Bacteroides Amylophilus Research Articles

How Many Ruminal Bacteria Are There?

With the development of strictly anaerobic techniques and habitat-simulating media, a variety of bacteria were isolated from the rumen in the 1940s and 1950s. Based on standard morphological and physiological characteristics, the microbial ecosystem of the rumen contains a very complex population of bacteria. In recent years, ruminal bacteria have been re-evaluated with newer, more objective, and genetically valid methods of classification. Ribosomes are complicated structures, and their DNA-encoding sequences are relatively free from selective pressure. Because ribosomes have evolved slowly, they provide a long-term natural history of evolution. The invariable and hypervariable regions of rRNA genes can be used to group bacteria into kingdoms, genera, and species. The 16s rRNA sequences have provided a basis for renaming some ruminal species (Bacteroides amylophilus is now Ruminobacter amylophilus and Bacteroides succinogenes is now Fibrobacter succinogenes) and for classifying at least one recently isolated ruminal bacterium (e.g., Clostridium aminophilum). The DNA:DNA hybridization is a more sensitive method of assessing bacterial relatedness than is 16S rRNA. Bacterial strains within a species should have a high degree of DNA:DNA homology, but some species of ruminal bacteria (e.g., Prevotella ruminicola and Butyrivibrio fibrisolvens) had highly unrelated strains. Studies of 16S rRNA and DNA:DNA hybridization indicate that the diversity of ruminal bacteria has been greatly underestimated. Traditional studies of phylogeny of ruminal bacteria were stymied by the fastidious growth requirements of many ruminal bacteria, and enumeration was tedious and inaccurate. Modern methods of bacterial classification do not require in vitro culture and have the potential of detecting even a single cell.

Identification of slowly metabolized sugars and sugar derivatives that could be used to establish new or modified microbial species in the rumen

For identification of compounds that could potentially be used to sustain a population of new or genetically modified organisms in the rumen, the rates of metabolism of several sugars and sugar derivatives were measured in ovine rumen fluid in vitro. Several sugars and sugar alcohols, including sorbitol, xylitol, dulcitol, arabinose, ribose, and maltitol, were degraded slowly and were therefore identified as candidates for use in this new manipulation strategy. None of the rumen bacteria,Bacteroides amylophilus WP91,Bacteroides ruminicola M384,Butyrivibrio fibrisolvens JW11,Lactobacillus casei LB17,Megasphaera elsdenii J1,Selenomonas ruminantium Z108, orStreptococcus bovis C277, grew on the above sugar alcohols, and onlyB. ruminicola M384 andL. casei LB17 grew significantly on the pentoses. The non-rumen strain,Escherichia coli ML308, grew rapidly on dulcitol and sorbitol; this suggests a possible role forE. coli in manipulation of rumen fermentation.

Proteolysis of Alcohol-Treated Soybean Meal Proteins by Bacteroides ruminicola, Bacteroides amylophilus, Pepsin, Trypsin, and in the Rumen of Steers

Sodium dodecyl sulfate-gel electrophoresis and cation exchange chromatography were used to examine degradation of treated and untreated soybean meal protein fractions by Bacteroides amylophilus H181, Bacteroides ruminicola B14, pepsin, trypsin, and intraruminally. Soybean meal treatments consisted of 30% vol/vol isopropanol, 40% propanol, or 50% ethanol at 22°C or 70% ethanol at 80°C. Water-soluble protein fractions were applied to a hydroxylapatite column and eluted with a discontinuous phosphate gradient of .03 to .27 and then .27 to 1.0 M. The four protein fractions with the highest absorbance at 276nm were dialyzed against distilled water prior to being subjected to enzymatic hydrolysis. Soybean meal treated with 40% propanol had the greatest reduction in absorbance of all effluents at 275nm, followed by soybean meal treated with 50% ethanol or 30% isopropanol. Comparison of electrophoretic patterns over time showed that B. amylophilus H181, degraded protein subunits more rapidly than B. ruminicola B14 Protein subunits with the highest molecular weights were the most rapidly degraded by B. amylophilus H181, B. ruminicola B14, pepsin, and trypsin. Hydroxylapatite chromatography of omasal fluid from steers supplemented with untreated soybean meal or soybean meal treated with 70% ethanol at 80°C indicated that no detectable soluble glycinin or conglycinin escaped ruminal degradation.

