Succinogenes S85 Research Articles

The role of ruminal carboxymethylcellulases in the degradation of β-glucans from cereal grain

The non-cellulolytic ruminal bacterium, Prevotella bryantii, grew rapidly on water soluble β-glucans, but a mutant deficient in carboxymethylcellulase (CMCase) activity could not. Native activity gels prepared with CMC and β-glucan indicated that the CMCase and β-glucanase activities migrated at similar rates. When a plasmid carrying the P. bryantii CMCase was transferred to Escherichia coli, the clone had CMCase and β-glucanase activities. P. bryantii grew on mixed β-1,3-1,4 glucans, but not on β-1,3 glucan, and similar results were obtained with the cellulolytic ruminal bacteria (Fibrobacter succinogenes S85, Ruminococcus flavefaciens FD1 and Ruminococcus albus B199). Mixed ruminal bacteria from cattle fed hay had twice as much CMCase activity as bacteria from cattle fed 90% cereal grain (P<0.05), and the CMCase and β-glucanase activities were highly correlated (r2=0.93) for the 22 samples tested. The CMCase and β-glucanase activities of mixed ruminal bacteria migrated slowly through polyacrylamide gels, but the migration distances were approximately the same. When β-glucan-utilizing ruminal bacteria were isolated from cattle fed hay or 90% cereal grain, 70 and 38% of the strains, respectively, had CMCase activity. A similar trend was observed with cellobiose-utilizing isolates (70 and 35%, respectively, were CMCase positive). All CMCase positive, cellobiose-utilizing ruminal bacteria could grow on β-glucan. CMCase activity was not strongly correlated with cellulose utilization, and less than 15% of the CMCase positive isolates grew on ball-milled cellulose. Based on these results, the cell-associated CMCases of ruminal bacteria provide a mechanism for utilizing water soluble β-glucans from cereal grains.

The role of ruminal carboxymethylcellulases in the degradation of β-glucans from cereal grain

The non-cellulolytic ruminal bacterium, Prevotella bryantii, grew rapidly on water soluble β-glucans, but a mutant deficient in carboxymethylcellulase (CMCase) activity could not. Native activity gels prepared with CMC and β-glucan indicated that the CMCase and β-glucanase activities migrated at similar rates. When a plasmid carrying the P. bryantii CMCase was transferred to Escherichia coli, the clone had CMCase and β-glucanase activities. P. bryantii grew on mixed β-1,3-1,4 glucans, but not on β-1,3 glucan, and similar results were obtained with the cellulolytic ruminal bacteria ( Fibrobacter succinogenes S85, Ruminococcus flavefaciens FD1 and Ruminococcus albus B199). Mixed ruminal bacteria from cattle fed hay had twice as much CMCase activity as bacteria from cattle fed 90% cereal grain ( P<0.05), and the CMCase and β-glucanase activities were highly correlated ( r 2=0.93) for the 22 samples tested. The CMCase and β-glucanase activities of mixed ruminal bacteria migrated slowly through polyacrylamide gels, but the migration distances were approximately the same. When β-glucan-utilizing ruminal bacteria were isolated from cattle fed hay or 90% cereal grain, 70 and 38% of the strains, respectively, had CMCase activity. A similar trend was observed with cellobiose-utilizing isolates (70 and 35%, respectively, were CMCase positive). All CMCase positive, cellobiose-utilizing ruminal bacteria could grow on β-glucan. CMCase activity was not strongly correlated with cellulose utilization, and less than 15% of the CMCase positive isolates grew on ball-milled cellulose. Based on these results, the cell-associated CMCases of ruminal bacteria provide a mechanism for utilizing water soluble β-glucans from cereal grains.

