporter called LacS that shares 95% identity with the LacS from Streptococcus thermophilus, a phylogenetically closely related organism. S. thermophilus releases galactose into the medium during growth on lactose. Expulsion of galactose is mediated via LacS and stimulated by phosphorylation of the transporter by HPr(HisϳP), a phosphocarrier of the phosphoenolpyruvate:sugar phosphotransferase transport system (PTS). Unlike S. thermophilus, S. salivarius grew on lactose without expelling galactose and took up galactose and lactose concomitantly when it is grown in a medium containing both sugars. Analysis of the C-terminal end of S. The effect of LacS phosphorylation on growth was studied with strain G71, an S. salivarius enzyme I-negative mutant that cannot synthesize HPr(HisϳP) or HPr(Ser-P)(HisϳP). These results indicated that (i) the wild-type and mutant strains had identical generation times on lactose, (ii) neither strain expelled galactose during growth on lactose, (iii) both strains metabolized lactose and galactose concomitantly when grown in a medium containing both sugars, and (iv) the growth of the mutant was slightly reduced on galactose.Streptococcus salivarius is the predominant bacterial species among the pioneer microorganisms that colonize the mouth (19). Acquisition of and competition for nutrients, particularly sugars, which serve as the major energy source for oral streptococci, constitute vital ecological determinants for the survival of oral bacteria. S. salivarius is able to metabolize a broad variety of sugars that can be grouped into two categories, non-PTS sugars, which are taken up by transport systems energized by proton motive force or ATP, and PTS sugars, which are transported by the phosphoenolpyruvate:sugar phosphotransferase system (PTS) (4,35,38). The PTS uses phosphoenolpyruvate (PEP) in a group translocation process to phosphorylate incoming mono-and disaccharides via a phosphoryl-transfer cascade involving the non-sugar-specific proteins, Enzyme I (EI) and HPr, and a family of sugar-specific enzyme II complexes (EII) (27). In gram-positive bacteria, the PTS controls sugar metabolism by regulating transporter activities and gene transcription via the protein HPr (6, 29). This protein can be phosphorylated by EI at the expense of PEP on a histidine at position 15, generating HPr(HisϳP), and by a ATP-dependent protein kinase/phosphorylase, called HPrK/P, on a serine at position 46, generating HPr(Ser-P) (6,8,29). Both HPr(HisϳP) and HPr(Ser-P) possess regulatory functions. HPr(HisϳP) accomplishes its regulatory functions by reversibly phosphorylating its targets, and HPr(Ser-P) accomplishes its regulatory functions by protein-protein interactions (7,13,31,42). In addition to the aforementioned phosphorylated forms of HPr, rapidly growing streptococcal cells contain substantial amounts of the doubly phosphorylated form HPr(Ser-P)(HisϳP), whose functions remain unclear (33,36,37).Lactose (milk sugar) is a disacc...

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The presence of two forms of the phosphocarrier protein HPr of the phosphoenolpyruvate: sugar phosphotransferase system in streptococci

Cited by 17 publications

References 25 publications

Diversity of Streptococcus salivarius ptsH Mutants That Can Be Isolated in the Presence of 2-Deoxyglucose and Galactose and Characterization of Two Mutants Synthesizing Reduced Levels of HPr, a Phosphocarrier of the Phosphoenolpyruvate:Sugar Phosphotransferase System

Diversity of Streptococcus salivarius ptsH Mutants That Can Be Isolated in the Presence of 2-Deoxyglucose and Galactose and Characterization of Two Mutants Synthesizing Reduced Levels of HPr, a Phosphocarrier of the Phosphoenolpyruvate:Sugar Phosphotransferase System

Distribution of proteins similar to IIIManH and IIIManL of the Streptococcus salivarius phosphoenolpyruvate:mannose-glucose phosphotransferase system among oral and nonoral bacteria

Phosphorylation of Streptococcus salivarius Lactose Permease (LacS) by HPr(His∼P) and HPr(Ser-P)(His∼P) and Effects on Growth

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