The effect of growth and starvation on the lysis of the ruminal cellulolytic bacterium Fibrobacter succinogenes.

Growing cultures of Fibrobacter succinogenes assimilated more ammonia than could be accounted for by cellular protein, RNA, or DNA and released large amounts of nonammonia nitrogen. The difference between net and true growth was most dramatic at low dilution rates, but mathematical derivations indicated that the lysis rate was a growth rate-independent function. The lysis rate was sevenfold greater than the true maintenance rate (0.07 h-1 versus 0.01 h-1). Because slowly growing cells had as much proton motive force and ATP as fast-growing cells, lysis was not a starvation response per se. Stationary-phase cells had a lysis rate that was 10-fold less than that of growing cells. Rapidly growing cells were not susceptible to phenylmethylsulfonyl fluoride, but phenylmethylsulfonyl fluoride increased the lysis rate of the cultures when they reached the stationary phase. This latter result indicated that autolysins of stationary-phase cells were being inactivated by a serine proteinase. When growing cells were treated with the glycolytic inhibitor iodoacetate, the proteinase-dependent transition to the stationary phase was circumvented, and the rate of lysis could be increased by as much as 50-fold.     

Effects of dilution rate and pH on the ruminal cellulolytic bacterium Fibrobacter succinogenes S85 in cellulose-fed continuous culture

The ruminal cellulolytic bacterium Fibrobacter succinogenes S85 was grown in cellulose-fed continuous culture at 22 different combinations of dilution rate (D, 0.014–0.076 h^-1) and extracellular pH (6.11–6.84). Effects of pH and D on the fermentation were determined by subjecting data on cellulose consumption, cell yield, product yield (succinate, acetate, formate), and soluble sugar concentrationto response surface analysis. The extent of cellulose conversion decreased with increasing D. First-order rate constants at rapid growth rates were estimated as 0.07–0.11 h^-1, and decreased with decreasing pH. Apparent decreases in the rate constant with increasing D was not due to inadequate mixing or preferential utilization of the more amorphous regions of the cellulose. Significant quantities of soluble sugars (0.04–0.18 g/l, primarily glucose) were detected in all cultures, suggesting that glucose uptake was rather inefficient. Cell yields (0.11–0.24 g cells/g cellulose consumed) increased with increasing D. Pirt plots of the predicted yield data were used to determined that maintenance coefficient (0.04–0.06 g cellulose/g cells · h) and true growth yield (0.23–0.25 g cells/g cellulose consumed) varied slightly with pH. Yields of succinate, the major fermentation endproduct, were as high as 1.15 mol/mol anhydroglucose fermented, and were slightly affected by dilution rate but were not affected by pH. Comparison of the fermentation data with that of other ruminal cellulolytic bacteria indicates that F. succinogenes S85 is capable of rapid hydrolysis of crystalline cellulose and efficient growth, despite a lower _max on microcrystalline cellulose.     

Effect of starvation on cytoplasmic pH, proton motive force, and viability of an acidophilic bacterium, Thiobacillus acidophilus.

The question of whether Thiobacillus acidophilus maintains its cytoplasmic pH at values close to neutrality by active or passive means was explored by subjecting the organism to long-term starvation (up to 22 days). Starving cells maintained a delta pH of 2 to 3 U throughout starvation, although cellular poly-beta-hydroxybutyric acid and ATP, the proton motive force, and culture viability were low or not detectable after 200 h. Cells exposed to azide or azide plus N,N'-dicyclohexylcarbodiimide immediately exhibited characteristics of cells starved for more than 200 h. Thus, a large delta pH in T. acidophilus was maintained in the absence of ATP, ATPase activity, respiration, significant levels of proton motive force, and cell viability and was therefore not dependent on chemiosmotic ionic pumping. The transition from a metabolically active to an inactive state was accompanied by a large increase in the positive membrane potential, which nearly completely compensated for the delta pH in the inactive cells. The longevity of the acidophile during starvation was comparable to that reported previously for neutrophiles, and the loss of viability occurred not because of the acidification of the cytoplasm but apparently because of energy depletion.     