Use of ratio of digested xylan to digested cellulose (X/C) as an index of fiber digestion in plant cell-wall material by ruminal microorganisms

Traits in digestion of plant cell-wall components of timothy ( Phleum pratense) hay were studied in pure cultures of ruminal fibrolytic microorganisms and in sacco. Four strains of rumen bacteria, Butyrivibrio fibrisolvens D1 (ATCC19171), Fibrobacter succinogenes S85 (ATCC19169), Prevotella ruminicola subsp. brevis 7-31, and Ruminococcus albus 7, and four strains of anaerobic fungi, Neocallimastix frontalis MCH3, Piromyces communis 93-3, Piromyces sp. P1 and polycentric fungus KP1, were used in this experiment. Each microorganism was inoculated on ground timothy hay (20 g l −1) in an anaerobic medium and incubated at 39°C. Dry matter loss of timothy hay and digested xylan to digested cellulose (X/C) ration of the substrate during incubation were determined. The X/C ratios in the cultures of Pre. ruminicola and B. fibrisolvens were greater than 0.5. Those of R. albus, Pir. communis, Piromyces sp. P1 and N. frontalis were between 0.3 and 0.5. Those of F. succinogenes and polycentric fungus KP1 were lower than 0.3. The X/C ratios observed in sacco were around 0.2 and close to those of F. succinogenes. The similarity of the digested X/C ratios between F. succinogenes and in sacco measurements suggests that F. succinogenes plays a predominant role in fiber digestion in the rumen.

A truncated bacterial xylanase expressed in mammalian cells exhibits increased sensitivity to proteases

A truncated xylanase gene from the ruminal bacterium Fibrobacter succinogenes S85 was expressed in Chinese hamster ovary (CHO) cells and pancreatic acinar 266–6 cells using the SV40 early promoter and the mouse Amy-2.2 promoter/enhancer, respectively. The enzyme produced in both systems was active and secreted into the medium. The xylanase secreted from CHO cells was highly glycosylated and became sensitive to protease digestion compared to the unglycosylated form. © Rapid Science Ltd. 1998

The adverse effect of nitrogen limitation and excess-cellobiose on Fibrobacter succinogenes S85

Fibrobacter succinogenes S85 cultures that were cellobiose-limited converted cellobiose to succinate and acetate, produced little glucose or cellotriose, maintained an intracellular ATP concentration of 4.1 mM and a membrane potential of 140 mV for 24 h, did not lyse at a rapid rate once they had reached sta- tionary phase, and had a most probable number of viable cells that was greater than 10 6 /ml. When the cel- lobiose concentration was increased 6-fold (5 mM to 30 mM), ammonia was depleted and the cultures left 10 mM cellobiose. Cultures provided with excess cello- biose produced succinate and acetate while they were growing, but there was little increase in fermentation acids after the ammonia was depleted and growth ceased. The stationary-phase, cellobiose-excess cultures had a lysis rate that was 7-fold faster than that of the cellobiose-limited cultures, and the most probable number was only 3.3 · 10 3 cells/ml. The stationary- phase, cellobiose-excess cultures had 2.5 times as much cellular polysaccharide as the cellobiose-limited cultures, but the intracellular ATP and membrane potential were very low (0.1 mM and 40 mV respectively). Met- hylglyoxal, a potentially toxic end-product of carbohy- drate fermentation, could not be detected, and fresh inocula grew rapidly in spent medium that was supple- mented with additional ammonia. Stationary-phase, cellobiose-excess cultures converted cellobiose to glucose and cellotriose, but the apparent Km of cellotriose for- mation was 15-fold lower than the Km of glucose pro- duction (0.7 mM compared to 10 mM).

Production of succinate from glucose, cellobiose, and various cellulosic materials by the ruminal anaerobic bacteria Fibrobacter succinogenes and Ruminococcus flavefaciens.

