Molecular Characterization and Expression of Pyruvate Formate-Lyase-Activating Enzyme in a Ruminal Bacterium, Streptococcus bovis

To clarify the significance of the activation of pyruvate formate-lyase (PFL) by PFL-activating enzyme (PFL-AE) in Streptococcus bovis, the molecular properties and gene expression of PFL-AE were investigated. S. bovis PFL-AE was deduced to consist of 261 amino acids with a molecular mass of 29.9 kDa and appeared to be a monomer protein. Similar to Escherichia coli PFL-AE, S. bovis PFL-AE required Fe²⁺ for activity. The gene encoding PFL-AE (act) was found to be polycistronic, and the PFL gene (pfl) was not included. However, the act mRNA level changed in parallel with the pfl mRNA level, responding to growth conditions, and the change was contrary to the change in the lactate dehydrogenase (LDH) mRNA level. PFL-AE synthesis appeared to change in parallel with PFL synthesis. Introduction of a recombinant plasmid containing S. bovis pfl and the pfl promoter into S. bovis did not affect formate and lactate production, which suggests that the activity of the pfl promoter is low. When the pfl promoter was replaced by the S. bovis ldh promoter, PFL was overexpressed, which caused an increase in the formate-to-lactate ratio. However, when PFL-AE was overexpressed, the formate-to-lactate ratio did not change, suggesting that PFL-AE was present at a level that was high enough to activate PFL. When both PFL-AE and PFL were overexpressed, the formate-to-lactate ratio further increased. It is conceivable that LDH activity is much higher than PFL activity, which may explain why the formate-to-lactate ratio is usually low.     

The effect of pH on the growth and metabolism of Streptococcus bovis in continuous culture

Streptococcus bovis H13/1 was grown anaerobically at pHs between 5.0 and 6.5 in a glucose-limited chemostat at a dilution rate of 0.05/h. The growth yield and the production of acetate, ethanol and formate decreased at pHs less than 6.5 whereas the production of lactate increased at the lower pH values. When a culture was subjected to sequential pH changes, growth yield and fermentation products were influenced not only by the pH existing in the culture medium but also by the metabolic activity of the cells at the preceding pHs in the sequence. The results are discussed in relation to the mechanisms available for the maintenance of pH homeo-stasis and for the metabolic control of fermentation pathways in Strep. bovis.     

The effect of pH on the growth and metabolism of Streptococcus bovis in continuous culture

Streptococcus bovis H13/1 was grown anaerobically at pHs between 5.0 and 6.5 in a glucose-limited chemostat at a dilution rate of 0.05/h. The growth yield and the production of acetate, ethanol and formate decreased at pHs less than 6.5 whereas the production of lactate increased at the lower pH values. When a culture was subjected to sequential pH changes, growth yield and fermentation products were influenced not only by the pH existing in the culture medium but also by the metabolic activity of the cells at the preceding pHs in the sequence. The results are discussed in relation to the mechanisms available for the maintenance of pH homeostasis and for the metabolic control of fermentation pathways in Strep. bovis.     

The effect of pH on the heat production and membrane resistance of Streptococcus bovis

Non-growing cultures of Streptococcus bovis JB1 which were incubated in 2-[N-moropholino] ethane-sulfonic acid (MES)-phosphate buffer (pH 6.8) and glucose (2 g/l) produced heat at a rate of 0.17 mW/mg protein, and this rate was proportional to the enthalpy change of the homolactic fermentation. Since the growth-independent heat production could be eliminated by dicyclohexylcarbodiimide (DCCD), an inhibitor of F₁F₀ ATPases, it appeared that virtually all of the energy was being used to counteract proton flux through the cell membrane. When the pH was decreased from 6.8 to 5.8, heat production and glucose consumption increased, the electrical potential () declined, the chemical gradient of protons (ZpH) increased, and there was a small increase in total protonmotive force (p). Further decreases in pH (5.8 to 4.5) caused a marked decrease in heat production and glucose consumption even though there was only a small decline in membrane voltage. Based on the enthalpy of ATP (4 kcal or 16.8 kJ/mol), it appeared that 38% of the wattage was passing through the cell membrane. The relationship between membrane voltage and membrane wattage or glucose consumption was non-linear (non-ohmic), and it appeared that the resistance of the membrane to current flow was not constant. Based on the electrical formula, resistance = voltage²/wattage and resistance = voltage/amperage, there was a marked increase in membrane resistance when the pH was less than 6.0. The increase in membrane resistance at low pH allowed S. bovis to maintain its membrane potential and expend less energy when its ability to ferment glucose was impaired.Abbreviations DCCD dicyclohexylcarbodiimide - MES 2-[N-moropholino] ethanesulfonic acid     

Effect of ionophores and pH on growth of Streptococcus bovis in batch and continuous culture.