Sequence of a 16S Ribosomal RNA Gene of Ruminobacter amylophilus: The Relation Between Homology Values and Similarity Coefficients

A 16S ribosomal RNA gene from Ruminobacter amylophilus (the former Bacteroides amylophilus ) was cloned, sequenced and the resulting primary structure compared to a variety of published homologous sequences. R. amylophilus shows the highest homology (87.6%) to Escherichia coli which is consistent with its current taxonomic position as a member of the gamma subdivision of purple phototrophic bacteria and their non-phototrophic relatives. Homology values obtained for R. amylophilus and representatives of the alpha, beta and delta subdivision range between 79.8 and 80.5%. Analyses of the 16S rDNA flanking regions comprises putative functional regions of a tandem promoter, and a RNase III recognition site. A single tRNA (gln) gene is located in the 16S 23S rDNA spacer. The relation between homology values and similarity coefficients (S AB -values) from total 16S rRNA sequences and 16S rRNA oligonucleotide cataloguing, respectively, is discussed.

The Membrane Potential in a Cytochrome-deficient Species of Bacteroides: Its Magnitude and Mode of Generation

The lipophilic cations butyltriphenylphosphonium (BTPP+), tetraphenylphosphonium (TPP+) and triphenylmethylphosphonium (TPMP+) were taken up into cells of Bacteroides amylophilus H18 under anaerobic conditions. Uptake was dependent on the presence of maltose together with both HCO− 3 and Na+; it was at a maximum at concentrations of ≥ 20 mm-HCO− 3 and ≥2 mm-Na+. The addition of 2-(n-heptyl)-hydroxyquinoline-N-oxide (HpHOQnO) or of an uncoupler of oxidative phosphorylation, or air, resulted in efflux of the lipophilic cations. From the binding behaviour and the physiological effects of the lipophilic cations, a membrane potential (Δψ) of 140 mV was estimated. There was no detectable ΔpH at an external pH of 6·7. The cytoplasmic Na+concentration was estimated to be 0·2 mm, indicating that B. amylophilus can maintain a Na+concentration gradient equivalent to at least 150 mV. Variation in the external Na+concentration (2 to 180 mm) had little influence on Δψ. High external concentrations of the fermentation products acetate, formate and succinate had little effect on the growth rate and the Δψ. The cytoplasmic ATP concentration decreased rapidly on addition of HpHOQnO oxide or of an uncoupler. The maximum internal ATP concentration was only maintained at an external concentration≥2mm-Na+. The NADH: fumarate reductase activity of vesicles of B. amylophilus was associated with alkalization of the suspension medium. The amount of H+taken up was in excess of the expected amount of scalar H+and was partially sensitive to uncoupler. It is concluded that the Δψ was generated via H+translocation driven primarily by the cytochrome-deficient NADH: fumarate reductase system. The transmembrane Na+gradient could be supported via the action of a Na+/2 H+antiporter.

Transfer of Bacteroides amylophilus to a new genus Ruminobacter gen. nov., nom. rev. as Ruminobacter amylophilus comb. nov.

Results of 16S ribosomal RNA cataloguing demonstrate the lack of close relationship between Bacteroides amylophilus ATCC 29744 and other members of the genus Bacteroides. B. amylophilus shows a remote relationship to purple sulfur bacteria and their non-phototrophic relatives (gamma subdivision, as defined by Woese et al., 1984b), while genuine members of Bacteroides cluster separately, forming one of the phylogenetically ancient groups of eubacteria. This finding is in accord with differences found between the chemotaxonomic properties of B. amylophilus and other Bacteroides species tested. On the basis of these results we propose that B. amylophilus be transferred to a new genus, Ruminobacter, as Ruminobacter amylophilus (Hamlin and Hungate 1956) comb. nov.

Proteolytic activity of the ruminal bacterium Butyrivibrio fibrisolvens.