The production of organic acids by two anaerobic ruminal bacteria Fibrobacter succinogenes S85 and Ruminococcus flavefaciens FD-1, was compared with glucose, cellobiose, microcrystalline cellulose, Walseth cellulose (acid swollen cellulose), pulped paper, and steam-exploded yellow poplar as substrates. The major end product produced by F. succinogenes from each of these substrates was succinate (69.5-83%), the principal secondary product was acetate (16-30.5%). Maximum succinate productivity ranged from 14.1 mg/L.h for steam-exploded yellow Poplar to 59.7 mg/L.h for pulped paper. For R. flavefaciens, the major end product from cellobiose, microcrystalline cellulose, and acid-swollen Walseth cellulose was acetate (39-46%), pulped paper and steam-exploded yellow poplar yielded succinate (42-54%) as the major product. Maximum succinate productivity by R. flavefaciens ranged from 9.21 mg/L.h for cellobiose to 43.1 mg/L.h for pulped paper. In general, much less succinate was produced at a lower maximum productivity by R. flavefaciens than by F. succinogenes under similar fermentation conditions. The maximum succinate productivities by these two organisms are comparable to the previously reported value of 59 mg/L.h for Anderobiospirillum succiniciproducens grown on glucose and corn steep liquor.

Effects of a Saccharomyces cerevisiae Culture on Ruminal Bacteria that Utilize Lactate and Digest Cellulose

The objective of this study was to determine the effects of a yeast (Saccharomyces cerevisiae) culture on lactate utilization and cellulose digestion by ruminal bacteria. Growth of Selenomonas ruminantium HD4 in medium that contained 5g/L of DL-lactate, Trypticase, and yeast extract was stimulated 7 and 15% by 1 and 5% (vol/vol) yeast culture filtrate, respectively. The 1 and 5% yeast culture filtrate stimulated growth of Selenomonas ruminantium H18 and Megasphaera elsdenii B159 and T81 on 5g/L of DL-lactate in medium without Trypticase or yeast extract. Growth of Fibrobacter succinogenes S85 and Ruminococcus albus B199 on 6g/L of cellobiose was stimulated by the addition of yeast culture filtrate to medium without Trypticase or yeast extract. The yeast culture filtrate increased the concentrations of acetate and total volatile fatty acids that were produced by Sel. ruminantium HD4 and increased the concentrations of propionate and total volatile fatty acids that were produced by Sel. ruminantium H18 but did not alter end-product formation of M. elsdenii or cellulolytic bacteria. Treatment with yeast culture increased the initial rate but not the extent of cellulose digestion by F. succinogenes S85 and Ruminococcus flavefaciens FD1. Collectively, these results suggest that yeast culture provides soluble growth factors (i.e., organic acids, B vitamins, and amino acids) that stimulate growth of ruminal bacteria that utilize lactate and digest cellulose.

Competition Between Ruminal Cellulolytic Bacteria for Adhesion to Cellulose

Competition for adhesion to cellulose among the three main ruminal cellulolytic bacterial species was studied using differential radiolabeling (14C/3H) of cells. When added simultaneously to cellulose, Ruminococcus flavefaciens FD1 and Fibrobacter succinogenes S85 showed some competition; however, both species were surpassed competitively by Ruminococcus albus 20. When R. flavefaciens FD1 and F. succinogenes S85 were already adherent, R. albus 20 adhesion occurred without inhibition but involved R. flavefaciens FD1 detachment.

Construction of a Fibrobacter succinogenes genomic map and demonstration of diversity at the genomic level.

The genomic cleavage map of the type strain Fibrobacter succinogenes S85 was constructed. The restriction enzymes AscI, AvrII, FseI, NotI, and SfiI generated DNA fragments of suitable size distribution that could be resolved by pulsed-field gel electrophoresis (PFGE). An average genome size of 3.6 Mb was obtained by summing the total fragment sizes. The linkages between the 15 AscI fragments of the genome were determined by combining two approaches: isolation of linking clones and cross-hybridization of restriction fragments. The genome of F. succinogenes was found to be represented by the single circular DNA molecule. Southern hybridization with specific probes allowed the eight genetic markers to be located on the restriction map. The genome of this bacterium contains at least three rRNA operons. PFGE of the other three strains of F. succinogenes gave estimated genome sizes close to that of the type strain. However, RFLP patterns of these strains generated by AscI digestion are completely different. Pairwise comparison of the genomic fragment distribution between the type strain and the three isolates showed a similarity level in the region of 14.3% to 31.3%. No fragment common to all of these F. succinogenes strains could be detected by PFGE. A marked degree of genomic heterogeneity among members of this species makes genomic RFLP a highly discriminatory and useful molecular typing tool for population studies.