Cellodextrin efflux by the cellulolytic ruminal bacterium Fibrobacter succinogenes and its potential role in the growth of nonadherent bacteria.

When glucose or cellobiose was provided as an energy source for Fibrobacter succinogenes, there was a transient accumulation (as much as 0.4 mM hexose equivalent) of cellobiose or cellotriose, respectively, in the growth medium. Nongrowing cell suspensions converted cellobiose to cellotriose and longer-chain cellodextrins, and in this case the total cellodextrin concentration was as much as 20 mM (hexose equivalent). Because cell extracts of glucose- or cellobiose-grown cells cleaved cellobioise and cellotriose by phosphate-dependent reactions and glucose 1-phosphate was an end product, it appeared that cellodextrins were being produced by a reversible phosphorylase reaction. This conclusion was supported by the observation that the ratio of cellodextrins to cellodextrins with one greater hexose [n/(n + 1)] was approximately 4, a value similar to the equilibrium constant (Keq) of cellobiose phosphorylase (J. K. Alexander, J. Bacteriol. 81:903-910, 1961). When F. succinogenes was grown in a cellobiose-limited chemostat, cellobiose and cellotriose could both be detected, and the ratio of cellotriose to cellobiose was approximately 1 to 4. On the basis of these results, cellodextrin production is an equilibrium (mass action) function and not just an artifact of energy-rich cultural conditions. Cellodextrins could not be detected in low-dilution-rate, cellulose-limited continuous cultures, but these cultures had a large number of nonadherent cells. Because the nonadherent cells had a large reserve of polysaccharide and were observed at all stages of cell division, it appeared that they were utilizing cellodextrins as an energy source for growth.(ABSTRACT TRUNCATED AT 250 WORDS)     

Structural analysis of the carbohydrate components of the outer membrane of the lipopolysaccharide-lacking cellulolytic ruminal bacterium Fibrobacter succinogenes S85.

The polysaccharides from the outer membrane of the Gram-negative ruminal bacterium Fibrobacter succinogenes were isolated by phenol/water extraction and separated by size-exclusion chromatography in the presence of deoxycholate detergent into a lower-molecular-mass fraction designated 'glycolipid' and a high-molecular-mass 'capsular polysaccharide' fraction. Both fractions lacked typical lipopolysaccharide components including 2-keto-3-deoxyoctulosonic acid and 3-hydroxy fatty acids. Carbohydrate components of these fractions were represented by two polysaccharides and one oligosaccharide (possibly glycolipid) with the following structures: : : where HEAEP is N-(2-hydroxyethyl)-2-aminoethylphosphonic acid, found for the first time in natural compounds. The polysaccharides contained pentadecanoic acid and anteisopentadecanoic acid, possibly present as the acyl components. All constituent monosaccharides except L-rhamnose had a D-configuration. In addition to having a structural role in the outer membrane, these polysaccharides may provide protection for this lipopolysaccharide-less bacterium in the highly competitive ruminal environment, as phosphonic acids covalently linked to membrane polymers have in the past been attributed the function of stabilizing membranes in the presence of phosphatases and lipases.     

Effect of phenolic monomers on the growth and beta-glucosidase activity of Bacteroides ruminicola and on the carboxymethylcellulase, beta-glucosidase, and xylanase activities of Bacteroides succinogenes

trans-p-Coumaric acid inhibited the growth of Bacteroides ruminicola on both cellobiose and glucose, while trans-ferulic acid and vanillin retarded growth. The phenolic monomers varied in their potential to inhibit the Bacteroides succinogenes beta-glucosidase, carboxymethylcellulase, and xylanase, with p-coumaric acid being the most inhibitory. The B. ruminicola beta-glucosidase was inhibited less than 10% by all three compounds.     