The proteolytic activity of Butyrivibrio fibrisolvens, a ubiquitously distributed bacterial species in the gastrointestinal tracts of ruminants and other mammals, was characterized. The relative proteolytic activity (micrograms of azocasein degraded per hour per milligram of protein) varied greatly with the strain: 0 to 1 for strains D1, D16f, E21C, and X6C61; 7 to 15 for strains IL631, NOR37, S2, LM8/1B, and X10C34; and 90 to 590 for strains 12, 49 H17C, CF4c, CF3, CF1B, and R28. The activity levels of the last group of strains were equal to or greater than those found with Bacteroides amylophilus or Bacteroides ruminicola. With the exception of strain R28 activity, 90% or more of the proteolytic activity was associated with the culture fluid and not the cells. Strain 49 produced proteolytic activity constitutively, but the level of activity (units per milligram of protein) was modulated by growth parameters. With various carbohydrates added to the growth medium, the proteolytic activities of strain 49 were positively correlated with the growth rate. However, when the growth rate varied with the use of different nitrogen sources, a similar correlation was not found. The highest activity level was observed with Casamino Acids (1 g/liter), but this level was reduced by ca. 70% with Trypticase (BBL Microbiology Systems, Cockeysville, Md.) or casein (1 g/liter) and by 85% with ammonium chloride (10 mM) as the sole nitrogen source. The addition of ammonium chloride (1 to 10 mM) to media with low levels of Casamino Acids or Trypticase resulted in lower proteolytic activities but not as low as seen when the complex nitrogen sources were increased to high levels (20 g/liter).(ABSTRACT TRUNCATED AT 250 WORDS)

A sodium-stimulated membrane-bound fumarate reductase system in Bacteroides amylophilus.

Membrane vesicles derived from whole cells of the strictly anaerobic rumen bacterium Bacteroides amylophilus exhibited fumarate reductase activity with NADH, FADH2, FMNH2, or reduced viologens as electron donors. The fumarate reductase system is most likely localized on the cytoplasmic side of the plasma membrane. Cytochromes and menaquinone were not detectable. The NADH-dependent activity was inactivated by oxygen, an endogenous protease, and by irradiation at 254 nm. The electron transport inhibitor HpHOQnO and Zn2+ were identified as strong inhibitors of the fumarate reductase reaction. Two types of functional SH-groups might be operative in this system as probed by ClHgSO3H. The oxidation of NADH by fumarate was stimulated by low concentrations of Na+. Concentrations of Na+ in the range of 4 to 30 mM had a pronounced influence on growth rate and cell yield of B. amylophilus. In the presence of 1 mM NaCl growth was observed only after a lag-period of 15 h.

The Role of Different Species of Bacteria in the Hydrolysis of Protein in the Rumen

Proteolytic strains of Bacteroides amylophilus, Bacteroides ruminicola, Butyrivibrio fibrisolvens, Butyriribrio alactacidigens, Selenomonas ruminantium var. Ruminantium, Se. ruminantium var. lactilytica and of the genera Eubacterium, Fusobacterium and Clostridium were isolated from the rumen of sheep. The location of the proteolytic enzymens, their activity against various substrates and their sensitivity to inhibitors were compared with the same properties of mixed bacteria prepared from strained rumen fluid, in order to assess the relative importance of the different species in the hydrolysis of protein in the rumen. Bacteroides ruminicola was judged by these criteria to be the most important of these proteolytic isolates in vivo. Other isolates of higher specific activity had enzymes with properties different from rumen fluid. Species of Butyrivibrio, Selenomonas and Clostridium of lower specific activity may also be important if present in sufficiently large numbers, as their activity was of the same type as the mixed population. Streptococcus bovis had a low proteolytic activity as determined by the conversion of 14C-labelled casein to TCA-soluble products, but was able to grow on casein, probably by virtue of its exceptionally high leucine aminopeptidase activity.

Influence of Culture Eh on the Growth and Metabolism of the Rumen Bacteria Selenomonas ruminantium, Bacteroides amylophilus, Bacteroides succinogenes and Streptococcus bovis in Batch Culture

One facultatively and three strictly anaerobic rumen bacteria were grown in pH-controlled anaerobic batch cultures in which the E h of the medium was regulated by the addition of titanium (III) citrate solution at values below −50 mV and potassium ferricyanide above −50 mV. Growth occurred over a wide range of E h, with the maximum limit being +360, + 250, +175 and +414 mV for Selenomonas ruminantium, Bacteroides amylophilus, Bacteroides succinogenes and the aerotolerant Streptococcus bovis, respectively. Changes in E h had little influence on the growth yield or ratios of fermentation end-products in these bacteria over a fairly wide range, although the specific growth rate of all species tended to decline at E h values above 0 mV. Abnormal, elongated forms of Sel. ruminantium and B. succinogenes predominated at high E h. It was concluded that O2, and not a high E h, is the toxic factor in ‘oxidized’ anaerobic growth medium, and that it would not normally be necessary to regulate E h closely in experiments where the growth and metabolism of these bacteria is under study, provided that O2-free conditions were maintained.