Batch cultures (pH 6.7) of Streptococcus bovis JB1 were severely inhibited by 1.25 and 5 microM lasalocid and monensin, respectively, even though large amounts of glucose remained in the medium. However, continuous cultures tolerated as much as 10 and 20 microM, respectively, and used virtually all of the glucose. Although continuous cultures grew with high concentrations of ionophore, the yield of bacterial protein decreased approximately 10-fold. When pH was decreased from 6.7 to 5.7, the potency of both ionophores increased, but lasalocid always caused a larger decrease in yield. The increased activity of lasalocid at pH 5.7 could largely be explained by an increased binding of the ionophore to the cell membrane. Because monensin did not show an increased binding at low pH, some other factor (e.g., ion turnover) must have been influencing its activity. There was a linear increase in lasalocid binding as the concentration increased, but monensin binding increased markedly at high concentrations. Based on the observations that (i) S. bovis cells bound significant amounts of ionophore (the ratio of ionophore to cell material was more important than the absolute concentration), (ii) batch cultures responded differently from continuous cultures, and (iii) pH can have a marked effect on ionophore activity, it appears that the term "minimum inhibitory concentration" may not provide an accurate assessment of microbial growth inhibition in vivo.     

Deoxyribonuclease Activity in Selenomonas ruminantium, Streptococcus bovis, and Bacteroides ovatus

Six Selenomonas ruminantium strains (132c, JW13, SRK1, 179f, 5521c1, and 5934e), Streptococcus bovis JB1, and Bacteroides ovatus V975 were examined for nuclease activity as well as the ability to utilize nucleic acids, ribose, and 2-deoxyribose. Nuclease activity was detected in sonicated cells and culture supernatants for all bacteria except S. ruminantium JW13 and 179f sonicated cells. S. ruminantium strains were able to utilize several deoxyribonucleosides, while S. bovis JB1 and B. ovatus V975 showed little or no growth on all deoxyribonucleosides. When S. ruminantium strains 5934e, 132c, JW13, and SRK1 were incubated in medium that contained 15 mm ribose, the major end products were acetate, propionate, and lactate. S. ruminantium 5521c1 and S. bovis JB1 did not grow on ribose, and none of the S. ruminantium strains or S. bovis JB1 grew on 15 mm 2-deoxyribose. In contrast, B. ovatus V975 was able to grow on ribose and 2-deoxyribose. In conclusion, all S. ruminantium strains, S. bovis JB1, and B. ovatus V975 had nuclease activity. However, not all bacteria were able to utilize deoxyribonucleosides, ribose, or 2-deoxyribose. Received: 9 February 2000 / Accepted: 27 March 2000     

Effect of Reduced H+-ATPase Activity on Acid Tolerance in Streptococcus bovis Mutants

In order to confirm that H⁺-ATPase plays an important role in the acid tolerance ofStreptococcus bovis , two mutants with low activities of H⁺-ATPase were isolated by use of ethyl methanesulfonate and neomycin resistance. The activity of H⁺-ATPase per cellular nitrogen was related to the lowest culture pH permitting growth. A mutant with little H⁺-ATPase activity (Mutant 2) was unable to grow below pH 5.5, which suggests that the intracellular pH should be maintained above 5.5 in S. bovis. Since lactate dehydrogenase activity, which is important for acid tolerance, was similar in parent and mutant strains, H⁺-ATPase activity is likely to affect acid tolerance. The amount of H⁺-ATPase protein as determined by Western-blot analysis with polyclonal antibody, was similar in Mutant 2 and its parent, indicating that H⁺-ATPase activity per enzyme protein is reduced by mutation. Probably, H⁺-ATPase synthesis was not changed by mutation. The gene encoding H⁺-ATPase of Mutant 2 had mutations at positions close to the ATP-binding motif A sequence in the β-subunit, which probably explains the reduced activity of H⁺-ATPase in this mutant. These results strongly support the assumption that H⁺-ATPase has a key role in the acid tolerance of S. bovis.     

