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.     

Characterization of the NADH dehydrogenase and fumarate reductase of Fibrobacter succinogenes subsp. succinogenes S85

Conditions promoting maximal in vitro activity of the particulate NADH:fumarate reductase from Fibrobacter succinogenes were determined. This system showed a pH optimum of 6.0 in K⁺ MES buffer only when salt (NaCl or KCl) was present. Salt stimulated the activity eightfold at the optimal concentration of 150m M. This effect was due to stimulation of fumarate reductase activity as salt had little effect on NADH: decylubiquinone oxidoreductase (NADH dehydrogenase). The stimulation of fumarate reductase by salt at pH 6.0 was not due to removal of oxaloacetate from the enzyme. Kinetic parameters for several inhibitors were also measured. NADH dehydrogenase was inhibited by rotenone at a single site with a K _i of 1 M. 2-Heptyl-4-hydroxyquinonline-N-oxide (HOQNO) inhibited NADH: fumarate reductase with a K _i of 0.006 M, but NADH dehydrogenase exhibited two HOQNO inhibition constants of approximately 1 M and 24 M. Capsaicin and laurylgallate each inhibited NADH dehydrogenase by only 20% at 100 M. NADH dehydrogenase gave K _m values of 1 M for NADH and 4 M for reduced hypoxanthine adenine dinucleotide.Published with the approval of the Director of the Agricultural Experiment Station, North Dakota State University, as journal article no. 2201     

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 extracellular pH and phenolic monomers on glucose uptake by Fibrobacter succinogenes S85

Little change in glucose uptake by Fibrobacter succinogenes S85 was observed when the extracellular pH was between 8.0 and 6.5, but uptake was reduced 42 and 65% at pH values of 5.0 and 4.0, respectively. p -Coumaric acid and vanillin were the strongest inhibitors of glucose uptake by F. succinogenes S85, while ferulic acid was less inhibitory. Collectively, these results help to explain, in part, the growth inhibition observed when F. succinogenes S85 is exposed to low extracellular pH and phenolic monomers.     

Mixotrophic and heterotrophic growth of Beggiatoa alba in continuous culture

Beggiatoa alba strain B18LD was grown in continuous culture under heterotrophic conditions on acetate or acetate and asparagine and under mixotrophic conditions on acetate plus either 1 mM sodium sulfide or 1 mM sodium thiosulfate. Considerable differences were observed between the yields and the cell compositions of heterotrophic and mixotrophic cultures at all dilution rates tested. The dry weight yield per gram acetate utilized was approximately three times higher in the acetate-sulfide mixotrophic culture than in the acetate heterotrophic culture, whereas the poly--hydroxybutyric acid and carbohydrate contents were much higher in the heterotrophic cultures. The high yields (0.52–0.75, corrected for the weight of the sulfur) obtained with the mixotrophic cultures imply that the acetate was utilized mainly for biosynthesis. Thus, the oxidation of sulfide supplied energy. The addition of catalase to the chemostat cultures increased yields slightly, but it was insufficient to explain the differences between the heterotrophic and the mixotrophic cultures.     

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)     

ATPase-dependent energy spilling by the ruminal bacterium,Streptococcus bovis

When the ruminal bacterium Streptococcus bovis was grown in batch culture with glucose as the energy source, the doubling time was approximately 21 min and the rate of bacterial heat production was proportional to the optical density (1.72 W/g protein). If exponentially growing cultures were treated with chloramphenicol, there was a decline in heat production, but the rate was greater than 0.30 W/g protein even after growth ceased. Since there was no heat production after glucose depletion, this growth-independent energy dissipation (spilling) was not simply due to endogenous metabolism. Stationary cells which were washed and incubated in nitrogen-free medium containing an excess of glucose produced heat at a rate of 0.17 W/g protein. Monensin and tetrachlorosalicylanilide (TCS), compounds which facilitate an influx of protons, caused a more than 2-fold increase in heat production. Dicyclohexylcarbodiimide (DCCD) virtually eliminated growth-independent heat production regardless of the mode of growth inhibition. Because DCCD had little effect on the glucose phosphotransferase system, it appeared that the combined action of proton influx and the membrane bound F₁F₀ proton ATPase was responsible for energy spilling.Abbreviations DCCD dicyclohexylcarbodiimide - TCS tetrachlorosalicylanilide     

