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

Abstract

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...

How Phosphotransferase System-Related Protein Phosphorylation Regulates Carbohydrate Metabolism in Bacteria

“…Wild-type S. salivarius cells immediately stop the uptake of the non-PTS sugar lactose or galactose when glucose or fructose is added to the growth medium and resume the metabolism of the non-PTS sugars only when glucose or fructose is exhausted (660). In contrast, the uptake of lactose or galactose by the ptsH mutants containing twofold-lower amounts of P-Ser-HPr was not affected by the presence of the PTS sugar glucose or fructose (870). To exert inducer exclusion, gram-positive bacteria therefore seem to require high levels of P-Ser-HPr.…”

“…An approximately twofold decrease in the P-Ser-HPr level was reported to lead to an almost complete loss of inducer exclusion in S. salivarius cells (870). The lower amount of P-Ser-HPr was due to specific point mutations in the ptsH gene or the ptsH leader sequence, which did not affect the uptake rate of PTS substrates, although the metabolism of PTS sugars was slowed.…”

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

“…Although there is no direct evidence that S. salivarius LacS is regulated by HPr(Ser-P), it was demonstrated that the I47T substitution in S. salivarius HPr inhibits the preferential metabolism of glucose and fructose over lactose (10), indicating that somehow HPr is involved in the regulation of LacS. Moreover, a CcpA binding site (cre sequence) has been identified in the promoter region of the S. salivarius gal operon (40), and reduction in the levels of intracellular HPr by a factor of 3 interferes with expression of the gal operon (33). Lastly, we cannot rule out that S. salivarius possesses a second, as-yet-unidentified galactose transporter that would allow growth of mutant G71 on galactose.…”

“…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).…”

“…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 disaccharide composed of glucose and galactose and is an important energy source for oral streptococci. It is taken up by S. salivarius via a non-PTS transport system (14) composed of a single membrane protein, lactose permease (LacS), that possesses an amino acid sequence that shares 95% identity with the sequence of Streptococcus thermophilus LacS (40).…”