Physiological characteristics of Streptococcus dysgalactiae and Streptococcus uberis and the effect of the lactoperoxidase complex on their growth in a chemically-defined medium and milk.

Aerobic or anaerobic degradation of glucose by Streptococcus dysgalactiae and Streptococcus uberis yielded products qualitatively similar to those observed previously for Streptococcus agalactiae. There were, however, quantitative differences. Though acetoin was formed during aerobic growth of Streptococcus uberis, there was none with Streptococcus dysgalactiae. Differences between Streptococcus dysgalactiae and Streptococcus uberis in their aerobic metabolism of glucose was in lower oxygen consumption (.5 mol/mol of glucose), greater conversion of glucose to lactic acid, and lower molar growth yields with Streptococcus uberis. Cell suspensions of Streptococcus uberis had strong peroxidase activity, and no hydrogen peroxide accumulated during the respiration on glucose. With Streptococcus dysgalactiae, there was more oxygen consumed during growth (1.5 mol/mol of glucose used), greater conversion of glucose to acetic and formic acids and carbon dioxide, and a cell yield of about 6 g of dry cells more per mole of glucose than with Streptococcus uberis. This increase in molar growth yield with Streptococcus dysgalactiae over Streptococcus uberis could be nearly all accounted for by differences in the amount of substrate level adenosine triphosphate generated. Cell suspensions oxidizing glucose accumulated hydrogen peroxide and showed no peroxidase activity. Streptococcus dysgalactiae showed the same growth relationships in three milk media as Streptococcus agalactiae, although growth and acid formation values were much lower. Growth inhibition by the lactoperoxidase complex was reversed with cystine. Acid formation by Streptococcus uberis was decreased by the lactoperoxidase complex and increased by the addition of cystine; however, neither appeared to affect the growth of the organism.

Cystine antagonism of the antibacterial action of lactoperoxidase-thiocyanate-hydrogen peroxide on Streptococcus agalactiae.

Cystine reduction in Streptococcus agalactiae, resulting in sulfhydryl formation, may account for antagonism of the antibacterial effect of lactoperoxidase-thiocyanate-hydrogen peroxide when cystine is present in excess of the amount needed for maximum growth. Accumulation of cystine by S. agalactiae and its reduction to form sulfhydryl compounds were demonstrated. The reduction of cystine appeared to occur by a couple reaction between glutathione reductase and glutathione-disulfide transhydrogenase activity, both of which were found in the supernatant fraction from cell homogenates. NADPH-specific glutathione reductase activity was found in the pellet and supernatant fractions from cell homogenates. Two sulfhydryls were formed for each mole of NADPH used during cystine reduction. The information presented offers a plausible explanation of how cystine, when present in excess of growth needs, may be reduced to generate sulfhydryl compounds which neutralize the antibacterial effect of lactoperoxidase-thiocyanate-hydrogen peroxide on S. agalactiae.

Antibacterial action of lactoperoxidase-thiocyanate-hydrogen peroxide on Streptococcus agalactiae.

Antibacterial activity of lactoperoxidase (LP)-thiocyanate (SCN)-hydrogen peroxide (H2O2) on Streptococcus agalactiae requires that the three reactants must be in contact with the cells simultaneously. Small but assayable amounts of LP adsorb to the cell surface and are not removed by washing. A diffusible antibacterial product of LP-SCN-H2O2 reaction was not found under our experimental conditions. Incubation of S. agalactiae cells with LP-H2O2 and 14C-labeled sodium SCN resulted in the incorporation of SCN into the bacterial protein. Most of the LP-catalyzed, incorporated SCN was released from the bacterial protein. Most of the LP-catalyzed, incorporated SCN was released from the bacterial protein with dithiothreitol. Cells that had their membrane permeability changed by treatment with Cetab or 80% ethanol incorporated more SCN than did untreated cells, i.e., approximately 1 mol of SCN for each mol of sulfhydryl group present in the reaction mixture. Alteration of membrane permeability caused protein sulfhydryls, normally protected by the cytoplasmic membrane, to become exposed to oxidation. The results suggest the LP-H2O2-catalyzed incorporation of SCN into the proteins of S. agalactiae by a mechanism similar to that reported for bovine serum albumin. Removal of reactive protein sulfhydryls from a functional role in membrane transport and in glucolysis in a likely cause of the antibacterial effect for S. agalactiae.

Lactoperoxidase, thiocyanate, and free cystine in bovine mammary secretions in early dry period and at the start of lactation and their effect on Streptococcus agalactiae growth.

The concentrations of lactoperoxidase (LP) and thiocyanate (SCN-) in the mammary secretions of 4 dairy cows in the early dry period were similar to or higher than concentrations in the milk before drying off. The concentrations of free cystine progressively increased in the secretions beginning 3 to 5 days after the last milking; the mean concentrations were 0.66 mumoles/L before drying off and 6.66 mumoles/L after drying off. The mean concentrations of free cysteine were 0.28 mumoles/L before drying off and 1.4 mumoles/L after drying off. The secretions, when diluted in steamed milk, showed greater stimulation of Streptococcus agalactiae growth as the drying-off period progressed. This increase in stimulatory activity was attributed primarily to the increased concentrations of cystine because cystine counteracts the LP/SCN-/hydrogen peroxide inhibitory system for S agalactiae. This effect on the LP system may account for any increase in susceptibility to S agalactiae under infection during the dry period. In 3 other cows, the mammary secretions on the day of calving had lower mean concentrations of LP, SCN-, and free cystine and cysteine than those obtained 4 to 5 days before, and 7 to 8 days after calving.

