Utilization of nucleic acids by Selenomonas ruminantium and other ruminal bacteria.

Species of ruminal bacteria were screened for the ability to grow in media containing RNA or DNA as the energy source. Bacteroides ruminicola D31d and Selenomonas ruminantium HD4, GA192, and D effectively used RNA for growth, but not DNA. B. ruminicola D31d was able grow on nucleosides but not on bases or ribose. The S. ruminantium strains were able to grow when provided with either nucleosides or ribose but not bases. Strains of S. ruminantium, but not B. ruminicola D31d, were also able to use nucleosides as nitrogen sources. These data suggest that RNA fermentation may be a general characteristic of S. ruminantium.     

Utilization of nucleic acids by Selenomonas ruminantium and other ruminal bacteria.

Species of ruminal bacteria were screened for the ability to grow in media containing RNA or DNA as the energy source. Bacteroides ruminicola D31d and Selenomonas ruminantium HD4, GA192, and D effectively used RNA for growth, but not DNA. B. ruminicola D31d was able grow on nucleosides but not on bases or ribose. The S. ruminantium strains were able to grow when provided with either nucleosides or ribose but not bases. Strains of S. ruminantium, but not B. ruminicola D31d, were also able to use nucleosides as nitrogen sources. These data suggest that RNA fermentation may be a general characteristic of S. ruminantium.     

Evidence for natural transfer of a tetracycline resistance gene between bacteria from the human colon and bacteria from the bovine rumen.

Previously, we demonstrated conjugal transfer of a specially constructed shuttle vector, pRDB5, from the human colonic anaerobe Bacteroides uniformis to the ruminal anaerobe Prevotella (Bacteroides) ruminicola B(1)4. We have now shown that naturally occurring gene transfer elements in Bacteroides species and Prevotella ruminicola can also be transferred between these two genera. A self-transmissible chromosomal element originally found in a clinical isolate of Bacteroides fragilis (Tcr Emr 12256) was transferred from B. uniformis 0061 to P. ruminicola B(1)4 and from P. ruminicola B(1)4 back to B. uniformis or to another human colonic species, Bacteroides thetaiotaomicron. Similarly, a conjugative plasmid (pRRI4) originally found in P. ruminicola 223 was transferred from P. ruminicola B(1)4 to B. uniformis or B. thetaiotaomicron. pRRI4 could be transferred from the colonic Bacteroides species only if the donor strain contained the Tcr Emr 12256 element in its chromosome. These results show that transfer of naturally occurring elements can be demonstrated under laboratory conditions. Evidence that such transfers may actually have occurred in nature came from our finding that the tetracycline resistance (Tcr) gene on the P. ruminicola plasmid pRRI4 hybridized on high-stringency Southern blots with the Tcr gene found on the Bacteroides Tcr elements. The presence of the same gene in such distantly related genera of bacteria is most likely to have occurred as a result of horizontal transfer.     

Evidence for natural transfer of a tetracycline resistance gene between bacteria from the human colon and bacteria from the bovine rumen.

Previously, we demonstrated conjugal transfer of a specially constructed shuttle vector, pRDB5, from the human colonic anaerobe Bacteroides uniformis to the ruminal anaerobe Prevotella (Bacteroides) ruminicola B(1)4. We have now shown that naturally occurring gene transfer elements in Bacteroides species and Prevotella ruminicola can also be transferred between these two genera. A self-transmissible chromosomal element originally found in a clinical isolate of Bacteroides fragilis (Tcr Emr 12256) was transferred from B. uniformis 0061 to P. ruminicola B(1)4 and from P. ruminicola B(1)4 back to B. uniformis or to another human colonic species, Bacteroides thetaiotaomicron. Similarly, a conjugative plasmid (pRRI4) originally found in P. ruminicola 223 was transferred from P. ruminicola B(1)4 to B. uniformis or B. thetaiotaomicron. pRRI4 could be transferred from the colonic Bacteroides species only if the donor strain contained the Tcr Emr 12256 element in its chromosome. These results show that transfer of naturally occurring elements can be demonstrated under laboratory conditions. Evidence that such transfers may actually have occurred in nature came from our finding that the tetracycline resistance (Tcr) gene on the P. ruminicola plasmid pRRI4 hybridized on high-stringency Southern blots with the Tcr gene found on the Bacteroides Tcr elements. The presence of the same gene in such distantly related genera of bacteria is most likely to have occurred as a result of horizontal transfer.     