In vivo 23Na nuclear magnetic resonance study of maintenance of a sodium gradient in the ruminal bacterium Fibrobacter succinogenes S85

Sodium gradients (DeltapNa) were measured in resting cells of Fibrobacter succinogenes by in vivo 23Na nuclear magnetic resonance using Tm(DOTP)5- [thulium(III) 1,4,7,10-tetraazacyclododecane-N',N",N"'-tetramethylenephosphonate] as the shift reagent. This bacterium was able to maintain a DeltapNa of -55 to -40 mV for extracellular sodium concentrations ranging from 30 to 200 mM. Depletion of Na+ ions during the washing steps led to irreversible damage (modification of glucose metabolism and inability to maintain a sodium gradient).     

A re-assessment of bacterial growth efficiency: the heat production and membrane potential of Streptococcus bovis in batch and continuous culture

Glucose-limited, continuous cultures (dilution rate 0.1 h^-1) of Streptococcus bovis JB1 fermented glucose at a rate of 3.9 mol mg protein^-1 h^-1 and produced acctate, formate and ethanol. Based on a maximum ATP yield of 32 cells/mol ATP (Stouthamer 1973) and 3 ATP/glucose, the theoretical glucose consumption for growth would have been 2.1 mol mg protein^-1 h^-1. Because the maintenance energy requirement was 1.7 mol/mg protein/h (Russell and Baldwin 1979), virtually all of the glucose consumption could be explained by growth and maintenance and the Y_ATP was 30. Glucose-limited, continuous cultures produced heat at a rate of 0.29 mW/mg protein, and this value was similar to the enthalpy change of the fermentation (0.32 mW/mg protein). Batch cultures (specific growth rate 2.0 h^-1) fermented glucose at a rate of 81 mol mg protein^-1 h^-1, and produced only lactate. The heat production was in close agreement with the theoretical enthalpy change (1.72 versus 1.70 mW/mg protein), but only 80% of the glucose consumption could be accounted by growth and maintenance. The Y_ATP of the batch cultures was 25. Nitrogen-limited, glucose-excess, non-growing cultures fermented glucose at a rate of 6.9 mol mg protein^-1 h^-1, and virtually all of the enthalpy for this homolactic fermentation could be accounted as heat (0.17 mW/mg protein). The nitrogenlimited cultures had a membrane potential of 150 mV, and nearly all of the heat production could be explained by a futile cycle of protons through the cell membrane (watts = amperes x voltage where H⁺/ATP was 3). The membrane voltage of the nitrogen-limited cells was higher than the glucose-limited continuous cultures (150 versus 80 mV), and this difference in voltage explained why nitrogen-limited cultures consumed glucose faster than the maintenance rate. Batch cultures had a membrane potential of 100 mV, and this voltage could not account for increased glucose consumption (more than growth plus maintenance). It appears that another mechanism causes the increased heat production and lower growth efficiency of batch cultures.     

A continuous culture study of the phosphorus nutrition of Rhizobium trifollii WU95, Rhizobium NGR234 and Bradyrhizobium CB756

With continuous cultures in a fully defined minimal salts medium steady states were achieved at both limiting and non-limiting concentrations of phosphate in the inflowing medium for Rhizobium trifolii WU95, cowpea Rhizobium NGR234, and Bradyrhizobium CB756.Millimolar growth yields obtained from P-limited cultures varied over 2-fold from 3.2 g dry weight·(mmol P)^-1 for WU95 to 5.3 g dry weight·(mmol P)^-1 for CB756 and 7.2 g dry weight·(mmol P)^-1 for NGR234.For both WU95 and NGR234 growth under P-excess conditions resulted in elevated levels of total biomass P and the storage compound polyphosphate, compared with P-limited cultures. However, P-limited cultures of these two strains still contained significant quantities of polyphosphate. The P-status for CB756 cultures did not affect either total biomass P or polyphosphate levels. Alkaline phosphatase was maximally derepressed in P-limited cultures of WU95 and NGR234. However, in CB756 alkaline phosphatase was not detected at significant levels regardless of its P supply.These data suggest that growth of rhizobia is controlled predominantly by the attainment of a critical internal P level.Abbreviation HEPES N-2-hydroxyethylpiperazine-N-2-ethane sulphonic acid     

