Denis Brochu

Quantitative determination of the intracellular concentration of the various forms of HPr, a phosphocarrier protein of the phosphoenolpyruvate: Sugar phosphotransferase system in growing cells of oral streptococci

A simple procedure for quantitative estimation of the different phosphorylated forms of the phosphocarrier protein HPr in growing cells of oral streptococci is described. The growth of the cells was rapidly stopped by acidification of the medium and concomitant addition of the ionophore Gramicidin D. This procedure inactivated Enzyme I, HPr(Ser) kinase, HPr(Ser-P) phosphatase, and the enzymes involved in the metabolism of the allosteric effectors as well as the substrates of HPr phosphorylation. The cellular concentrations of HPr (His approximately P), HPr (Ser-P), HPr (His approximately P) (Ser-P), and free HPr were then determined by crossed immunoelectrophoresis.

Replacement of isoleucine‐47 by threonine in the HPr protein of Streptococcus salivarius abrogates the preferential metabolism of glucose and fructose over lactose and melibiose but does not prevent the phosphorylation of HPr on serine‐46

Phosphorylation of HPr on a serine residue at position 46 (Ser‐46) by an ATP‐dependent protein kinase has been reported in several Gram‐positive bacteria, and the resulting intermediate, HPr(Ser‐P), has been shown to mediate inducer exclusion in lactococci and lactobacilli and catabolite repression in Bacillus subtilis and Bacillus megaterium. We report here the phenotypic properties of an isogenic spontaneous mutant (G22.4) of Streptococcus salivarius ATCC 25975, in which a missense mutation results in the replacement of isoleucine at position 47 (Ile‐47) by threonine (Thr) in HPr. This substitution did not prevent the phosphorylation of HPr on Ser‐46, nor did it impede the phosphorylation of HPr on His‐15 by EI or the transfer of the phosphoryl group from HPr(His∼P) to other PTS proteins. However, the I47T substitution did perturb, in glucose‐grown but not in galactose‐grown cells, the cellular equilibrium between the various forms of HPr, resulting in an increase in the amount of free HPr at the expense of HPr(His∼P)(Ser‐P); the levels of HPr(His∼P) and HPr(Ser‐P) were not affected. Growth on melibiose was virtually identical for the wild‐type and mutant strains, whereas the generation time of the mutant on the other sugars tested (glucose, fructose, mannose, lactose and galactose) increased 1.2‐ to 1.5‐fold. The preferential metabolism of PTS sugars (glucose and fructose) over non‐PTS sugars (lactose and melibiose) that is observed in wild‐type cells was abolished in cells of mutant G22.4. Moreover, α‐ and β‐galactosidases were derepressed in glucose‐ and fructose‐grown cells of the mutant. The data suggest that HPr regulates the preferential metabolism of PTS sugars over the non‐PTS sugars, lactose and melibiose, through the repression of the pertinent catabolic genes. This HPr‐dependent repression, however, seems to occur solely when cells are growing on a PTS sugar.

Surface location of HPr, a phosphocarrier of the phosphoenolpyruvate: sugar phosphotransferase system in Streptococcus suis

HPr is a low-molecular-mass phosphocarrier protein of the bacterial phosphoenolpyruvate (PEP): sugar phosphotransferase system (PTS) found in the cytoplasm or associated with the inner surface of the cytoplasmic membrane. Treatment of Streptococcus suis cells with a Sorvall Omnimixer, a technique used to extract cell surface components, resulted in the extraction of a major protein with a molecular mass of 9 kDa. Several lines of evidence suggested that this protein was HPr: (i) the S. suis protein showed homology over the first 35 N-terminal amino acid residues with the HPrs of Streptococcus salivarius and Streptococcus mutans, including the signature sequence for the site of PEP-dependent phosphorylation; (ii) it cross-reacted with the S. salivarius anti-HPr antibody preparation; (iii) it could be phosphorylated by enzyme I at the expense of PEP, and by a membrane-associated kinase at the expense of ATP; and (iv) it possessed phosphocarrier activity when used as a source of HPr in an in vitro PTS assay. The data suggested that a portion of the cellular HPr is associated with the external cell surface in S. suis, a result that was confirmed by immunogold electron microscopy. The cellular HPr of S. suis consisted of two forms that could be distinguished by the presence or the absence of the N-terminal methionine. Amino acid sequence analysis indicated that the cell-surface-associated HPr of S. suis lacked the N-terminal methionine residue.

Alterations in the cellular envelope of spontaneous IIIManL-defective mutants of Streptococcus salivarius

In Streptococcus salivarius, the phosphoenolpyruvate: mannose phosphotransferase system (PTSMan) transports and concomitantly phosphorylates mannose, glucose, fructose and 2-deoxyglucose. PTSMan consists of a membrane Enzyme II and two forms of Enzyme III (IIIMan) having molecular masses of 38.9 kDa (IIIManH) and 35.2 kDa (IIIManL) respectively. We have previously reported the isolation of spontaneous mutants lacking IIIManL, and showed that they exhibited abnormal growth when cultured in mixtures of sugars containing glucose. The mutants also synthesize several cytoplasmic glucose-repressible proteins during growth on glucose and some of them constitutively express a fructose PTS which is induced by fructose in the parental strain. We have now investigated the properties and composition of the cellular envelope of three S. salivarius IIIManL-defective mutants (strains A37, B31 and G29) after growth on glucose. The mutants have altered sensitivity to various toxic compounds that interfere with cell-envelope functions. The mutants also exhibited altered membrane-protein profiles when analysed by two-dimensional PAGE and modified total lipid and phosphorus contents and lipid/protein ratio. In one mutant (strain G29), the proportion of the phospholipids separated by TLC was different from the parental strain. Electron microscopy indicated that one mutant (strain A37) possessed more fimbriae than the parental strain. The results suggested that these IIIManL-defective mutants were affected in a global regulatory gene controlling several cellular or physiological functions, many of these being related to the cellular envelope.

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

In streptococci, HPr, a phosphocarrier of the phosphoenolpyruvate:sugar phosphotransferase transport system (PTS), undergoes multiple posttranslational chemical modifications resulting in the formation of HPr(His approximately P), HPr(Ser-P), and HPr(Ser-P)(His approximately P), 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 alpha-galactosidase, beta-galactosidase, and galactokinase when the cells were grown at the expense of a PTS sugar but did not affect the synthesis of alpha-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).