Catalytic properties of the cellulose-binding endoglucanase F from Fibrobacter succinogenes S85.

The celF gene from the predominant cellulolytic ruminal bacterium Fibrobacter succinogenes encodes a 118.3-kDa cellulose-binding endoglucanase, endoglucanase F (EGF). This enzyme possesses an N-terminal cellulose-binding domain and a C-terminal catalytic domain. The purified catalytic domain displayed an activity profile typical of an endoglucanase, with high catalytic activity on carboxymethyl cellulose and barley beta-glucan. Immunoblotting of EGF and the formerly characterized endoglucanase 2 (EG2) from F. succinogenes with antibodies prepared against each of the enzymes demonstrated that EGF and EG2 contain cross-reactive epitopes. This data in conjunction with evidence that the proteins are the same size, share a 19-residue internal amino acid sequence, possess similar catalytic properties, and both bind to cellulose allows the conclusion that celF codes for EG2.

Effect of silica on the colonization of rice straw by ruminal bacteria

The relationship between silica distribution and microbial colonization in the cuticle of rice straw was examined using scanning electron microscopy and energy dispersive X-ray microanalysis (EDX). Rice straw stems cut into 1 -cm lengths were either treated with 40 g NH3 kg−1 dry matter (DM) or left untreated. Microbial colonization was examined using cultures of Fibrobacter succinogenes S85 grown on rice straw as the sole source of carbohydrate. The nylon bag technique was also used to study the colonization of rice straw by mixed ruminal microorganisms and to determine in sacco dry matter disappearance (ISDMD). Examination by EDX revealed the outer (abaxial) surface of the leaf sheath to be virtually covered by silica, with high levels of accumulation within distinctly arranged opaline bodies (phytoliths). Silica was distributed diffusely over most of the inner (adaxial) surface of the leaf sheath, but phytoliths were absent and some regions were devoid of silica. Ammonia treatment dramatically increased the ISDMD of rice straw, but did not alter the content or distribution of silica on either surface of the leaf sheath. Trichomes contained no silica and were major sites of invasion and colonization in the intact cuticle of rice straw. Cuticular damage and loss were more prevalent in treated than in untreated rice straw, and underlying tissues were found to be devoid of silica and extensively colonized. The improved digestibility of ammonia-treated rice straw may be partially due to a weakening of the adhesion between the cuticle and underlying tissues. Detachment of the cuticle removes this barrier to digestion and exposes underlying silica-free tissues to bacterial colonization.

Chemical and Biological Assays to Evaluate Bacterial Inhibition by Tannins

The interactions among purified tannins from three different plants with five strains of ruminal bacteria were investigated. Tannins were purified from crude quebracho (Schinopsis balansae spp.), desmodium (Desmodium ovalifolium), and myrtle (Mirtus communis). The ruminal bacteria studied were Streptococcus bovis JB1, Ruminococcus albus 8, Fibrobacter succinogenes S85, Prevotella ruminicola H14, and a recently isolated tannin-tolerant bacterium. Bacterial binding to tannins, rate of tannin binding, and the inhibitory concentrations for each bacterium to each tannin were evaluated. The effects of tannins on glucose utilization and fermentation product formation also were studied. Tannin binding was rapid with little additional binding occurring 10 min after tannin addition. There was variation among bacteria in the amount of tannins bound and in the amount of tannin required to inhibit growth. Condensed tannins from myrtle and desmodium had the highest binding capacity and were most inhibitory to microbes. Absorbance readings from the total phenolics assay were highest for myrtle and lowest for quebracho. Similarly, readings for condensed tannins were highest for desmodium, which also had the largest polymer size. Myrtle and quebracho gave the largest precipitate rings in the radial diffusion assay, a biological assay that measures precipitation of bovine serum albumin by tannins. The relative absorbance values obtained from the total phenolics assay were the most useful predictor of tannin inhibition of microbial growth. The acid–butanol assay and the radial diffusion assay using bovine serum albumin as the precipitable protein were much less useful predictors of tannin biological activity against the bacteria studied.