Nutritional Interdependence Among Rumen Bacteria, Bacteroides amylophilus, Megasphaera elsdenii , and Ruminococcus albus

Nutritional interdependence among three representatives of rumen bacteria, Bacteroides amylophilus, Megasphaera elsdenii, and Ruminococcus albus, was studied with a basal medium consisting of minerals, vitamins, cysteine hydrochloride, and NH(4). B. amylophilus grew well in the basal medium supplemented with starch and produced branched-chain amino acids after growth ceased. When cocultured with B. amylophilus in the basal medium supplemented with starch and glucose, amino acid-dependent M. elsdenii produced an appreciable amount of branched-chain fatty acids, which are essential growth factors for cellulolytic R. albus. A small addition of starch (0.1 to 0.3%) to the basal medium containing glucose and cellobiose brought about successive growth of the three species in the order of B. amylophilus, M. elsdenii, and R. albus, and successive growth was substantiated by the formation of branched-chain amino acids and fatty acids in the culture. Supplementation with 0.5% starch, however, failed to support the growth of R. albus. On the basis of these results, the effects of supplementary starch or branched-chain fatty acids on cellulose digestion in the rumen was discussed.

Ammonia Saturation Constants for Predominant Species of Rumen Bacteria

Ammonia saturation constants were determined for representative pure cultures of predominant, anaerobic, fermentative rumen bacteria. Based on growth experiments with ammonia limited continuous cultures, average estimates for ammonia saturation constants of Bacteroides amylophilus and Bacteroides ruminicola were 10.5 and 23.5μM ammonia-nitrogen, respectively. With ammonia-limited linear-growth cultures, the estimates for the ammonia saturation constants of B. amylophilus, B. ruminicola, and Selenomonas ruminantium were, respectively, 6, 33.5, and 18μM. ammonia nitrogen. By a third method, which involved estimation of ammonia concentration in the medium when the growth rate of ammonia-limited batch cultures reached half maximal, the ammonia saturation constant was determined for the species mentioned as well as Megaspaera elsdenii and Ruminococcus flavefaciens. Except for M. elsdenii, saturation constants of the other bacteria were less than 50μM ammonia nitrogen. An organism with a saturation constant for ammonia of 50μM growing in a medium containing 1mM ammonia should achieve 95% of its maximum specific growth rate. Many of the predominant species of rumen bacteria are efficient scavengers of ammonia.

Degradation of soluble and insoluble proteins by Bacteroides amylophilus protease and by rumen microorganisms.

Various soluble and insoluble proteins (6.25 mg) were incubated at 37 C with partially purified protease from Bacteroides amylophilus (156 micrograms) in 2.0 ml of .1 M potassium phosphate buffer, pH 7.6, for 2, 4, 6 and 18 hr, and the liberated amino acids were determined by the ninhydrin method. Results showed that (1) although soluble, serum albumin and ribonuclease A were resistant to hydrolysis; (2) soluble and insoluble proteins of soybean meal were hydrolyzed at almost identical rates; (3) soluble proteins from soybean meal, rapeseed meal and casein were hydrolyzed at different rates, and (4) treatment of resistant proteins (serum albumin, ribonuclease A and insoluble fish meal and rapeseed meal proteins) with mercaptoethanol in 8 M urea or oxidation with performic acid rendered these proteins susceptible to hydrolysis. It is concluded that (1) solubility or insolubility of a protein is not by itself an indication of the protein's resistance or susceptibility to hydrolysis by rumen bacterial protease; (2) structural characteristics of the properties which renders feed protein resistant to degradation is the presence of crosslinking disulfide bonds.

