SUMMARYA chemically defined culture medium was used to study the effect of purified lactoperoxidase and thiocyanate on the growth of several cultures of Streptococcus pyogenes and S. agalactiae. While not inhibited by either component alone, S. pyogenes growth was completely inhibited when both components were present in the medium. The growth inhibition was annulled completely by glutathione, thioglycollic (mercaptoacetic) acid or catalase. S. pyogenes glyceraldehyde phosphate dehydrogenase was inhibited by lactoperoxidase when hydrogen peroxide was present. The inhibition was annulled with cysteine and glutathione which suggested t h i s dehydrogenase to be a possible site of inhibition. The inhibition was postulated to be a peroxidatic conversion of essential enzymic sulphydryl p u p s to inactive disulphide groups, thus interfering with the energy metabolism of S. pyogenes. With S. agalactiae cultures a delay in growth inhibition up to 6 hr resulted instead of complete growth inhibition.Catalase neutralized this effect. The extent of growth inhibition was greatest in those strains which were unable to adapt to an oxidative pathway for their energy supply. In becoming independent of the fermentative pathway, the cultures were no longer as sensitive to peroxidase, thiocyanate and hydrogen peroxide. The necessity for thiocyanate in the inhibitory system is not yet clear. Thiouracil and thiourea were ineffective replacements for thiocyanate.

Transport of 2-deoxyglucose or glucose in Streptococcus agalactiae was strongly inhibited if the cells were first exposed to a combination of lactoperoxidase-thiocyanate-hydrogen peroxide (LP-complex). The inhibition was completely reversible with dithiothreitol. N-ethylmaleimide and p-chloromercuribenzoate inhibited sugar transport, and the inhibition was also reversible with dithiothreitol. Sodium fluoride also inhibited sugar transport. Glucolysis was completely inhibited, and dithiothreitol completely reversed the inhibition. Phosphoenolpyruvate-dependent phosphotransferase activity in S. agalactiae was not strongly inhibited by the LP-complex. Interference of the entry of glucose into cells of S. agalactiae by the LP-complex could well account for its growth inhibitory properties with this organism. The inhibition of glucose transport by the LP-complex and its reversibility with dithiothreitol suggest the modification of functional sulfhydryl groups in the cell membrane as a cause of transport inhibition.

In a complex medium with the energy source as the limiting nutrient factor and under anaerobic growth conditions, Streptococcus agalactiae fermented 75% of the glucose to lactic acid and the remainder to acetic and formic acids and ethanol. By using the adenosine triphosphate (ATP) yield constant of 10.5, the molar growth yield suggested 2 moles of ATP per mole of glucose from substrate level phosphorylation. Under similar growth conditions, pyruvate was fermented 25% to lactic acid, and the remainder was fermented to acetic and formic acids. The molar growth yield suggested 0.75 mole of ATP per mole of pyruvate from substrate level phosphorylation. Under aerobic growth conditions about 1 mole of oxygen was consumed per mole of glucose; about onethird of the glucose was converted to lactic acid and the remainder to acetic acid, acetoin, and carbon dioxide. Molar growth yields indicated 5 moles of ATP per mole of glucose. Estimates based on products of glucose degradation suggested that about one-half of the ATP was derived from substrate level phosphorylation and one-half from oxidative phosphorylation. Addition of 0.5 M 2,4-dinitrophenol reduced the growth yield to that occurring in the absence of oxygen. Aerobic pyruvate degradation resulted in 30% of the substrate becoming reduced to lactic acid and the remainder being converted to acetic acid and carbon dioxide, with small amounts of formic acid and acetoin. The molar growth yields and products found suggested that 0.70 mole of ATP per mole of pyruvate resulted from substrate level phosphorylation and 0.4 mole per mole of pyruvate resulted from oxidative phosphorylation.Crude cell-free extracts of Streptococcus agalactiae possess a reduced nicotinamide adenine dinucleotide (NADH) oxidase. The oxidase activity was enhanced considerably when the cells were grown under aerobic conditions (11). To evaluate the energy value of the NADH oxidase pathway, adenosine triphosphate (ATP) formation during NADH oxidation was measured with crude cell-free extracts (12). The results obtained suggested that oxidative phosphorylation occurred. The yield of ATP was low, and synthesis could be prevented by exclusion of 02 or by the use of respiratory poisons. It seemed desirable to confirm the results by an in vivo method. By a comparison of cellular yields per mole of substrate used under aerobic and anaerobic growth conditions, a more reliable measure of the energy value of the oxidative metabolic pathway should be obtainable. Bauchop and 96