Bacteroides Ruminicola Research Articles

The Role of Menaquinone and b-Type Cytochrome in Anaerobic Reduction of Fumarate by NADH in Membrane Preparations from Bacteroides ruminicola Strain B14

Summary The electron transfer system catalysing reduction of fumarate by NADH under anaerobic conditions in membrane preparations of Bacteroides ruminicola strain B 14 was inhibited by 2-n-heptyl-4-hydroxyquinoline N-oxide. Reduction of b-type cytochrome by NADH under anaerobic conditions was much slower than reduction of fumarate by NADH, and was more sensitive to 2-n-heptyl-4-hydroxyquinoline N-oxide inhibition. Thus b-type cytochrome is probably not an intermediate in the reduction of fumarate by NADH under the in vitro assay conditions used. Extraction of quinone from the membrane and subsequent reincorporation showed that menaquinone (vitamin K2) is an obligatory intermediate in the reduction of both fumarate and b-type cytochrome by NADH, which is consistent with the 2-n-heptyl-4-hydroxyquinoline N-oxide inhibition results. A tentative scheme for electron transport in B. ruminicola is presented.

Characterization of the predominant bacteria occurring in the rumen of goats (Capra hircus).

A total of 44 strains of bacteria were isolated from rumen contents of the goat. Based on morphology, Gram stain, anaerobiosis, motility, and fermentation end products, they were grouped into 11 different types. For each type, all or representative strains were characterized in detail. The type, number of strains characterized over total number of strains, and identification were as follows: type 1, 6/21, atypical Butyrivibrio fibriosolvens; type 2, 6/9, atypical Butyrivibrio fibrisolvens; type 3, 3/3, genus uncertain; type 4, 2/2, genus uncertain; type 5, 3/3, Streptoccous bovis; type 6, 1/1, Butyrivibrio fibrisolvens; type 7, 1/1, Bacteroides ruminicola subsp. ruminicola; type 8, 1/1, Bacteroides ruminicola subsp. brevis; type 9, 1/1, family Peptococcaceae, genus uncertain; type 10, 1/1, genus Bacteroides; type 11, 1/1, genus Bacteroides. About 70% of the isolated strains were classified as Butyrivibrio, which is quite high compared with previous studies in cattle on similar rations. Of the 30 strains listed as type 1 and 2, the 12 studied further were characterized as atypical Butyrivibrio fibrisolvens, which differed from the species description primarily by their inability to hydrolyze starch and lack of gas production.

Urease assay and urease-producing species of anaerobes in the bovine rumen and human feces.

A growth medium and test were developed for rapid detection of urease in fermentative anaerobic bacteria. Using nonselective rumen fluid roll-tube agar medium and the new test, it was confirmed that Peptostreptococcus productus is often the most numerous urease-forming species in human feces. Also, some fecal strains of Ruminococcus albus, Clostridium innocuum, and Clostridium beijerinckii produced urease. Single strains of Fusobacterium prausnitzii, Coprococcus catus, and Streptococcus mitis that were strongly ureolytic on isolation later lost this ability. Urease activity was also detected in many strains of nonselectively isolated rumen species. They include Succinivibrio dextrinosolvens, Treponema sp., Ruminococcus bromii (not previously known to be present in the rumen), Butyrivibrio sp., Bifidobacterium sp., Bacteroides ruminicola, and P. productus. Most P. productus strains contain urease; however, the uniformity of this feature in the other species noted above is not known. The urease in many of these species was not detected if the growth medium contained 0.2% or more (each) yeast extract and Trypticase.

Metabolism and growth yields in Bacteroides ruminicola strain b14

Metabolism of D-glucose by Bacteroides ruminicola subsp. brevis, strain B14, has been examined. Growth yield studies gave molar growth yields, corrected for storage polysaccharide, of approximately 66 g (dry weight)/mol of glucose fermented. The storage polysaccharide amounted to about 14% of the total dry weight, or 55% of the total cellular carbohydrate, at full growth. After correcting glucose utilization for incorporation into cellular carbohydrate, measurement of product formation showed that 1.1 succinate, 0.8 acetate, and 0.35 formate are produced and 0.5 CO2 net is taken up during the fermentation of 1 glucose under the conditions used. The implication of these results with respect to adenosine 5'-triphosphate (ATP) molar growth yield calculations is discussed. If substrate-level phosphorylation reactions alone are responsible for ATP generation, then the ATP molar growth yield must be about 23 g (dry weight)/mol of ATP. Alternatively, if anaerobic electron transfer-linked phosphorylation also occurs, the ATP molar growth yield will be lower.