Glucose transport in Streptococcus agalactiae and its inhibition by lactoperoxidase-thiocyanate-hydrogen peroxide.

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.

Effects of nutritional characteristics of Streptococcus agalactiae on inhibition of growth by lactoperoxidase-thiocyanate-hydrogen peroxide in chemically defined culture medium.

Five cultures of Streptococcus agalactiae have an absolute requirement for L-cystine to grow in a chemically defined medium. The L-cystine could be replaced with cysteine, glutathione, or the disulfide form of glutathione. Dithiothreitol could not substitute for the sulfur-containing amino acids of glutathione; hence, the growth requirement appears to be truly nutritional. Growth was maximum with 4 to 5 mug of L-cystine per ml. If the concentration of L-cystine was no greater than 4 to 5 mug/ml, complete growth inhibition could be obtained by the addition of lactoperoxidase, thiocyanate, and H2O2. The growth inhibition, however, was nullified by additions of L-cystine 10-fold or more in excess of the concentration needed for maximum growth. During the aerobic degradation of glucose by cell suspensions, H2O2 accumulation could be shown with cultures 317 and 11-13, the only cultures the growth of which was inhibited without addition of exogenous H2O2. All of the cultures had varying degrees of peroxidase activity. The balance between H2O2 generation and peroxidase activity of the culture evidently determined whether growth could be inhibited with lactoperoxidase and thiocyanate without H2O2 addition. The growth yeilds per 0.5 mol of the disulfide forms (cystine and oxidized glutathione) were 1.5 and 1.9 times greater than that per 1 mol of the sulfhydryl forms (cysteine and glutathione).

Effect of uncoupling agents and respiratory inhibitors on the growth of Streptococcus agalactiae.

2,4-Dinitrophenol, dicoumarol, carbonylcyanide, m-chlorophenyl-hydrazone and pentachlorophenol all depressed aerobic molar growth yields of Streptococcus agalactiae to values equal to, or less than, those supported by substrate level phosphorylation. When the only source of energy was from substrate phosphorylation (anaerobic growth conditions), there was also a severe depression of the molar growth yield by the same four uncoupling agents. These results indicate that the effect of these agents is to uncouple both substrate and oxidative phosphorylation in S. agalactiae. Amytal inhibited glucose utilization, reduced the amount of O(2) used per mole of substrate and reduced the molar cell yield to that supported by substrate phosphorylation. Atebrin inhibited the respiration rate, but final O(2) consumed per mole of substrate was unchanged, and the respiration was coupled to biosynthesis. Rotenone had no effect on respiration, substrate utilization, or on molar growth yields.

Glucose degradation, molar growth yields, and evidence for oxidative phosphorylation in Streptococcus agalactiae.

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 one-third 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.

Phosphorylation and the reduced nicotinamide adenine dinucleotide oxidase reaction in Streptococcus agalactiae.

Cell-free extracts from aerobically grown Streptococcus agalactiae cells possess a reduced nicotinamide adenine dinucleotide (NADH) oxidase which is linked to oxygen. It is inhibited by cyanide, although cytochromes evidently are not involved. Adenosine triphosphate (ATP) formation occurs during the reaction, but 66 to 75% of the total ATP is formed nonoxidatively. The remaining 25 to 35% of the ATP formation is related to the oxidation of NADH. The formation of ATP in the oxidative reaction can be prevented by excluding oxygen or adding cyanide to prevent NADH oxidation. It can also be prevented by adding methylene blue or pyruvate, which bypasses electron transport to oxygen, but does not interfere with NADH oxidation. Potential sources of ATP, such as glycolysis, the pyruvate oxidase reaction, or the oxidative pentose cycle, are not present, and the high nonoxidative ATP formation is ascribed to the adenylate kinase reaction. The reaction requires adenosine diphosphate (ADP) as a phosphate acceptor. NADH oxidation is independent of ADP. Antimycin A, amytal, and 2,4-dinitrophenol decreased, but did not prevent, oxidative formation of ATP. P:O ratios ranged from 0.15 to 0.25. All of the oxidative activity was in the soluble portion of the cell-free extracts.

Aerobic metabolism of Streptococcus agalactiae.

Streptococcus agalactiae cultures possess an aerobic pathway for glucose oxidation that is strongly inhibited by cyanide. The products of glucose oxidation by aerobically grown cells of S. agalactiae 50 are lactic and acetic acids, acetylmethylcarbinol, and carbon dioxide. Glucose degradation products by aerobically grown cells, as percentage of glucose carbon, were 52 to 61% lactic acid, 20 to 23% acetic acid, 5.5 to 6.5% acetylmethylcarbinol, and 14 to 16% carbon dioxide. There was no evidence for a pentose cycle or a tricarboxylic acid cycle. Crude cell-free extracts of S. agalactiae 50 possessed a strong reduced nicotinamide adenine dinucleotide (NADH(2)) oxidase that is also cyanide-sensitive. Dialysis or ultrafiltration of the crude, cell-free extract resulted in loss of NADH(2) oxidase activity. Oxidase activity was restored to the inactive extract by addition of the ultrafiltrate or by addition of menadione or K(3)Fe(CN)(6). Noncytochrome iron-containing pigments were present in cell-free extracts of S. agalactiae. The possible participation of these pigments in the respiration of S. agalactiae is presently being studied.