Resistance of Streptococcus bovis to acetic acid at low pH: relationship between intracellular pH and anion accumulation

Streptococcus bovis JB1, an acid-tolerant ruminal bacterium, was able to grow at pHs from 6.7 to 4.5, and 100 mM acetate had little effect on growth rate or proton motive force across the cell membrane. When S. bovis was grown in glucose-limited chemostats at pH 5.2, the addition of sodium acetate (as much as 100 mM) had little effect on the production of bacterial protein. At higher concentrations of sodium acetate (100 to 360 mM), production of bacterial protein declined, but this decrease could largely be explained by a shift in fermentation products (acetate, formate, and ethanol production to lactate production) and a decline in ATP production (3 ATP per glucose versus 2 ATP per glucose). YATP (grams of cells per mole of ATP) was not decreased significantly even by high concentrations of acetate. Cultures supplemented with 100 mM sodium acetate took up [14C]acetate and [14C]benzoate in accordance with the Henderson-Hasselbalch equation and gave similar estimates of intracellular pH. As the extracellular pH declined, S. bovis allowed its intracellular pH to decrease and maintained a relatively constant pH gradient across the cell membrane (0.9 unit). The decrease in intracellular pH prevented S. bovis from accumulating large amounts of acetate anion. On the basis of these results it did not appear that acetate was acting as an uncoupler. The sensitivity of other bacteria to volatile fatty acids at low pH is explained most easily by a high transmembrane pH gradient and anion accumulation.     

Molar Growth Yield of Streptococcus faecalis on Pyruvate

Y(pyruvate) was 17.3, similar to Y(arginine), for Streptococcus faecalis 6783 grown statically in a complex medium in 1 atm of air.     

Resistance of Streptococcus bovis to acetic acid at low pH: relationship between intracellular pH and anion accumulation.

Streptococcus bovis JB1, an acid-tolerant ruminal bacterium, was able to grow at pHs from 6.7 to 4.5, and 100 mM acetate had little effect on growth rate or proton motive force across the cell membrane. When S. bovis was grown in glucose-limited chemostats at pH 5.2, the addition of sodium acetate (as much as 100 mM) had little effect on the production of bacterial protein. At higher concentrations of sodium acetate (100 to 360 mM), production of bacterial protein declined, but this decrease could largely be explained by a shift in fermentation products (acetate, formate, and ethanol production to lactate production) and a decline in ATP production (3 ATP per glucose versus 2 ATP per glucose). YATP (grams of cells per mole of ATP) was not decreased significantly even by high concentrations of acetate. Cultures supplemented with 100 mM sodium acetate took up [14C]acetate and [14C]benzoate in accordance with the Henderson-Hasselbalch equation and gave similar estimates of intracellular pH. As the extracellular pH declined, S. bovis allowed its intracellular pH to decrease and maintained a relatively constant pH gradient across the cell membrane (0.9 unit). The decrease in intracellular pH prevented S. bovis from accumulating large amounts of acetate anion. On the basis of these results it did not appear that acetate was acting as an uncoupler. The sensitivity of other bacteria to volatile fatty acids at low pH is explained most easily by a high transmembrane pH gradient and anion accumulation.     

Identification of intracellular amylase activity in Streptococcus bovis and Streptococcus salivarius

The ruminal bacterium Streptococcus bovis has been demonstrated to produce an extracellular amylase activity. We previously reported on the cloning of a gene from S. bovis encoding for what was initially believed to be the extracellular amylase. DNA sequence analyses indicated that the amylase produced by the cloned gene did not match the N-terminus amino acid sequence of the purified extracellular amylase and contained no apparent leader sequence for secretion. Analyses of crude extracts demonstrated the presence of an intracellular amylase in S. bovis JB1 that differed in molecular weight (56,000) from that of the extracellular amylase (70,000). The 56,000 molecular weight amylase was identical to the amylase produced by Escherichia coli containing the cloned amylase gene. Low levels of intracellular amylase activity were also detected in other strains of S. bovis and also Streptococcus salivarius. Introduction of the plasmid pVA838 containing the cloned amylase gene into S. bovis and S. sanguis resulted in enhanced intracellular amylase production by both organisms. The amylase gene has been sequenced, and analysis of the deduced amino acid sequence for the amylase indicates a high degree of similarity with secreted amylases from Bacillus species.Names are necessary to report factually on available data; however, the USDA neither guarantees nor warrants the standard of the product, and the use of the name by USDA implies no approval of the product to the exclusion of others that may also be suitable.     