Some Effects of Phosphate and Volatile Fatty Acid Salts on the Growth of Rumen Bacteria

SUMMARY: Bacterial cultures in media with a low (0·38%, w/v) initial glucose concentration showed much smaller pH changes during growth than cultures grown in media with excess (1·25%, w/v) glucose. Increasing the concentration of the phosphate or volatile fatty acid salt (VFA salt, i.e. acetate, propionate or butyrate) in the media had no beneficial effect on cultures of Streptococcus bovis or Lactobacillus plantarum which had only minor falls in pH during growth (final pH ≥ 6·0), but increased the dry weight yield (but not the molar growth yield) of cultures which had major pH falls (final pH ≤ 4·6) during growth. The improvements in dry weight yield could be correlated with increases in the pH buffering capacity of media caused by the increased phosphate or VFA salt concentration. The response of Bacteroides ruminicola to increases in the phosphate concentration of media was qualitatively similar to that of S. bovis. However, VFA salts (particularly acetate) always decreased both dry weight and molar growth yield. The effects of VFA salts on three other rumen bacteria, Butyrivibrio fibrisol-vens, Megasphaera elsdenii and Veillonella alcalescens, were varied. The possible mechanisms and ecological implications of the effects of these compounds are discussed.

Effect of actinomycin D and oxygen on the ribonucleic acid synthesis of an anaerobic gram-negative bacterium.

The synthesis of ribonucleic acid by whole cells of Bacteroides ruminicola is not sensitive to actinomycin D, but it is sensitive to actinomycin D in the presence of ethylenediaminetetraacetate. Ribonucleic acid synthesis by whole cells of this gram-negative anaerobic bacterium is also totally inhibited by oxygen.

Accumulation of phosphatidylserine in strictly anaerobic lactate fermenting bacteria

Phosphatidylserine has been found as a minor phospholipid constituent in most bacteria investigated so far [l-4] . Recently we demonstrated [5] that this phospholipid is a major membrane component of Megasphaera elsdenii, a strictly anaerobic rumen bacterium. Both phosphatidylserine and phosphatidylethanolamine, the only other major phospholipid present in this organism, were found to comprise high levels of plasmalogen analogues. A limited search among other predominant rumen bacteria, such as Ruminccoccus albus, R. falvefaciens, Bacteroides succinogenes, B. amylophilus, Eubacterium cellulosolvens, E. ruminantium and Methanobacterium ruminantium showed that these organisms contain only trace amounts (less than 1%) of phosphatidylserine (unpublished results). Also from the extensive studies of Rizza et al. [6] and Lev and Milford [7] on the lipids of Bacteroides melaninogenicus, of Kunsman [S] on Bacteroides ruminicola and of Makula and Finnerty [9] on Desulphovibrio species, it is clear that the unique simple polar lipid composition of M. elsdenii is not a general characteristic of anaerobic rumen or intestinal bacteria. One of the major functions of M. elsdenii is the fermentation of lactate produced by other microorganisms in the rumen [lo] . It was thought of interest to investigate whether the uncommon accumulation of phosphatidylserine in M. elsdenii may be related to the capability of this organism to ferment lactate. In this paper we report that three other strictly anaerobic bacteria which are known

The Establishment of Bacteroides ruminicola in a Gnotobiotic Lamb

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Cell envelope morphology of rumen bacteria.

The cell walls of three species of rumen bacteria (Bacteroides ruminicola, Bacteroides succinogenes, and Megasphaera elsdenii) were studied by a variety of morphological methods. Although all the cells studied were gram-negative and had typical cytoplasmic membranes and outer membranes, great variation was observed in the thickness of their peptidoglycan layers. Megasphaera elsdenii evidenced a phenomenally thick peptidoglycan layer whose participation in septum formation was very clearly seen. All species studied have cell wall "coats" external to the outer membrane. The coat of Bacteroides ruminicola is composed of large (approximately 20 nm) globules that resemble the protein coats of other organisms, whereas the coat of Bacteroides succinogenes is a thin and irregular carbohydrate coat structure. Megasphaera elsdenii displays a very thick fibrillar carbohydrate coat that varies in thickness with the age of the cells. Because of the universality of extracellular coats among rumen bacteria we conclude that the production of these structures is a protective adaptation to life in this particular, highly competitive, environment.

