Streptococcus pyogenes pregrown on lactose took up glucose, lactose, or methyl beta-D-thiogalactopyranoside (MeSGal or TMG) by a phosphoenolpyruvate-dependent phosphotransferase system. MeSGal accumulated in the cell as MeSGal-phosphate (MeSGalP). Three effects were noted when various sugars were added to MeSGal preloaded cells: (i) no decrease in intracellular MeSGalP concentration after addition of fructose, sucrose, o-nitrophenyl-beta-D-galactoside, glycerol, 6-deoxyglucose, alpha-methyl D-glucoside, 2-deoxygalactose, glucose 1-phosphate, or glucose 6-phosphate; (ii) slow loss of preaccumulated MeSGalP evoked by lactose, 2-deoxy-D-glucose, or unlabeled MeSGal; and (iii) a short lag followed by extremely rapid expulsion of intracellular MeSGalP elicited by glucose or mannose and a slower expulsion elicited by glucosamine. The expelled compound was free MeSGal, indicating the involvement of dephosphorylation in the expulsion mechanism. Deoxyglucose inhibited the expulsion evoked by mannose, and prepoisoning of cells with fluoride or arsenate prevented the glucose-dependent expulsion. The expulsion is due to activation of an expulsion mechanism rather than to turnover of MeSGalP and leak of internal MeSGal with concomitant inhibition of MeSGal influx. The results suggest the need for phosphotransferase-dependent translocation of a preferential sugar or accumulation of the sugar catabolite for expulsion activation. The significance of the expulsion mechanism in synthesis regulation of enzymes involved in carbohydrate utilization is proposed.

A stabilized L-form of Streptococcus pyogenes continues to synthesize glycerol teichoic acid. This polymer was obtained from S. pyogenes and its L-form, treated in identical fashion, and compared. Highly purified glycerol teichoic acid from only the L-form was found to be devoid of D-alanine and to have a shorter chain length. Otherwise, the glycerol teichoic acid from these two organisms was found to be a 1, 3-phosphodiester-linked glycerophosphate polymer substituted with D-glucose. Evidence is presented that most, if not all, of the glycerol teichoic acid in.this streptococcus lies between the wall and membrane. A possible need for the continued synthesis of a minute amount of glycerol teichoic acid by this L-form for survival is discussed in terms of the known function of teichoic acids in bacteria.MATERIALS AND METHODS Bacteria, media, and cell fractionation. S. pyogenes, type 12, and its stabilized L-form were used (7). S. pyogenes (5 to 8 liters) was incubated at 37 C for 16 h and harvested when in late stationary phase by centrifugation (17,000 x g) at 4 C. The streptococcus was cultured in brucella broth (Pfizer Diagnostics, Brooklyn, N.Y.) containing either bovine serum albumin, fraction V (8 g/liter; Armour Pharmaceutical Co., Chicago, Ill.) or supplemented with (in grams per liter): yeast extract, 8; sodium acetate, 10; glucose, 19; and KH2P04, 4.5, with the pH adjusted to 7.3. L-form cells were cultured in brucella broth containing bovine serum albumin with the addition of 3% NaCl (7) with and without penicillin G (1,000 U/ml). Where indicated, L-form and coccal media were supplemented, except that yeast extract was omitted. Supplemented medium readily caramelized when autoclaved. Thus, precautions had to be taken (i.e., rapid cooling after autoclaving) to prevent excessive caramelization. An alternative procedure, referred to as noncaramelized medium, denotes the addition of the supplement to the autoclaved basal medium after sterilization by membrane filtration (Millipore Corp.; 934 on July 31, 2020 by guest