Response surface analysis of the effects of pH and dilution rate on Ruminococcus flavefaciens FD-1 in cellulose-fed continuous culture.

The ruminal cellulolytic bacterium Ruminococcus flavefaciens FD-1 was grown in cellulose-fed continuous culture with 20 different combinations of pH and dilution rate (D); the combinations were selected according to the physiological pH range of the organism (6.0 to 7.1) and growth rate of the organism on cellulose (0.017 to 0.10 h-1). A response surface analysis was used to characterize the effects of pH and D on the extent of cellulose consumption, growth yield, soluble sugar concentration, and yields of fermentation products. The response surfaces indicate that pH and D coordinately affect cellulose digestion and growth yield in this organism. As expected, the net cellulose consumption increased with increasing D while the fraction of added cellulose that was utilized decreased with increasing D. The effect of changes in pH within the physiological range on cellulose consumption was smaller than that of changes in D. Cellulose degradation was less sensitive to low pH than to high pH. At low Ds (longer retention times), cellulose degradation did not follow first-order kinetics. This decreased rate of cellulose digestion was not due to poor mixing, limitation by other medium components, or preferential utilization of the more amorphous fraction of the cellulose. The cell yield increased from 0.13 to 0.18 mg of cells per mg of cellulose with increasing Ds from 0.02 to 0.06 h-1 and decreased when the pH was shifted from the optimum of 6.5 to 6.8. The effect of pH on cell yield increased with increasing D. The reduced cell yield at low pH appears to be due to both an increase in maintenance energy requirements and a decrease in true growth yield.     

Response surface analysis of the effects of pH and dilution rate on Ruminococcus flavefaciens FD-1 in cellulose-fed continuous culture.

The ruminal cellulolytic bacterium Ruminococcus flavefaciens FD-1 was grown in cellulose-fed continuous culture with 20 different combinations of pH and dilution rate (D); the combinations were selected according to the physiological pH range of the organism (6.0 to 7.1) and growth rate of the organism on cellulose (0.017 to 0.10 h-1). A response surface analysis was used to characterize the effects of pH and D on the extent of cellulose consumption, growth yield, soluble sugar concentration, and yields of fermentation products. The response surfaces indicate that pH and D coordinately affect cellulose digestion and growth yield in this organism. As expected, the net cellulose consumption increased with increasing D while the fraction of added cellulose that was utilized decreased with increasing D. The effect of changes in pH within the physiological range on cellulose consumption was smaller than that of changes in D. Cellulose degradation was less sensitive to low pH than to high pH. At low Ds (longer retention times), cellulose degradation did not follow first-order kinetics. This decreased rate of cellulose digestion was not due to poor mixing, limitation by other medium components, or preferential utilization of the more amorphous fraction of the cellulose. The cell yield increased from 0.13 to 0.18 mg of cells per mg of cellulose with increasing Ds from 0.02 to 0.06 h-1 and decreased when the pH was shifted from the optimum of 6.5 to 6.8. The effect of pH on cell yield increased with increasing D. The reduced cell yield at low pH appears to be due to both an increase in maintenance energy requirements and a decrease in true growth yield.     