Comparison of the Sphingolipid Content of Rumen Bacteroides Species

Ten strains of Bacteroides ruminicola were found to contain phosphosphingolipids. Four strains of Bacteroides amylophilus and one strain each of Bacteroides succinogenes and Bacteroides sp. were devoid of phosphosphingolipids.

Localization of alkaline phosphatase in three gram-negative rumen bacteria.

Of the three species (Bacteroides ruminicola, B. succinogenes, and Megasphaera elsdenii) of anaerobic gram-negative rumen bacteria studied, only B. ruminicola produced significant amounts of alkaline phosphatase. This enzyme, which is constitutive, showed a greater affinity for p-nitrophenylphosphate than for sodium-beta-glycerophosphate and was shown to be located exclusively in the periplasmic space of log-phase cells. Small amounts of this enzyme were released from these cells in stationary-phase cultures, but washing in 0.01 M MgCl(2) and the production of spheroplasts by using lysozyme in 0.01 M MgCl(2) did not release significant amounts of the enzyme. Exposure to 0.2 M MgCl(2) did not release significant amounts of the periplasmic alkaline phosphatase of the cell, and when these cells were spheroplasted with lysozyme in 0.2 M MgCl(2) only 25% of the enzyme was released. Spheroplasts were formed spontaneously in aging cultures of B. ruminicola, but even these cells retained most of their periplasmic alkaline phosphatase. It was concluded that the alkaline phosphatase of B. ruminicola is firmly bound to a structural component within the periplasmic area of the cell wall and that the enzyme is released in large amounts only when the cells break down. The behavior of alkaline phosphatase in this bacterium contrasts with that of conventional periplasmic enzymes of aerobic bacteria, which are released upon conversion into spheroplasts by lysozyme and ethylenediaminetetraacetic acid and by other types of cell wall damage. All three species of bacteria studied here, as well as bacteria found in mixed populations in the rumen, have thick, complex layers external to the double-track layer of their cell walls. In addition, B. ruminicola produces a loose extracellular material.

Spheroplast formation by an anaerobic gram-negative bacterium Bacteroides ruminicola.

The spontaneous formation of spheroplasts was observed in stationary phase cultures and in aging cultures of Bacteroides ruminicola when grown in synthetic or a rumen fluid medium. Unlike most other gram-negative bacteria, spheroplasts of this organism also were formed when log-phase cells were treated with lysozyme in the presence of 0.01 M Mg2+ in 0.01 M Tris, pH 8.4.

Characterization of the Lipids of Six Strains of Bacteroides ruminicola

The lipids of six strains of Bacteroides ruminicola were characterized. The nonpolar lipid accounted for 6 to 24% of the total lipid and was composed of diglycerides, triglycerides, and free fatty acids. The phospholipid fraction contained phosphatidylethanolamine, phosphosphingolipids, and trace quantities of phosphatidic acid. In three strains the phosphosphingolipid fraction made up more than half of the total lipid. The fatty acids in the nonpolar, acyl- and phosphosphingolipid consisted of a homologous series of branched and normal chains from 12 to 19 carbons. The long-chain base isolated from the phosphosphingolipids consisted of a homologous series of branched and normal chains from 14 to 24 carbons.

Effect of acetohydroxamic acid on growth and volatile fatty acid production by rumen bacteria.

