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-3-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 PTSmutants did not display diauxic growth in glucoselactose mixtures and, in fact, transported the disaccharide preferentially.The underlying mechanisms of regulation exerted by the phosphoenolpyruvate (PEP)-dependent glucose phosphotransferase system (glc-PTS) on potential substrates involve inhibition of transport as well as repression of gene expression for key metabolic enzymes. These mechanisms are termed inducer exclusion and catabolite repression, respectively, and have long been recognized as being the result of PTS-mediated uptake affecting the metabolism of non-PTS sugars (17). Saier has proposed a unifying hypothesis to explain these two distinct regulatory mechanisms (20). He proposes that key PTS proteins also function as regulators of adenylate cyclase and the non-PTS permeases by means of a phosphorylation-dephosphorylation mechanism. Central to this scheme is the enzyme III (EIII) of the high-affinity glc-PTS of enteric species which is suggested to be the regulatory protein linking PTS-mediated transport with regulation of transport of non-PTS sugars (20,21).More poorly understood, however, is the preferential utilization by enteric species of glucose over other PTS substrates, such as sorbitol (16) and fructose (9). Since all major sugars catabolized by Streptococcus mutans are translocated by PTS (7,9), the physiological basis for the preferential utilization of one PTS substrate over another is relevant to our understanding of transport regulation in this species. A regulatory role for the glc-PTS of S. mutans has been proposed, but has been somewhat controversial. Hamilton and Lo (7) observed a diauxic effect when this species was grown in the presence of glucose and lactose, resulting from the apparent inhibition of the induction of the lactose (lac) PTS by glucose. However, this "glucose effect" was atypical since it was dependent on the glucose concentration. Slee and Tanzer (25) also demonstrated biphasic growth when S. mutans was grown in mixtures of glucose and sucrose. Recently, Dills and Seno (4) found that glucose repressed the utilization of sorbitol and mannitol by S. mutans 6715.

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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 [I4C]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.

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, a-methylglucose is not taken up by strain GS5. The transport of [14C]mannose and ['4C]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 PTSmutant 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.

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