Molecular beacons: trial of a fluorescence-based solution hybridization technique for ecological studies with ruminal bacteria.

Molecular beacons are fluorescent probes developed for solution rather than membrane hybridization. We have investigated the utility of these probes to study rumen microbial ecology. Two cellulolytic species, Ruminococcus albus and Fibrobacter succinogenes, were tested. Membrane and solution hybridizations gave similar results in competition experiments with cocultures of R. albus 8 and F. succinogenes S85.

Kinetics of Cellulose Digestion by Fibrobacter succinogenes S85

Growing cultures of Fibrobacter succinogenes S85 digested cellulose at a rapid rate, but nongrowing cells and cell extracts did not have detectable crystalline cellulase activity. Cells that had been growing exponentially on cellobiose initiated cellulose digestion and succinate production immediately, and cellulose-dependent succinate production could be used as an index of enzyme activity against crystalline cellulose. Cells incubated with cellulose never produced detectable cellobiose, and cells that were preincubated for a short time with thiocellobiose lost their ability to digest cellulose (competitive inhibition [K(infi)] of only 0.2 mg/ml or 0.56 mM). Based on these results, the crystalline cellulases of F. succinogenes were very sensitive to feedback inhibition. Different cellulose sources bound different amounts of Congo red, and the binding capacity was HCl-regenerated cellulose > ball-milled cellulose > Sigmacel > Avicel > filter paper. Congo red binding capacity was highly correlated with the maximum rates of metabolism of cellulose digestion and inversely related to K(infm). Congo red (250 (mu)g/ml) did not inhibit the growth of F. succinogenes S85 on cellobiose, but this concentration of Congo red inhibited the rate of ball-milled cellulose digestion. A Lineweaver-Burk plot of ball-milled cellulose digestion rate versus the amount of cellulose indicated that Congo red was a competitive inhibitor of cellulose digestion (K(infi) was 250 (mu)g/ml).

Competition for cellulose among three predominant ruminal cellulolytic bacteria under substrate-excess and substrate-limited conditions.

Three predominant ruminal cellulolytic bacteria (Fibrobacter succinogenes S85, Ruminococcus flavefaciens FD-1, and Ruminococcus albus 7) were grown in different binary combinations to determine the outcome of competition in either cellulose-excess batch culture or in cellulose-limited continuous culture. Relative populations of each species were estimated by using signature membrane-associated fatty acids and/or 16S rRNA-targeted oligonucleotide probes. Both F. succinogenes and R. flavefaciens coexisted in cellulose-excess batch culture with similar population sizes (58 and 42%, respectively; standard error, 12%). By contrast, under cellulose limitation R. flavefaciens predominated (> 96% of total cell mass) in coculture with F. succinogenes, regardless of whether the two strains were inoculated simultaneously or whether R. flavefaciens was inoculated into an established culture of F. succinogenes. The predominance of R. flavefaciens over F. succinogenes under cellulose limitation is in accord with the former's more rapid adherence to cellulose and its higher affinity for cellodextrin products of cellulose hydrolysis. In batch cocultures of F. succinogenes and R. albus, the populations of the two species were similar. However, under cellulose limitation, F. succinogenes was the predominant strain (approximately 80% of cell mass) in cultures simultaneously coinoculated with R. albus. The results from batch cocultures of R. flavefaciens and R. albus were not consistent within or among trials: some experiments yielded monocultures of R. albus (suggesting production of an inhibitory agent by R. albus), while others contained substantial populations of both species. Under cellulose limitation, R. flavefaciens predominated over R. albus (85 and 15%, respectively), as would be expected by the former's greater adherence to cellulose. The retention of R. albus in the cellulose-limited coculture may result from a combination of its ability to utilize glucose (which is not utilizable by R. flavefaciens), its demonstrated ability to adapt under selective pressure in the chemostat to utilization of lower concentrations of cellobiose, a major product of cellulose hydrolysis, and its possible production of an inhibitory agent.

Competition for cellobiose among three predominant ruminal cellulolytic bacteria under substrate-excess and substrate-limited conditions.