The mechanism of methyl-beta-D-thiogalactoside-phosphate (TMG-P) expulsion from Streptococcus pyogenes was studied. The expulsion elicited by glucose was not due to exchange vectorial transphosphorylation between the expelled TMG and the incoming glucose since more beta-galactoside was displaced than glucose taken up, and the stoichiometry between TMG and glucose transport was inconstant. Instead, two distinct and sequential reactions, intracellular dephosphorylation of TMG-P followed by efflux of free TMG, mediated the expulsion. This was shown by temporary accumulation of free TMG effected by competitive inhibition of its efflux and by the aid of arsenate, which arrested dephosphorylation of TMG-P but did not affect efflux of free TMG formed intracellularly before arsenate addition. The competitive inhibition of TMG efflux by its structural analogs suggests that a transport protein facilitates the expulsion. Iodoacetate or fluoride prevented TMG-P dephosphorylation and its expulsion. However, provision of ATP via the arginine deiminase pathway restored these activities in the presence of the glycolytic inhibitors and stimulated expulsion in their absence. Other amino acids tested did not promote this restoration, and canavanine or norvaline severely inhibited it. Arginine without glucose neither elicited the dephosphorylation nor evoked the expulsion of TMG-P. Ionophores or ATPase inhibitors did not prevent the expulsion as elicited by glucose or its restoration by arginine. The results suggest that activation of the dephosphorylation-expulsion mechanism occurs independently of a functional glycolytic pathway, requires ATP provision, and is possibly due to protein phosphorylation controlled by a yet unknown metabolite. The in vivo phosphorylation of a protein (approximate molecular weight - 10,000) under the conditions of expulsion was demonstrated.

The ratio of teichoic acid to lipoteichoic acid (LTA) in a strain of Streptococcus agalactiae type III was found to be 8:1, with the total amount of LTA being 0.1% of the dry weight of the organism. Purified teichoic acid contained D-alanine and possibly a small amount of D-glucose and was approximately 22 glycerol phosphate units in length. The linkage between each of these units was 1-3. In addition, LTA contained a complex lipid, more glucose, and an unusually high content of a short-chain fatty acid, tridecanoic acid. This LTA was cytotoxic for a variety of human cell monolayers in tissue culture, including one derived from the human central nervous system. Established human cells were more sensitive than primary cell monolayers to this LTA, with as little as 12.5 ,ug of LTA per ml being cytotoxic for HeLa cells. Teichoic acid (250 Fig/ml) was nontoxic under identical conditions. These cytotoxicity results suggest an LTA involvement in group B streptococcal pathogenesis. Also, the first model system for the study of group B streptococcal adherence to primary human embryonic amnion cells in tissue culture is detailed. This system was used to quantitate pronounced differences in tissue tropism between S. agalactiae and Streptococcus pyogenes and showed enhanced binding by this group A coccus over that of S. agalactiae for amnion cell monolayers. The adherence of both streptococcal species to only a portion (40%) of these amnion cells suggested that host cell receptor expression may vary for primary cells in vitro. Finally, this strain of S. agalactiae was shown to adhere to amnion cells by a non-LTA-mediated mechanism. The possibility of an LTA-mediated versus a protein-mediated adherence mechanism for host cells that is related to the virulence of S. agalactiae is discussed. Group B streptococci are important pathogens of infants during the neonatal period. These organisms also produce disease in adults (37). Of all the serotypes known, type III strains account for the largest proportion of infections in humans (16). Studies of the physiology of this pathogen have dealt with cell wall composition (9), extracellular enzymes (31), and the release of soluble antigens (11). However, except for an earlier study by McCarty (22) which established the presence of a glycerol-type teichoic acid (TA) in a group B streptococcus, serotype unknown, the TA of these cocci has not been detailed. The important role of lipoteichoic acid (LTA) in bacterial adherence to host cells (3, 4) and its putative role in immunological and immunopathological (35) maladies is now well established. More recently, the ability of LTA from a group A streptococcus to destroy * Corresponding author. MATERIALS AND METHODS Bacteria. S. agalactiae type III, a human isolate designated DS2242-77, was obtained from Richard R. Facklam, Centers for Disease Control, Atlanta, Ga. The Streptococcus pyogenes type 12 used as a control has been described

An osmotically fragile L-form of Streptococcus pyogenes, type 12, was quickly rendered osmotically stable by decreasing the sodium chloride content of the growth medium and with the temporary use of oleic acid. The change from osmotic-fragility to stability was accompanied by changes in cell yield, generation time, saturated/unsaturated fatty acid ratio of the membrane, and cytoplasmic protein composition. Finally, this resulting osmotically stable L-form survived and was capable of rapidly destroying Girardi human heart cells in tissue culture.