Effect of growth conditions on levels of components of the phosphoenolpyruvate:sugar phosphotransferase system in Streptococcus mutans and Streptococcus sobrinus grown in continuous culture

Abstract

The membrane-bound, sugar-specific enzyme II The phosphoenolpyruvate:sugar phosphotransferase system (PTS) concomitantly catalyzes the transport and phosphorylation of mono-and disaccharides in many bacteria (23,25). The overall reaction catalyzed by the PTS requires the membrane protein enzyme II (EII) and the general spluble proteins, enzyme I (El) and HPr, and in many instances a sugar-specific, soluble protein called enzyme III, or simply III (22,23,25). The oral streptococci transport a variety of sugars via the PTS (1, 13, '26, 29), and recent studies (7,18,19,30,32,33) have been directed at the isolation and characterization of the components from these bacteria, particularly Streptococcus mutans.Over the past decade, continuous-culture studies with S. mutans Ingbritt have demonstrated that the glucose-PTS is repressed by a variety of changes in the growth environment under conditions in which glucose uptake and metabolism was either unaffected or even enhanced. For example, changes in growth conditions to acidic pHs (12), increased growth rates (4), limiting glucose to excess glucose (4), or limiting sucrose (3) resulted in repression of glucose-PTS activity when assayed with decryptified cells. These and other results (8,27) have led to the suggestion, that the organi §m possesses an alternate, non-PTS transport system supplementing the glucose-PTS, which is active at low pH, at high growth rates, and in the presence of low concentra-* Corresponding author.tions of sucrose and excess glucose. Subsequent research with S. mutans and other strains of oral streptococci have indicated that the alternate, non-PTS transport system is driven by the gradient energy in t-he proton motive force (10,14,16,17,21).We have now undertaken to study the components of the glucose-PTS in chemostat-grown cells of S. mutans Ingbritt in an effort to determine which element of the system is subject to regulation by environmental factors. In addition, experiments have been conducted with chemostat cultures of glucose-grown S. mutans DR0001 and lactose-grown Streptococcus sobrinus (formerly S. mutans 27352 [33]) to determine whether these same factors influence the components of the PTS in these organisms. Previous research with S. mutans 27352 has demonstrated that transport of lactose by the organism requires the induction of not only a specific EII for lactose (EIILaC) but also the soluble component 111Lac (33), a factor which appears to be an integral part of the lactose-PTS in a variety of strains of S. mutans (34). Furthermore, repression of EII activity for glucose and fructose has also been reported with S. mutans and S. sobrinus growing on lactose in batch cultures (34). The activity of EII was measured in isolated membrane preparations in the presence of excess' EI and HPr while the concentration of the latter components and 111Lac were determined by immunoelectrophoresis. Our results indicate that the general proteins HPr, El, and I11Lac are relatively immune to changes in environmental conditions; however, synthesi...

Up-regulation of competence- but not stress-responsive proteins accompanies an altered metabolic phenotype in Streptococcus mutans biofilms

“…Notwithstanding, neither this nor previous 2-DGE analyses (Len et al, 2003(Len et al, , 2004b have identified the integral cytoplasmic membrane glucose transporters EII man and EII glc , or the glucose permease of S. mutans required for uptake of glucose (Cvitkovitch et al, 1995;Vadeboncoeur et al, 1991) due to their hydrophobic structure. As a consequence, any change that the level of expression of these transporters may have on glucose uptake and subsequent metabolic rate cannot be surmised.…”

Vesicles prepared from Streptococcus mutans demonstrate the presence of a second glucose transport system

“…The culture was maintained and assessed for purity as described previously (Hamilton & Ellwood, 1978). Cultures were grown in chemostats at a dilution rate ( D ) of 0.1 h-' in semi-defined medium (Hamilton & Buckley, 1991) under the following conditions: condition A (5 mM glucose at pH 7*0), which results in optimal glucose-PTS activity (PTS-0 cells), and condition B (50 mM glucose pH 5.5), which results in repression of the glucose-PTS (PTS-R cells) (Hamilton et a/., , 1989Vadeboncoeur et a/., 1987Vadeboncoeur et a/., , 1991.…”

Proteome analysis of Streptococcus mutans metabolic phenotype during acid tolerance

“…As S. mutans growing in continuous culture is devoid of measurable glucose PEP-PTS activity at pH 5?0 (Vadeboncoeur et al, 1991), phosphoenolpyruvate is not required to initiate the phosphorylation cascade for this glucose uptake system. The levels of the final three enzymes in glycolysis at pH 5?0 were consistent with a reduced pool of phosphoenolpyruvate, since the reduced levels of enolase, in conjunction with an overall 2?8-fold increase in the four isoforms of pyruvate kinase, would be expected to convert 2-phosphoglycerate to pyruvate without any build-up of phosphoenolpyruvate.…”

“…Two such systems exist in S. mutans: EII man , which has been well characterized, and EII glc , the existence of which is primarily based on physiological observations (Vadeboncoeur, 1984;Néron & Vadeboncoeur, 1987). However, continuous-culture studies suggest that S. mutans is completely devoid of EII man and EII glc activity at pH 5?0 (Vadeboncoeur et al, 1991). At this acid pH, the bacterium preferentially utilizes a non-PTS glucose permease system (Hamilton & St Martin, 1982;Dashper & Reynolds, 1990;Buckley & Hamilton, 1994), while maintaining a more alkaline cytoplasm by increasing the level of its proton-translocating F 1 F 0 -ATPase (Hamilton & Buckley, 1991;Cvitkovitch et al, 1995).…”