The ruminal cellulolytic bacteria Ruminococcus flavefaciens FD-1 and Fibrobacter succinogenes S85 coexisted in substrate-excess coculture with about equal population size, but R. flavefaciens outcompeted F. succinogenes for cellobiose in the substrate-limited cocultures whether the two strains were coinoculated or a steady-state culture of F. succinogenes was challenged by R. flavefaciens. This outcome of competition between these two strains is due to a classical pure and simple competition mechanism based on affinity for cellobiose. Although the population size of F. succinogenes was much higher (> 70%) than that of another cellulolytic species, Ruminococcus albus 7 in substrate-excess coculture, F. succinogenes was replaced by a population of R. albus in the substrate-limited coculture in both coinoculation and challenge experiments. R albus outcompeted F. succinogenes, apparently due to selection in the chemostat of a population of R. albus with a higher affinity for cellobiose. R. albus also outcompeted R. flavefaciens under substrate-limited conditions.

Re-investigation of glucose metabolism in Fibrobacter succinogenes, using NMR spectroscopy and enzymatic assays. : Evidence for pentose phosphates phosphoketolase and pyruvate formate lyase activities

The glucose metabolism of Fibrobacter succinogenes S85 was studied in detail; key intermediates and alternative pathways were evidenced by NMR and/or enzymatic assays. A high phosphoketolase activity was detected in four strains of Fibrobacter under strictly anaerobic conditions, with ribose-5-phosphate as substrate, no activity was evidenced with fructose-6-phosphate. This is the first report of a pentose phosphates phosphoketolase in bacteria unable to use pentoses. In contrast, the Entner-Doudoroff pathway and the oxidative branch of the pentose phosphate pathway could not be evidenced. Incubation of living cells of F. succinogenes with Na 2 13CO 3 confirmed the incorporation of 13CO 2 in the carboxylic group of succinate. The presence of fumarase was evidenced by in vivo 13C-NMR using 2-heptyl-4-hydroxyquinoline-N-oxide (HQNO); the enzyme showed a high reversibility under physiological conditions. The production of formate from glucose catabolism was evidenced by enzymatic assay and by NMR and a pyruvate formate lyase activity was detected using strictly anaerobic conditions.

Simultaneous metabolism of glucose and cellobiose in F. succinogenes S85 studied by in vivo 13C-NMR. Evidence of glucose 6-phosphate accumulation

Simultaneous metabolism of glucose and cellobiose in F. succinogenes S85 studied by in vivo ¹³C-NMR. Evidence of glucose 6-phosphate accumulation

Endoglucanase G from Fibrobacter succinogenes S85 belongs to a class of enzymes characterized by a basic C-terminal domain.

A 3.6-kb fragment of the Fibrobacter succinogenes S85 DNA was sequenced and found to contain two open reading frames (ORFs) on the same strand separated by 242 nucleotide bases. The translated protein from ORF1 had a predicted mass of 52.3 kDa. In a region of 320 amino acid overlap, it shares a 35% identity with the b-chain of the glutamate synthase of Escherichia coli. The ORF2 protein encodes a 519 residue protein designated CelG. It consists of an ORF of 1557 bp, encoding a polypeptide of 54.5 kDa. The N-terminal region, which contains the catalytic domain, is linked to a C-terminal basic domain, which has a predicted isoelectric point of 10.8. The catalytic domain in endoglucanase G (CelG) is homologous to the family 5 (A) cellulases. The enzyme has an apparent mass of 55 kDa, a pH optimum of 5.5, and temperature optimum of 25 degrees C. It had a specific activity of 16.5 mumols x min(-1) x mg-1 on barley b-glucan and produced a mixture of cellooligosaccharides from the hydrolysis of acid swollen cellulose and cellooligosaccharides. Antiserum raised against the purified form of CelG in E. coli failed to react with proteins from the native organism when grown on either glucose or crystalline cellulose, but reverse transcription and polymerase chain reaction techniques using RNA from the native organism demonstrated that the celG gene was expressed constitutively. Its distribution amongst subspecies of Fibrobacter was restricted to F. succinogenes S85.

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

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.