Assimilation of Ammonia by Bacteroides amylophilus in Chemostat Cultures

Summary: The size of the amino acid pool in Bacteroides amylophilus, a rumen bacterium, was limited by the availability of ammonia in the growth medium. Alanine was the major pool constituent irrespective of the growth-limiting nutrient. In steady-state ammonia-limited chemostat cultures, glutamine synthetase (GS) activity was tenfold higher and glutamate dehydrogenase (GDH) activity up to fivefold lower than in maltose-limited cultures. No glutamate synthase (GOGAT) activity was demonstrated. When excess ammonia was pulsed into an ammonia-limited chemostat culture, the glutamine pool expanded rapidly and GS was inactivated. This was consistent with the observation that, in a number of bacteria, GS functions to assimilate ammonia when the prevailing concentration is low. However, GDH was always very active in extracts of B. amylophilus and its Michaelis constant for ammonia was relatively low (1 to 2 mm). This suggested that GDH could continue to function bio-synthetically when GS was derepressed.

Growth and energy production in Bacteroides amylophilus

The molar growth yield (Ym) of Bacteroides amylophilus strain WP91 on maltose was 68±2 g/mol when determined from batch cultures at the peaks of maximal growth. Continued incubation led to considerable cell lysis. When calculated from batch cultures in exponential phase (specific growth rate, μ=0.57 h-1) Ym was 101 g/mol. The maximum value of Ym in maltose-limited chemostat cultures at the maximum dilution rate (D) attainable (D=μ=0.39 h-1) was about 79 g/mol. Ammonia-Fmited chemostat cultures metabolized maltose with a much reduced efficiency and this was associated with a difference in morphology and chemical composition of the cells. The theoretical maximum molar growth yields (Ymmax) were 55 and 114 g/mol for ammonia- and maltose-limited growth respectively. However, if account was taken of extracellular nitrogen-containing material in ammonia-limited cultures, Ymmaxbecame 60. The maintenance coefficient (ms), estimated from the lines relating the specific rate of maltose consumption (qm) and D (where ms=qm at D=0), was 7.4±0.6×10-4 mol maltose/g x h for both nutrient limitations. A difference in maintenance energy demand, independent of growth-rate, could not account, therefore, for the observed differences in Ym between ammonia- and maltose-limited growth.

TECHNIQUE FOR FRACTIONATION OF BACTERIA IN RUMEN MICROBIAL ECOSYSTEM. III. ATTACHMENT OF BACTERIA ISOLATED FROM BOVINE RUMEN TO STARCH GRANULES IN VITRO AND ELUTION OF BACTERIA ATTACHED THEREFROM

The attaching ability of a number of bacteria, isolated from bovine rumen and animal intestine, to starch granules was examined in vitro. Among bacteria examined in the present investigation, strains possessing the attaching ability to starch were those belonging to an untypable Bacteroides sp., Bacteroides amylophilus, Bacteroides oralis, Bacteroides ruminicola ss. ruminicola, an untypable Bifidobacterium sp., Bifidobacterium thermophilum, Bifidobacterium pseudolongum, and Bifidobacterium longum.The reaction temperature had a profound effect on the attachment of bacteria to starch. All of bacteria listed above attached well to starch at 38°. Strains of the untypable Bacteroides sp., B. oralis, B. thermophilum, B. pseudolongum, and B. longum attached to starch even at the reaction temperature of 4°. On the other hand, those of B. amylophilus did not attach to starch at 4°.The activity of several hydrolases of various kinds of bacteria was examined. The amylase activity of cells of bacteria capable of attaching to starch was remarkably high, compared with that of bacteria incapable of attaching. Bacteria such as B. fibrisolvens and Streptococcus bovis, which were incapable of attaching to starch but possessed intense amylolytic activity, secreted extracellularly large parts of amylase produced by them. Only Peptostreptococcus productus among examined bacteria exhibited remarkably intense urease activity. There was no significant difference in the specific activity of the remaining three hydrolases between bacteria capable and incapable of attaching to starch. The attachment of bacteria to starch was inhibited by various carbohydrates. Carbohydrates like dextrin, amylose, amylopectin, soluble starch, and derivatives of starch except carboxymethyl-starch inhibited the attachment of bacteria to starch. The degree of this inhibition by these carbohydrates depended on their concentrations. Other carbohydrates examined, including derivatives of cellulose and dextran, did not inhibit significantly the attachment of bacteria to starch.An attempt was made to detach or elute bacteria from starch by some means. Cells of B. amylophilus, once attached to starch at 38°, were detached by the cooling treatment at 4°. Although both cells of B. thermophilum and B. pseudolongum were not detached from starch by the cooling treatment at 4°, they were eluted from starch with a solution of dextrin or partially-hydrolysed hydroxypropyl-starch. Cells of Bacteroides sp. 7-4, once attached to starch, were not detached either by the cooling treatment at 4° or by the elution treatment with a solution of above two carbohydrates.