Effect of Dichloroacetate on Regional Energy Metabolites and Pyruvate Dehydrogenase Activity During Ischemia and Reperfusion in Gerbil Brain

The objective of this study was to determine whether administration of dichloroacetate (DCA), an activator of pyruvate dehydrogenase (PDH), improves recovery of energy metabolites following transient cerebral ischemia. Gerbils were pretreated with DCA, and cerebral ischemia was produced using bilateral carotid artery occlusion for 20 min, followed by reperfusion up to 4 h. DCA had no effect on the accumulation of lactic acid and the decrease in ATP and phosphocreatine (PCr) during the 20-min insult, nor on the recovery of these metabolites measured at 20 and 60 min reperfusion. However, at 4 h reperfusion, levels of ATP and PCr were significantly higher in DCA-treated animals than in controls, as PCr exhibited a secondary decrease in caudate nucleus of control animals. PDH was markedly inhibited at 20 min reperfusion in both groups, but was reactivated to a greater extent in DCA-treated animals at 60 min and 4 h reperfusion. These results demonstrate that DCA had no effect on the initial recovery of metabolites following transient ischemia. However, later in reperfusion, DCA enhanced the postischemic reactivation of PDH and prevented the secondary failure of energy metabolism in caudate nucleus. Thus, inhibition of PDH may limit the recovery of energy metabolism following cerebral ischemia.     

Factors Affecting the Antibacterial Activity of the Ruminal Bacterium,Streptococcus bovis HC5

Streptococcus bovis HC5 inhibits a variety of S. bovis strains and other Gram-positive bacteria, but factors affecting this activity had not been defined. Batch culture studies indicated that S. bovis HC5 did not inhibit S. bovis JB1 (a non-bacteriocin-producing strain) until glucose was depleted and cells were entering stationary phase, but slow-dilution-rate, continuous cultures (0.2 h⁻¹) had as much antibacterial activity as stationary-phase batch cultures. Because the activity of continuous cultures (0.2–1.2 h⁻¹) was inversely related to the glucose consumption rate, it appeared that the antibacterial activity was being catabolite repressed by glucose. When the pH of continuous cultures (0.2 h⁻¹) was decreased from 6.7 to 5.4, antibacterial activity doubled, but this activity declined at pH values less than 5.0. Continuous cultures (0.2 h⁻¹) that had only ammonia as a nitrogen source had antibacterial activity, and large amounts of Trypticase (10 mg ml⁻¹) caused only a 2.0-fold decline in the amount of HC5 cell-associated protein that was needed to prevent S. bovis JB1 growth. Because S. bovis HC5 was able to produce antibacterial activity over a wide range of culture conditions, there is an increased likelihood that this activity could have commercial application. Received: 6 February 2002 / Accepted: 27 March 2002     

Peptide and amino acid transport in Streptococcus bovis

Summary In amino acid transport studies with Streptococcus bovis using ¹⁴C-labelled amino acids, it has been shown that between 87% and 95% of cell-associated radioactivity was located in the cytosol. In similar studies with unlabelled peptides, most test peptide associated with S. bovis was truly intracellular. Using sodium dodecyl sulphate-polyacrylamide gel electrophoresis, the proteolytic activity in S. bovis was found to be largely cell-associated and of the serine-protease type, but stimulated by dithiothreitol. A wide range of extracellular peptide hydrolysing activities was demonstrated against the pentapeptide Leu-Trp-Met-Arg-Phe, which was completely hydrolysed to eight products after 10 min incubation. Some of this pentapeptide was transported intact, indicating the existence of mechanisms for the transport of peptides up to 751 Da. In studies with Arg-Phe-Ala, only Phe (F) and Ala (A), and to a much lesser extent Phe-Ala (FA) were transported after extracellular hydrolysis to FA, Arg (R), F and A. In this case, amino acid transport was much more predominant than peptide transport. The extent and nature of peptide transport was affected by the addition of protease inhibitors. Offprint requests to: R. I. Mackie     

Effect of pH on the activity of bovicin HC5, a bacteriocin from Streptococcus bovis HC5

The bacteriocin, bovicin HC5, catalyzed potassium efflux from Streptococcus bovis JB1, and this activity was highly pH dependent. When the pH was near neutral, glucose-energized cells were not affected by bovicin HC5, but the intracellular steady-state concentration of potassium decreased at acidic pH values. The idea that pH was affecting bovicin HC5 binding was supported by the observation that acidic pH also enhanced the efflux of potassium from non-energized cells that had been loaded with potassium. The relationship between bovicin HC5 concentration and potassium depletion was a saturation function, but cooperativity plots indicated that the binding of one bovicin molecule to the cell membrane facilitated the binding of another.