Use of a modified Bacteroides-Prevotella shuttle vector to transfer a reconstructed beta-1,4-D-endoglucanase gene into Bacteroides uniformis and Prevotella ruminicola B(1)4.

A carboxymethyl cellulase (CMCase) gene from Prevotella ruminicola B(1)4 was reconstructed by adding a cellulose binding domain from a Thermomonospora fusca cellulase and was conjugally transferred from Escherichia coli to Bacteroides uniformis 0061 by using a chloramphenicol and tetracycline resistance shuttle vector (pTC-COW). pTC-COW was specifically constructed to facilitate conjugal transfer of vectors from B. uniformis donors to P. ruminicola recipients. B. uniformis transconjugants containing CMCase constructs cloned into pTC-COW expressed Cmr, but they did not produce the reconstructed CMCase until a xylanase promoter from P. ruminicola 23 was added upstream of the CMCase (pTC-XRCMC). The xylanase promoter allowed the B. uniformis transconjugants to produce large amounts of the reconstructed CMCase, which was present on the outside surface of the cells. Although the reconstructed CMCase alone did not allow B. uniformis to grow on acid-swollen cellulose, rapid growth was observed when two exocellulases were added to the culture supernatant. Under these conditions, the reconstructed CMCase permitted faster growth than the wild-type CMCase. The frequency of transfer of pTC-XRCMC from B. uniformis to P. ruminicola B(1)4 was increased 100-fold when strictly anaerobic conditions, nitrocelluose filters (cell immobilization), and more stringent selections were employed. Although the P. ruminicola B(1)4 (pTC-XRCMC) transconjugates expressed Tcr and had DNA that hybridized with a probe to the shuttle vector, these transconjugants did not produce detectable levels of the reconstructed CMCase even when xylan was the carbon source. On the basis of these results, it appears that not all of the promoters recognized by B. uniformis and P. ruminicola 23 are functional in P. ruminicola B(1)4. However, the results with B. uniformis suggest that the introduction of a P. ruminicola B(1)4 promoter should allow expression of the reconstructed CMCase in P. ruminicola B(1)4.     

Enzymes associated with metabolism of xylose and other pentoses by Prevotella (Bacteroides) ruminicola strains, Selenomonas ruminantium D, and Fibrobacter succinogenes S85.

Prevotella (Bacteroides) ruminicola strains B(1)4 and S23 and Selenomonas ruminantium strain D used xylose as the sole source of carbohydrate for growth, whereas Fibrobacter succinogenes was unable to metabolize xylose. Prevotella ruminicola strain B(1)4 exhibited transport activity for xylose. In contrast, F. succinogenes lacked typical xylose uptake activity but did exhibit low binding potential for the sugar. Prevotella ruminicola strains B(1)4 and S23 as well as S. ruminantium D showed low xylose isomerase activities but higher xylulokinase activities, using assays that gave high activities for these enzymes in Escherichia coli. Xylose isomerase appeared to be produced constitutively in these ruminal bacteria, but xylulokinase was induced to varying degrees with xylose as the source of carbohydrate. Fibrobacter succinogenes lacked xylose isomerase and xylulokinase. All three species of ruminal bacteria possessed transketolase, xylulose-5-phosphate epimerase, and ribose-5-phosphate isomerase activities. Neither P. ruminicola B(1)4 nor F. succinogenes S85 showed significant phosphoketolase activity. The data indicate that F. succinogenes is unable to either actively uptake or metabolize xylose as a result of the absence of functional xylose permease, xylose isomerase, and xylulokinase activities, although it and both P. ruminicola and S. ruminantium possess the essential enzymes of the nonoxidative branch of the pentose phosphate cycle.     

Interaction of ruminal bacteria in the production and utilization of maltooligosaccharides from starch.