Influence of acetate on the growth of Candida utilis in continuous culture

Candida utilis was grown on acetate in chemostat cultures that were, successively, carbon and ammonia-limited (30° C; pH 5.5). With carbon(acetate)-limited cultures, the specific rate of oxygen consumption (q _{O 2}) was not a linear function of the growth rate but was markedly stimulated at the higher dilution rates, thus effecting a marked decrease in the Y _O value. This increased respiration rate, and decreased yield value, correlated closely with a marked increase in the extracellular acetate concentration. Under ammonia-limiting conditions, very low Y _O values were found, generally comparable with those found with carbon-limited cultures growing at the higher dilution rates, but these varied markedly with the extracellular acetate concentration. Thus, when the unused acetate concentration was raised progressively from about 5 g/l to about 21 g/l, the Y _O value decreased non-linearly from 11.4 to 5.8. When the extracellular acetate concentration was further increased to 25 g/l, growth was inhibited and the culture washed out. This relationship between respiration rate and the extracellular concentration of unused acetate was also markedly influenced by the culture pH value. Thus, with a fixed extracellular acetate concentration (16±2g/l) and dilution rate (0.14 h^–1), lowering the culture pH value progressively from 6.9 to 5.1 effected a marked and progressive increase in the respiration rate. Further lowering of the culture pH to 4.8, however, caused a complete collapse of respiration. In contrast to this situation, progressively lowering the pH value of an acetatelimited culture from 6.9 to 4.5 affected only slightly the culture respiration rate, and growth was possible even at a pH value of 2.5. These results are discussed in the context of the possible mechanisms whereby acetate exerts its toxic effect on the growth of C. utilis.     

A continuous culture study of an ATPase-negative mutant of Escherichia coli

For anaerobic glucose-limited chemostat cultures of Escherichia coli a value of 8.5 was found for Y _ATP ^max . For anaerobic glucose- or ammoniumlimited chemostat cultures of the ATPase-negative mutant M2-6 of E. coli Y _ATP ^max values of 17.6 and 20.0 were found, respectively. From these data it can be concluded that in the wild type during anaerobic growth 51–58% of the total ATP production is used for energetization of the membrane. Using the Y _ATP values obtained in the anaerobic experiments a P/O ratio of 1.46 could be calculated for aerobic experiments with the wild type. It is concluded that from the energy obtained by respiration in wild type E. coli about 60% is used for membrane energetization and only about 40% for the actual formation of ATP. No dramatic difference in the maintenance requirement for ATP or glucose has been observed between glucose- and ammonium-limited chemostat cultures of the mutant. The large difference in maintenance requirement observed for such cultures of the wild type is therefore supposed to be made possible by ATP hydrolysis by the ATPase.     

The role of energy-spilling reactions in the growth ofKlebsiella aerogenes NCTC 418 in aerobic chemostat culture

When cell-saturating amounts of glucose and phosphate were added to steady state cultures ofKlebsiella aerogenes that were, respectively, glucose-and phosphate-limited, the organisms responded immediately with an increased oxygen consumption rate. This suggested that in neither case was glucose transport the rate-limiting process, and also that organisms must posses effective mechanisms for spilling the excess energy initially generated when a growth-limitation is temporarily relieved.Steady state cultures of mannitol- or glucose-limited organisms also seemingly generated energy at a greater rate than was required for cell synthesis since gluconate-limited cultures consumed oxygen at a lower rate, at each corresponding growth rate, than did mannitol- or glucose-limited cultures, and there-fore expressed a higherY _o value. Thus, mannitol- and glucose-limitations must be essentially carbon (and not energy) limitations. The excess energy generated by glucose metabolism is one component of maintenance and could be used at lower growth rates to maintain an increased solute gradient across the cell membrane, imposed by the addition of 2%, w/v, NaCl to the growth environment.The maintenance rates of oxygen consumption ofK. aerogenes also could be caused to increase by adding glucose discontinuously (drop-wise) to a glucose-limited chemostat culture, or by exchanging nitrate for ammonia as the sole utilizable nitrogen source.The significance of these findings to an assessment of the physiological factors circumscribing energy-spilling reactions in aerobic cultures ofK. aerogenes is discussed.     