Six strains of anaerobic rumen bacteria were grown in a rumen fluid medium containing different concentrations of the urease inhibitor, acetohydroxamic acid. Bacteroides succinogenes S85 failed to grow in the presence of 1 × 10−2 M acetohydroxamic acid whereas growth by Selenomonas ruminantium GA192 was not affected when the initial concentration was 5 × 10−2 M. In cultures of the remaining strains, acetohydroxamic acid extended the lag phase without changing the growth rate in the log phase. The order of decreasing overall sensitivity to acetohydroxamic acid by the strains in terms of growth was Bacteroides succinogenes S85 [Formula: see text]Bacteroides ruminicola 23 > Butyrivibrio fibrisolvens D1 ≥ Butyrivibrio sp. C3 > Megasphaera (Peptostreptococcus) elsdenii B159 > Selenomonas ruminantium GA192. Washed cell suspensions of Butyrivibrio fibrisolvens D1, Megasphaera elsdenii B159, and Selenomonas ruminantium GA192, but of none of the other strains were capable of degrading acetohydroxamic acid. In the presence of 5 × 10−3 M acetohydroxamic acid, the extent of glucose, starch, and xylan fermentation by the strains was modified. The amounts and molar proportions of volatile fatty acids produced from these substrates were also changed. In Bacteroides ruminicola 23, the presence of acetohydroxamic acid resulted in an 89% reduction in glucose fermentation and a shift in volatile fatty acid production from propionate to acetate. Butyrivibrio fibrisolvens D1 and Butyrivibrio sp. C3 fermented more glucose, but less xylan, when acetohydroxamic acid was present; with glucose as substrate, volatile fatty acid production shifted markedly from n-butyrate to propionate. The results support the conclusion drawn from previous studies with the mixed rumen microbiota, that the potential effect of the compound upon rumen microbial activities in vivo is not limited to inhibition of urease activity.

Fermentation of isolated pectin and pectin from intact forages by pure cultures of rumen bacteria.

Studies on the rate and extent of galacturonic acid and isolated pectin digestion were carried out with nine strains of rumen bacteria (Butyrivibrio fibrisolvens H10b and D16f, Bacteroides ruminicola 23 and D31d, Lachnospira multiparus D15d, Peptostreptococcus sp. D43e, B. succinogenes A3c, Ruminococcus flavefaciens B34b, and R. albus 7). Only three strains, 23, D16f, and D31d, utilized galacturonic acid as a sole energy source, whereas all strains except A3c and H10b degraded (solubilized) and utilized purified pectin. Nutrient composition of the basal medium and separate sterilization of the substrate affected the rate and extent of fermentation for both substrates. Pectin degradation and utilization were measured with two maturity stages each of intact bromegrass and alfalfa. For bromegrass I, all strains tested (B34b, 23, D16f, D31d, D15d, and D43e) degraded a considerable amount of pectin and, with the exception of B34b, utilized most of what was degraded. Similar, but lower, results were obtained with bromegrass II, except for the two strains of B. ruminicola, 23 and D31d, which were unable to degrade and utilize pectin from this forage. All strains were able to degrade and utilize pectin from both maturity stages of alfalfa; however, values were considerably lower for strains 23 and D31d. Synergism studies, in which a limited utilizing strain, B34b, was combined with the limited degrading strain, D31d, resulted in a slight increase in degradation and a very marked increase in utilization of the pectin in all four forages. Similar results were obtained on both alfalfa substrates with a combination of strains B34b and D16f; however, no increases were observed with this combination on bromegrass.

Fermentation of Isolated Pectin and Pectin from Intact Forages by Pure Cultures of Rumen Bacteria1

Studies on the rate and extent of galacturonic acid and isolated pectin digestion were carried out with nine strains of rumen bacteria (Butyrivibrio fibrisolvens H10b and D16f, Bacteroides ruminicola 23 and D31d, Lachnospira multiparus D15d, Peptostreptococcus sp. D43e, B. succinogenes A3c, Ruminococcus flavefaciens B34b, and R. albus 7). Only three strains, 23, D16f, and D31d, utilized galacturonic acid as a sole energy source, whereas all strains except A3c and H10b degraded (solubilized) and utilized purified pectin. Nutrient composition of the basal medium and separate sterilization of the substrate affected the rate and extent of fermentation for both substrates. Pectin degradation and utilization were measured with two maturity stages each of intact bromegrass and alfalfa. For bromegrass I, all strains tested (B34b, 23, D16f, D31d, D15d, and D43e) degraded a considerable amount of pectin and, with the exception of B34b, utilized most of what was degraded. Similar, but lower, results were obtained with bromegrass II, except for the two strains of B. ruminicola, 23 and D31d, which were unable to degrade and utilize pectin from this forage. All strains were able to degrade and utilize pectin from both maturity stages of alfalfa; however, values were considerably lower for strains 23 and D31d. Synergism studies, in which a limited utilizing strain, B34b, was combined with the limited degrading strain, D31d, resulted in a slight increase in degradation and a very marked increase in utilization of the pectin in all four forages. Similar results were obtained on both alfalfa substrates with a combination of strains B34b and D16f; however, no increases were observed with this combination on bromegrass.