The degradation and utilization of starch by three amylolytic and one nonamylolytic species of ruminal bacteria were studied. Pure cultures of Streptococcus bovis JB1, Butyrivibrio fibrisolvens 49, and Bacteroides ruminicola D31d rapidly hydrolyzed starch and maltooligosaccharides accumulated. The major starch hydrolytic products detected in S. bovis cultures were glucose, maltose, maltotriose, and maltotetraose. In addition to these oligosaccharides, B. fibrisolvens cultures produced maltopentaose. The products of starch hydrolysis by B. ruminicola were even more complex, yielding glucose through maltotetraose, maltohexaose, and maltoheptaose but little maltopentaose. Selenomonas ruminantium HD4 grew poorly on starch, digested only a small portion of the available substrate, and generated no detectable oligosaccharides as a result of cultivation in starch containing medium. S. ruminantium was able to grow on a mixture of maltooligosaccharides and utilize those of lower degree (less than 10) of polymerization. A coculture system containing S. ruminantium as a dextrin-utilizing species and each of the three amylolytic bacteria was developed to test whether the products of starch hydrolysis were available for crossfeeding to another ruminal bacterium. Cocultures of S. ruminantium and S. bovis contained large numbers of S. bovis but relatively few S. ruminantium and exhibited little change in the pattern of maltooligosaccharides observed for pure cultures of S. bovis. In contrast, S. ruminantium was able to compete with B. fibrisolvens and B. ruminicola for these growth substrates. When grown with B. fibrisolvens, S. ruminantium grew to high numbers and maltooligosaccharides accumulated to a much lesser degree than in cultures of B. fibrisolvens alone. S. ruminantium-B. ruminicola cultures contained large numbers of both species, and maltooligosaccharides never accumulated in these cocultures.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cloning and expression in Escherichia coli of a xylanase gene from Bacteroides ruminicola 23

A gene coding for xylanase activity in the ruminal bacterial strain 23, the type strain of Bacteroides ruminicola, was cloned into Escherichia coli JM83 by using plasmid pUC18. AB. ruminicola 23 genomic library was prepared in E. coli by using BamHI-digested DNA, and transformants were screened for xylanase activity on the basis of clearing areas around colonies grown on Remazol brilliant blue R-xylan plates. Six clones were identified as being xylanase positive, and all six contained the same 5.7-kilobase genomic insert. The gene was reduced to a 2.7-kilobase DNA fragment. Xylanase activity produced by the E. coli clone was found to be greater than that produced by the original B. ruminicola strain. Southern hybridization analysis of genomic DNA from the related B. ruminicola strains, D31d and H15a, by using the strain 23 xylanase gene demonstrated one hybridizing band in each DNA.     

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.     

Interaction of ruminal bacteria in the production and utilization of maltooligosaccharides from starch.

The degradation and utilization of starch by three amylolytic and one nonamylolytic species of ruminal bacteria were studied. Pure cultures of Streptococcus bovis JB1, Butyrivibrio fibrisolvens 49, and Bacteroides ruminicola D31d rapidly hydrolyzed starch and maltooligosaccharides accumulated. The major starch hydrolytic products detected in S. bovis cultures were glucose, maltose, maltotriose, and maltotetraose. In addition to these oligosaccharides, B. fibrisolvens cultures produced maltopentaose. The products of starch hydrolysis by B. ruminicola were even more complex, yielding glucose through maltotetraose, maltohexaose, and maltoheptaose but little maltopentaose. Selenomonas ruminantium HD4 grew poorly on starch, digested only a small portion of the available substrate, and generated no detectable oligosaccharides as a result of cultivation in starch containing medium. S. ruminantium was able to grow on a mixture of maltooligosaccharides and utilize those of lower degree (less than 10) of polymerization. A coculture system containing S. ruminantium as a dextrin-utilizing species and each of the three amylolytic bacteria was developed to test whether the products of starch hydrolysis were available for crossfeeding to another ruminal bacterium. Cocultures of S. ruminantium and S. bovis contained large numbers of S. bovis but relatively few S. ruminantium and exhibited little change in the pattern of maltooligosaccharides observed for pure cultures of S. bovis. In contrast, S. ruminantium was able to compete with B. fibrisolvens and B. ruminicola for these growth substrates. When grown with B. fibrisolvens, S. ruminantium grew to high numbers and maltooligosaccharides accumulated to a much lesser degree than in cultures of B. fibrisolvens alone. S. ruminantium-B. ruminicola cultures contained large numbers of both species, and maltooligosaccharides never accumulated in these cocultures.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cloning and expression in Escherichia coli of a xylanase gene from Bacteroides ruminicola 23.