Cloning, nucleotide sequence and expression of the gene encoding the cellulose-binding protein 1 (CBP1) of Fibrobacter succinogenes S85

Abstract The nucleotide sequence of the gene encoding the Fibrobacter succinogenes S85 cellulose-binding protein 1 (CBP1) has been determined. The gene encodes a protein of 1054 amino acids with a molecular mass of 118614. The deduced amino acid sequence of CBPl showed an extensive similarity to the cellulose-binding domain of an endoglucanase (EGCCD) from Clostridium cellulolyticum and contained the reiterated regions. The cloned gene was inserted into an expression vector, pRSETA, and was expressed in E. coli as a fused protein with the peptide consisting of six consecutive histidine residues. The fused protein was detected by immunoblotting using antiserum against CBP1, and exhibited the cellulose-binding activities.     

Production of oligosaccharides and cellobionic acid by <Emphasis Type="Italic">Fibrobacter succinogenes</Emphasis> S85 growing on sugars,cellulose and wheat straw

Extracellular culture fluid of Fibrobacter succinogenes S85 grown on glucose, cellobiose, cellulose or wheat straw was analysed by 2D-NMR spectroscopy. Cellodextrins did not accumulate in the culture medium of cells grown on cellulose or straw. Maltodextrins and maltodextrin-1P were identified in the culture medium of glucose, cellobiose and cellulose grown cells. New glucose derivatives were identified in the culture fluid under all the substrate conditions. In particular, a compound identified as cellobionic acid accumulated at high levels in the medium of F. succinogenes S85 cultures. The production of cellobionic acid (and cellobionolactone also identified) was very surprising in an anaerobic bacterium. The results suggest metabolic shifts when cells were growing on solid substrate cellulose or straw compared to soluble sugars.     

Influence of culture conditions on the cell yield and amylases biosynthesis in continuous culture by Schwanniomyces castellii

Schwanniomyces castellii excreted -amylase and amyloglucosidase into the medium in the presence of starch. The biosynthesis and the rate of excretion were influenced by dissolved oxygen (specially for -amylase), pH of the culture and dilution rate. The cell yield observed (0.59) remained constant up to D=0.35h^-1 with starch as substrate. But in the case of growth on glucose, the yield observed was equal to 0.62 up to a dilution rate of D=0.18 h^-1. Beyond this value Y x/s decreased and ethanol was produced. The onset of fermentation dependend partly on the nature of the substrate and not only on the environment in particular on the quantity of dissolved oxygen present.     

A continuous culture study of the bioenergetic aspects of growth and production of exocellular protease in Bacillus licheniformis

Summary Bacillus licheniformis S 1684 is able to produce an alkaline serine protease exocellularly. In glucose-limited chemostat cultures the specific rate of protease production was maximal at a -value of 0.22. Above this growth rate protease production was repressed. Dependent on 10–20% of the glucose input was used for exocellular product formation. The degree of reduction of exocellular products was 4.1.Maximum molar growth yields were high and indicate a high efficiency of growth. The values of Y _glu ^max and YO ₂ ^max were 83.8 and 53.3, respectively. When Y _glu ^max was corrected for the amount of glucose used for product formation a value of 100.3 was obtained. These high maximum molar growth yields are most probably caused by a high Y _ATP ^max . Anaerobic batch experiments showed a Y _ATP of 14.6.Sometimes the used strain was instable in cell morphology and protease production. Non-protease producing cells most probably develop from producing cells by mutation in the rel-gene. Producing cells most probably are relaxed (rel ^-) and non-producing cells stringent (rel ⁺).Glossary specific growth rate (h^-1) - Y _sub growth yield permol substrate (g biomass/mol) - Y ^max maximum molar growth yield, corrected for maintenance requirements (g biomass/mol) - Y ^max(corr) Y ^max corrected for product formation (g biomass/mol) - m _sub maintenance requirements (mol/g biomass·h) - m _sub(corr) maintenance requirements corrected for product formation (mol/g biomass·h) - Y _c fraction of organic substrate converted in biomass - z fraction of organic substrate converted in exocellular products - d fraction of organic substrate converted in CO₂ (g mol/g atom C) - Crec% carbon recovery % - average degree of reduction of exocellular products - P/O amount of ATP produced during electron-transport of 2 electrons to oxygen