Designation of Neotype Strains for Bacteroides amylophilus Hamlin and Hungate 1956 and Bacteroides succinogenes Hungate 1950

Abstract Bacteroides amylophilus ATCC 29744 and B. succinogenes ATCC 19169 are described and here designated the neotype strains of these two species.

Production of branched-chain volatile fatty acids by certain anaerobic bacteria.

Net production of isobutyric acid, isovaleric acid, and 2-methylbutyric acid by cultures of Bacteroides ruminicola and Megasphaera elsdenii on media that contained Trypticase or casein hydrolysate continued (up to 5 days) after growth had ceased. Only trace quantities of these acids were produced in a medium that contained a mixture of amino acids that did not include the branched-chain amino acids. M. elsdenii produced increased quantities of the branched-chain fatty acids in a medium that contained Trypticase when glucose was reduced or eliminated from the culture medium. However, B. ruminicola produced increased quantities of branched-chain fatty acids and of phenylacetic acid from Trypticase when glucose was supplied at 3 mg/ml rather than at 1 mg/ml. Single strains of Streptococcus bovis, Selenomonas ruminantium, Bacteroides amylophilus, and Butyrivibrio fibrisolvens did not produce branched-chain fatty acids.

Purification and some properties of an extracellular alpha-amylase from Bacteroides amylophilus.

A medium was developed to obtain maximum yields of extracellular amylase from Bacteroides amylophilus 70. Crude enzyme preparation, obtained by ammonium sulfate precipitation of cell-free broth, contained six amylolytic isoenzymes that were detected by isoelectric focusing and polyacrylamide gel electrophoresis. One of these amylases was purified by diethylaminoethyl-Sephadex A-50 ion-exchange chromatography and Sephadex G-200 gel filtration techniques. Some properties of the purified extracellular alpha-amylase were: optimum pH, 6.3; optimum temperature, 43 degrees C: PH stability range, 5.8 to 7.5; isoelectric point, pH 4.6; molecular weight, 92,000 (by sodium dodecyl sulfatedisc gel electrophoresis); and sugars causing inhibition, cyclomaltoheptaose, cyclomaltohexaose, and alpha-d-phenylglucoside. In addition, Ca2+ and Co2+ were strong activators,and Hg2+ was a strong inhibitior; all other cations were slightly stimulatory. Dialysis against 0.01 M ethylenediaminetetraacetic acid caused a 58% loss of activity that was restored to 92% of the original by the addition of 0.04 M Ca2+. The enzyme affected a blue-value-reducing-value curve characteristic of alpha-type amylases. The relative rates of hydrolysis of amylose, soluble starch, amylopectin, and dextrin were 100, 97, 92, and 60%, respectively; Michaelis constants for these substrates were 18.2, 18.7, 18.2, and 16.7 mumol of d-glucosidic bond/liter, respectively. The enzyme degraded maize (corn) starch granules to some extent and had relatively little activity on potato starch granules.

The cell-bound protease of Bacteroides amylophilus H18

Most of the cell-bound protease activity of Bacteroides amylophilus was attached to lysozyme spheroplasts (79%) and to the ruptured spheroplast envelope (48%). Protease activity also remained bound to mechanically ruptured cell envelopes from which it could be liberated by n-butanol treatment.Butanol-liberated protease activity was purified 80-fold, contained 23% RNA, and was excluded from G-200 Sephadex. Small molecular weight protease activity could not be liberated from this complex by treatment with acetone, EDTA, or urea. RNA could not be separated from the protease activity by treatment with streptomycin sulfate, MgCl2, or sodium dodecyl sulfate but was separated from the protein by phenol extraction. Protamine sulfate precipitation gave variable results. It is concluded that the cell-bound protease remains firmly bound to cellular components from which it cannot be liberated easily.