Energy charge,phosphorylation potential and proton motive force in chloroplasts

Adenylate concentrations were measured in intact chloroplasts under a variety of conditions. Energy charge was significant in the dark and increased in the light, but remained far below values expected from observed phosphorylation potentials in broken chloroplasts, which were 80 000 M^?1 or more in the light. With nitrite as electron acceptor, phosphorylation potentials in intact chloroplasts were about 80 M^?1 in the dark and only 300 M^?1 in the light. Similar phosphorylation potentials were observed, when oxaloacetate, phosphoglycerate or bicarbonate were used as substrates. ΔG′_ATP was ?42 kJ/mol in darkened intact chloroplasts, ?46 kJ/mol in illuminated intact chloroplasts and ?60 kJ/mol in illuminated broken chloroplasts. Uncoupling by NH₄Cl, which stimulated electron transport to nitrite or oxaloacetate and decreased the proton gradient, failed to decrease the phosphorylation potential of intact chloroplasts. Also, it did not increase the quantum requirement of CO₂ reduction. It is concluded that the proton motive force as conventionally measured and phosphorylation potentials are far from equilibrium in intact chloroplasts. The insensitivity of CO₂ reduction and of the phosphorylation potential to a decrease in the proton motive force suggests that intact chloroplasts are over-energized even under low intensity illumination. However, such a conclusion is at variance with available data on the magnitude of the proton motive force.     

Estimation of the cytoplasmic pH of Coxiella burnetii and effect of substrate oxidation on proton motive force.

The magnitude of the proton motive force generated during in vitro substrate oxidation by Coxiella burnetii was examined. The intracellular pH of C. burnetii varied from about 5.1 to 6.95 in resting cells over an extracellular pH range of 2 to 7. Similarly, delta psi varied from about 15 mV to -58 mV over approximately the same range of extracellular pH. Both components of the proton motive force increased during substrate oxidation, resulting in an increase in proton motive force from about -92 mV in resting cells to -153 mV in cells metabolizing glutamate at pH 4.2. The respiration-dependent increase in proton motive force was blocked by respiratory inhibitors, but the delta pH was not abolished even by the addition of proton ionophores such as carbonyl cyanide-m-chlorophenyl hydrazone or 2,4-dinitrophenol. Because of this apparently passive component of delta pH maintenance, the largest proton motive force was obtained at an extracellular pH too low to permit respiration. C. burnetii appears, therefore, to behave in many respects like other acidophilic bacteria. Such responses are proposed to contribute to the extreme resistance of C. burnetii to environmental conditions and subsequent activation upon entry into the phagolysosome of eucaryotic cells in which this organism multiplies.     

Generation of a large, protonophore-sensitive proton motive force and pH difference in the acidophilic bacteria Thermoplasma acidophilum and Bacillus acidocaldarius.

The mechanism by which acidophilic bacteria generate and maintain their cytoplasmic pH close to neutrality was investigated. For this purpose we determined the components of proton motive force in the eubacterium Bacillus acidocaldarius and the archaebacterium Thermoplasma acidophilum. After correction for probe binding, the proton motive force of untreated cells was 190 to 240 mV between external pH 2 and 4. Anoxia diminished total proton motive force and the transmembrane pH difference by 60 to 80 mV. The protonophore 2,4-dinitrophenol abolished the total proton motive force almost completely and diminished the transmembrane pH difference by at least two units. However, even after correction for probe binding, protonophore-treated cells maintained a pH difference of approximately one unit.     

Effect of phenolic monomers on the growth and beta-glucosidase activity of Bacteroides ruminicola and on the carboxymethylcellulase, beta-glucosidase, and xylanase activities of Bacteroides succinogenes.

trans-p-Coumaric acid inhibited the growth of Bacteroides ruminicola on both cellobiose and glucose, while trans-ferulic acid and vanillin retarded growth. The phenolic monomers varied in their potential to inhibit the Bacteroides succinogenes beta-glucosidase, carboxymethylcellulase, and xylanase, with p-coumaric acid being the most inhibitory. The B. ruminicola beta-glucosidase was inhibited less than 10% by all three compounds.     