Pure and Mixed Continuous Culture of Two Rumen Anaerobes

This project was designed to determine if Bacteroides ruminicola, while growing in a continuous culture, would liberate sufficient amino acids to sustain the simultaneous growth of a rumen organism which required an amino acid.Initially pure cultures of Bacteroides ruminicola, grown on cellobiose-casein media, solubilized the casein in the media while utilizing peptides and possibly ammonia as nitrogen sources.Pure cultures of Ruminococcus flavefaciens would only grow on the same medium if methionine was added, indicating a requirement for this amino acid. Methionine was not required as the major nitrogen source for Ruminococcus flavefaciens since large amounts of ammonia disappeared from the media during growth.Both organisms were then added to the same culture vessel with a cellobiose-casein medium without free methionine. Bacteroides ruminicola, the faster growing organism, predominated throughout the culture period. After 96 hr, however, Ruminococcus flavefaciens accounted for 20 to 40% of the total bacterial count, indicating Bacteroides ruminicola liberated sufficient methionine to support the growth of Ruminococcus flavefaciens.

Serology of rumen bacteroides.

SUMMARY: The serological relationships of 26 strains of rumen Bacteroides species comprising Bacteroides ruminicola, B. amylophilus and B. succinogenes were examined using precipitation, agglutination and fluorescent antibody techniques. With precipitation techniques two types of acid-soluble thermostable antigens were detected, a strain-specific antigen and an antigen demonstrating wider serological relationships. With B. amylophilus and B. succinogenes this latter antigen was species-specific. With B. ruminicola, although eight out of 12 strains possessed the same species-specific antigen, there was evidence of two other serological groups within the species. Agglutination tests detected only the strain-specific antigens. Fluorescent antibody tests detected species-specific antigens of B. amylophilus and B. succinogenes but with B. ruminicola only the strain-specific antigens could be demonstrated. Preliminary work with B. ruminicola indicated that both kinds of antigen were polysaccharides. Eleven out of 20 freshly isolated strains of B. ruminicola reacted with the B. ruminicola antiserum. When 30 strains of eight other species of Bacteroides were tested against the species-specific antiserum of the different rumen species of Bacteroides, four strains of B. oralis and one strain of B. melanogenicus were found to have a common antigen with that of B. ruminicola, and another strain of B. oralis had a common antigen with another B. ruminicola strain. No other cross-reactions were observed.

The biosynthesis of valine from isobutyrate by peptostreptococcus elsdenii and Bacteroides ruminicola.

1. Growing cultures of Peptostreptococcus elsdenii and Bacteroides ruminicola incorporate (14)C from [1-(14)C]isobutyrate into the valine of cell protein. With P. elsdenii some of the (14)C is also incorporated into leucine. 2. Crude cell-free extracts of both organisms in the presence of glutamine, carbon dioxide and suitable sources of energy and electrons incorporate (14)C from [1-(14)C]isobutyrate into valine but not into leucine. 3. With extracts of P. elsdenii treated with DEAE-cellulose the reaction is dependent on ATP, CoA, thiamin pyrophosphate, molecular hydrogen and a low-potential electron carrier (ferredoxin, flavodoxin or benzyl viologen). 4. The same extracts incorporate (14)C from NaH(14)CO(3) into valine in the presence of isobutyrate plus ATP, CoA, glutamine and ferredoxin; isobutyryl-CoA or isobutyryl phosphate plus CoA will replace the isobutyrate plus CoA and ATP. With acetyl phosphate in place of isobutyryl phosphate, (14)C is incorporated into alanine. With isovalerate or 2-methylbutyrate in place of isobutyrate, (14)C is incorporated into leucine and isoleucine respectively. 5. When carrier 2-oxoisovalerate is added to the carboxylating system (14)C from [1-(14)C]isobutyrate passes into the oxo acid fraction. 6. It is concluded that these two organisms form valine from isobutyrate by the sequence isobutyrate-->isobutyryl-CoA-->2-oxoisovalerate-->valine and that the reductive carboxylation of isobutyrate is catalysed by a system similar to the pyruvate synthetase of clostridia and photosynthetic bacteria.

Biosynthesis of α-Ketoglutarate by the Reductive Carboxylation of Succinate in Bacteroides ruminicola

Biosynthesis of α-Ketoglutarate by the Reductive Carboxylation of Succinate in <i>Bacteroides ruminicola</i>