Glucose phosphoenolpyruvate-dependent phosphotransferase system of Streptococcus mutans GS5 studied by using cell-free extracts.

The glucose phosphotransferase system (PTS) of Streptococcus mutans GS5 has been partially characterized, using fractions derived from cells treated with the muramidase mutanolysin. Membranes retained functional PTS enzymes for the phosphoenolpyruvate-dependent phosphorylation of glucose, fructose, and mannose. This was confirmed by assaying membranes directly for enzyme I (EI) and enzyme IIglc (EIIglc) by employing specific phosphoryl-exchange reactions for each factor. Membranes prepared from glucose PTS- mutants, however, were either deficient in glucose phosphorylation or reflected the "leakiness" displayed by whole cells. Mutant membranes were unable to catalyze the glucose:glucose 6-phosphate transphosphorylation reaction, indicating a defective EIIglc in these fractions. Although total cellular EI activities in the mutant clones were about the same as that measured for the wild-type strain by employing the pyruvate:phosphoenolpyruvate phosphoryl-exchange reaction, mutant membranes were found to possess less than 10% of the specific EI activity of wild-type membranes. The cytoplasmic fractions of mutants, however, displayed markedly increased specific activities for this enzyme when compared with wild-type extracts. These results strongly suggest a molecular association of EI with a normal membrane protein, perhaps EIIglc, that is absent in mutants. This would explain the absence of fructose PTS activity in glucose PTS- mutant membranes despite the fact that whole cells of these clones are normal for this transport function.

Transport of glucose and mannose by a common phosphoenolpyruvate-dependent phosphotransferase system in Streptococcus mutans GS5.

Decryptified cells of Streptococcus mutans GS5 transport glucose, mannose, and fructose by constitutive phosphoenolpyruvate-dependent phosphotransferase systems (PTSs). Although the non-metabolizable glucose analog 2-deoxyglucose is transported by a PTS, alpha-methylglucose is not taken up by strain GS5. The transport of [14C]mannose and [14C]glucose was almost totally blocked by the heterologous sugars, indicating that these substrates may share a common PTS permease. [14C]fructose transport, however, was not inhibited by large excesses of glucose, indicating the existence of a separate fructose PTS. All "tight" glucose PTS- mutant clones studied were also unable to transport mannose, whereas some "leaky" glucose PTS- clones also were leaky for mannose phosphorylation. Fructose transport in most of these mutant strains was unimpaired, indicating that genetic lesions did not involve soluble (cytoplasmic) PTS components.

Role of the phosphoenolpyruvate-dependent glucose phosphotransferase system of Streptococcus mutans GS5 in the regulation of lactose uptake.

When Streptococcus mutans GS5 was grown in equimolar (5 mM) amounts of glucose and lactose, a classical diauxic growth curve was obtained. Glucose was taken up during the first growth phase, followed by a 60-min lag, and then lactose was transported. Synthesis of lactose phosphotransferase system (PTS) enzymes was repressed until the complete exhaustion of glucose, indicative of an inducer exclusion mechanism of repression. The enzyme phospho-beta-galactosidase, however, was found in small amounts even in the presence of glucose. Repression was not observed when GS5 was grown in equimolar amounts of fructose and lactose. Although fructose was taken up preferentially, synthesis of the lactose PTS occurred from the onset of growth in these sugars. It is proposed that a component of the glucose PTS may be a regulatory factor in lactose transport. Glucose PTS- mutants did not display diauxic growth in glucoselactose mixtures and, in fact, transported the disaccharide preferentially.

Mutanolysin-induced spheroplasts of Streptococcus mutants are true protoplasts.

A method is described for the preparation of protoplasts of Streptococcus mutans BHT. The muralytic enzyme mutanolysin was prepared free of contaminating proteinases and shown to completely dissolve cell walls of this strain. Whole cells were converted to stabilizable protoplasts by using the enzyme in an isotonic medium containing 40% raffinose. Experiments using [3H]thymidine and [14C]leucine as cytoplasmic pool markers revealed only minimal (10%) leakage during a 1-h incubation. Examination by electron microscopy revealed the apparent absence of structural cell wall on the enlarged spherical bodies. Quantitative chemical analyses of membranes prepared by lysing protoplasts demonstrated only very small amounts of rhamnose and trace amounts of galactose. These sugars are the principal components of the BHT cell wall polysaccharide. Also, there were only small amounts of peptidoglycan components (e.g., N-acetylglucosamine) in the purified membranes obtained by this method.