The effect of ionophores on proton flux in the ruminal bacterium,streptococcus bovis

NonenergizedStreptococcus bovis cells, which were washed in potassium-phosphate buffer and incubated in Tris buffer containing 200mm potassium chloride (pH 6.5), did not take up tetraphenylphosphonium ion (TPP⁺), but the same cells took up TPP⁺ when they were incubated in Tris buffer lacking potassium. This result indicated that passive potassium diffusion was creating an electrical potential () across the cell membrane. Neither cells took significant amounts of 9-aminoacridine (9-AA), an intracellular pH marker. Cells that were incubated in Tris buffer and treated with carbonyl cyanidem-chlorophenylhydrazone (CCCP) took up 9-AA, and this result indicated that this protonophore was facilitating proton influx. The ionophores monensin and lasalocid also caused 9-AA uptake, and it appeared that they were responsible for or responsive to potassium/proton antiport. However, there was also a rapid accumulation of 9-AA when the cells were treated with valinomycin, a potassium uniporter that cannot translocate protons. This latter result indicated that potassium efflux was associated with another avenue of proton influx (e. g., potassium/proton symport). Because cells treated with dicyclohexyl carbodiimide (DCCD) also exhibited valinomycin-dependent 9-AA uptake, it is unlikely that the F₁F₀ATPase or ATP formation was responsible for proton flux across the cell membrane.     

Effects of K+ and Na+ on the proton motive force of respiring Escherichia coli at alkaline pH.

The role of K+ and Na+ in the maintenance of the proton motive force (delta p) was studied in Escherichia coli incubated in alkaline media. Cells respiring in Tris buffer (pH 7.8) that contained less than 100 microEq of K+ and Na+ per liter had a normal delta p of about -165 mV. At pH 8.2, however, the delta p was reduced significantly. The decrease in delta p at pH 8.2 was due to a marked decrease in the transmembrane potential (delta psi), while the internal pH remained at 7.5 to 7.7. When KCl or NaCl, but not LiCl or choline chloride, was added to the cells, the delta psi rose to the values seen at an external pH of 7.8. In addition, choline chloride inhibited the enhancement of delta psi by K+. None of the salts had a significant effect on the internal pH. The effects can be attributed to alterations of K+ or Na+ cycling in and out of the cells via the known K+ and Na+ transport systems.     

Simultaneous measurements of proton motive force, delta pH, membrane potential, and H+/O ratios in intact Escherichia coli.

An instrument is described that enables the simultaneous monitoring of proton motive force (PMF), membrane potential (delta psi), the delta pH across a membrane, oxidase activity, proton movements, and H+/O ratios. We have studied the relationship existing among these parameters of energy transduction as a critical condition is changed during an experiment. The major findings are: (a) In the pH range of 4.5 to 7.5, increasing the external pH causes an increase in delta psi, internal pH, and oxidase activity, a decrease in H+/O ratio, and a peak-plateau in PMF from pH 5.5 to 6.6 where delta pH is converted to delta psi. (b) An increase in [K+] from 1 to 100 mM, in the presence of 0.5 microM valinomycin, causes the conversion of delta psi to delta pH, a gradual decline in PMF and an increase in H+/O ratio, internal pH, and oxidase activity. (c) Increasing valinomycin concentration from 0 to 2.5 microM, in the presence of 50 mM [K+], causes a decline in delta psi from 125 to 0 mV, and an increase in delta pH from 35 to 70 mV. From 2.5 to 10 microM, the delta pH and the PMF (which it solely represents), stay constant, H+/O ratio increases mainly from 0 to 0.5 microM and much more slowly from 2.5 to 10 microM. (d) Oxygen at only 10% of its concentration in air-saturated buffer can support the generation of 90% or more of the delta psi, delta pH, and PMF generated in an air-saturated solution. (e) The return of extruded protons to the cell (referred to here as "suck-back") represents a complicated process driven by delta psi and influenced by a variety of factors. (f) H+/O ratios measured by the kinetic technique used here are much higher than those measured by standard oxygen pulse techniques.     

The interaction between electron transfer,proton motive force and solute transport in bacteria

The properties of proton solute symport have been studied inStreptococcus cremoris, Rhodopseudomonas sphaeroides andEscherichia coli. In the homolactic fermentative organismS. cremoris the efflux of lactate is a membrane proteinmediated process, which can lead to the generation of a proton motive force. These observations support the energy-recycling model that postulates the generation of metabolic energy by end-product efflux. Studies with oxidants and reductants and specific dithiol reagents inE. coli membrane vesicles demonstrated the presence of two redox-sensitive dithiol-disulphide groups in the transport proteins of proline and lactose. The redox state of these groups is controlled by the redox potential of the environment and by the proton motive force. One redox-sensitive group is located at the inner surface, the other at the outer surface of the membrane. InRps. sphaeroides andE. coli the activity of several transport proteins depends on the activity of the electron transfer systems. On the basis of these results a redox model for proton solute transport coupled in parallel to the electron transfer system is postulated.     

Measurement of components of proton motive force and related parameters in steady,microaerobic conditions

An experimental system is described for the simultaneous measurement of components of the proton motive force and other energy-related activies in microorganisms under steady defined microaerobic conditions. Oxygen is supplied in a solution containing an O₂-carrier such as myoglobin or leghaemoglobin, to a stirred reaction chamber in which a suspension of the microorganism is required aboce a membrane filter whic is pervious to the carrier. The rate of O₂ consumption is regulated by the rate at which the solution is pumped through the chamber. The concentration of free O₂ in the chamber and the rate of its consumption are calculated from the pumping rate and the decline in the relative oxygenation of the carrier measured spectrophotometrically in the effluent solution. The uptake by the microorganisms of radioactively labelled probes for ΔpH and Δψ is calculated following their injection into the reaction chamber and monitoring of continuously collected fractions of effluent solution, after it has passed through the spectrophometer. An example of the use of the system is given.The use of this system is advocated for many microaerobic applications since most of the measurements can be made without perturbing the steady state until the final shape of the suspension is collected.     

Effect of pH and Monensin on Glucose Transport by Fibrobacter succinogenes, a Cellulolytic Ruminal Bacterium

Fibrobacter succinogenes S85, a cellulolytic ruminal bacterium, required sodium for growth and glucose uptake. Cells which were deenergized with iodoacetate (500 μM) could not take up [¹⁴C]glucose. However, deenergized cells which were treated with valinomycin, loaded with potassium, and diluted into sodium or sodium plus potassium to create an artificial electrical gradient (ΔΨ) plus a chemical gradient of sodium (ΔpNa) or ΔpNa alone transported glucose at a rapid rate. Cells which were loaded with potassium plus sodium and diluted into sodium (ΔΨ with sodium, but no ΔpNa) also took up glucose at a rapid rate. Potassium-loaded cells that were diluted into buffers which did not contain sodium (ΔΨ without sodium) could not take up glucose. An artificial ZΔpH which was created by acetate diffusion could not drive glucose transport even if sodium was present. The maximum rate and affinity of glucose transport (pH 6.7) were 62.5 nmol/mg of protein per min and 0.51 mM, respectively. S85 was unable to grow at a pH of less than 5.5, and there was little glucose transport at this pH. When the extracellular pH was decreased, the glucose carrier was inhibited, intracellular pH declined, the cells were no longer able to metabolize glucose, and ΔΨ declined. Monensin (1 μM) or lasalocid (5 μM) decreased intracellular ATP and dissipated both the ΔΨ and ΔpNa. Since there was no driving force for transport, glucose transport was inhibited. These results indicated that F. succinogenes used a pH-sensitive sodium symport mechanism to take up glucose and that either a ΔΨ or a ΔpNa was required for glucose transport.     

Uniport of anionic citrate and proton consumption in citrate metabolism generates a proton motive force in Leuconostoc oenos.

The mechanism and energetics of citrate transport in Leuconostoc oenos were investigated. Resting cells of L. oenos generate both a membrane potential (delta psi) and a pH gradient (delta pH) upon addition of citrate. After a lag time, the internal alkalinization is followed by a continuous alkalinization of the external medium, demonstrating the involvement of proton-consuming reactions in the metabolic breakdown of citrate. Membrane vesicles of L. oenos were prepared and fused to liposomes containing cytochrome c oxidase to study the mechanism of citrate transport. Citrate uptake in the hybrid membranes is inhibited by a membrane potential of physiological polarity, inside negative, and driven by an inverted membrane potential, inside positive. A pH gradient, inside alkaline, leads to the accumulation of citrate inside the membrane vesicles. Kinetic analysis of delta pH-driven citrate uptake over a range of external pHs suggests that the monovalent anionic species (H2cit-) is the transported particle. Together, the data show that the transport of citrate is an electrogenic process in which H2cit- is translocated across the membrane via a uniport mechanism. Homologous exchange (citrate/citrate) was observed, but no evidence for a heterologous antiport mechanism involving products of citrate metabolism (e.g., acetate and pyruvate) was found. It is concluded that the generation of metabolic energy by citrate utilization in L. oenos is a direct consequence of the uptake of the negatively charged citrate anion, yielding a membrane potential, and from H(+)-consuming reactions involved in subsequent citrate metabolism, yielding a pH gradient. The uptake of citrate is driven by its own concentration gradient, which is maintained by efficient metabolic breakdown (metabolic pull).     

Physiological properties of Cellulomonas fermentans,a mesophilic cellulolytic bacterium

Summary The fermentation of cellobiose, glucose and cellulose MN 300 by Cellulomonas fermentans was studied. The molar growth yields (i.e. grams of cells per mole of hexose equivalent) were similar on cellobiose and cellulose at low sugar consumption levels (47.8 and 46.5 respectively), but was lower on glucose (38.0). The occurrence of cellobiose phosphorylase activity, detected in cellobiose- and cellulose-grown cells, might explain this result. The specific growth rates measured in cultures on cellobiose, glucose and cellulose were 0.055 h^-1, 0.040 h^-1 and 0.013 h^-1 respectively. Growth inhibition was observed, and a drop in Y_H occurred after relatively low but different quantities of hexose were consumed (2.2 mM, 5 mM and 8 mM hexose equivalent with cellulose, glucose and cellobiose respectively), which coincided with a change in the fermentative metabolism from a typical mixed acid metabolism (1 ethanol, 1 acetate and 2 formate synthesized by consumed hexose) to a more ethanolic fermentation. When growth ceased in cellulose cultures, consumption of cellulose continued, as did production of ethanol.Molar growth yields of C. fermentans were similar in anaerobic and aerobic cellobiose cultures (47.8 g/mol and 42.2 g/mol respectively). Specific growth rates were also quite similar under both culture conditions (0.055±0.013 h^-1 and 0.070±0.007 h^-1 respectively). Aerobic metabolism was studied using ¹⁴C glucose. During the exponential growth phase, acetate, succinate and nonidentified compound(s) accumulated in the supernatant, but no ¹⁴CO₂ was produced. During the stationary phase, acetate was oxidized and ¹⁴CO₂ produced, but without any further biomass synthesis. It seems that a blocking of metabolite oxidation may have occurred in C. fermentans except in the case of acetate, but acetate oxidation was apparently not coupled with production of energy utilizable in biosynthesis.