A gene coding for xylanase activity in the ruminal bacterial strain 23, the type strain of Bacteroides ruminicola, was cloned into Escherichia coli JM83 by using plasmid pUC18. AB. ruminicola 23 genomic library was prepared in E. coli by using BamHI-digested DNA, and transformants were screened for xylanase activity on the basis of clearing areas around colonies grown on Remazol brilliant blue R-xylan plates. Six clones were identified as being xylanase positive, and all six contained the same 5.7-kilobase genomic insert. The gene was reduced to a 2.7-kilobase DNA fragment. Xylanase activity produced by the E. coli clone was found to be greater than that produced by the original B. ruminicola strain. Southern hybridization analysis of genomic DNA from the related B. ruminicola strains, D31d and H15a, by using the strain 23 xylanase gene demonstrated one hybridizing band in each DNA.     

Effect of sodium monensin and cinnamaldehyde on the growth and phenotypic characteristics of Prevotella bryantii and Prevotella ruminicola

Two representative strains of Gram-negative rumen bacteria from the genus Prevotella were used as model organisms in order to evaluate the effect of cinnamaldehyde (the secondary metabolite found in extracts of the Cinnamomum family) vs. sodium monensin on growth, cell size and cell protein production. Prevotella bryantii B(1)4 was found to be remarkably more resistant to the action of both compounds than Prevotella ruminicola 23. The approximate IC(50) concentrations of sodium monensin influenced the increase in cell size of both strains during growth, which was much more pronounced in the case of the B(1)4 strain. A similar effect was observed in strain B(1)4 when 1.438 mmol/L cinnamaldehyde was added to the growth medium, indicating a possible interference with cell division. The action of cinnamaldehyde on P. bryantii B(1)4 was concentration-dependent, in contrast to the effect observed on P. ruminicola 23.     

Effects of potassium ion concentrations on the antimicrobial activities of ionophores against ruminal anaerobes

The antimicrobial activities of monensin and lasalocid against representative strains of ruminal bacteria were evaluated in medium containing three different concentrations of potassium (1.3, 7.9, or 23.3 mM). The growth of Eubacterium ruminantium was inhibited by low concentrations of ionophores (less than or equal to 0.16 mg/liter), while the strain of Streptococcus bovis tested was resistant to high concentrations of ionophores (40 mg/liter) at all potassium concentrations tested. The MICs of the ionophores for strains of Bacteroides succinogenes, Butyrivibrio fibrisolvens, Ruminococcus albus, and Ruminococcus flavefaciens and for one strain of Bacteroides ruminicola increased with increasing potassium concentrations in the medium. High concentrations of ionophores (40 mg/liter) decreased the maximum cell yields or increased the lag times or both in cultures of one strain of Bacteroides ruminicola and two strains of Selenomonas ruminantium but did not completely inhibit the growth of these organisms. Increased potassium concentrations in the medium (from 7.9 to 23.3 mM) decreased the lag times or increased the cell yields or both when these three strains were grown in ionophore-containing medium, while the activities of lasalocid and monensin against these organisms were enhanced in the medium containing low potassium concentrations (1.3 mM). The data from this study suggest that extracellular potassium concentrations may influence the antimicrobial activities of ionophores in the rumen.     

Conjugal transfer of a shuttle vector from the human colonic anaerobe Bacteroides uniformis to the ruminal anaerobe Prevotella (Bacteroides) ruminicola B(1)4.

Prevotella ruminicola (formerly Bacteroides ruminicola) is an anaerobic, gram-negative, polysaccharide-degrading bacterium which is found in the rumina of cattle. Since P. ruminicola is thought to make an important contribution to digestion of plant material in rumina, the ability to alter this strain genetically might help improve the efficiency of rumen fermentation. However, previously there has been no way to introduce foreign DNA into P. ruminicola strains. In this study we transferred a shuttle vector, pRDB5, from the colonic species Bacteroides uniformis to P. ruminicola B(1)4. The transfer frequency was 10(-6) to 10(-7) per recipient. pRDB5 contains sequences from pBR328, a cryptic colonic Bacteroides plasmid pB8-51, and a colonic Bacteroides tetracycline resistance (Tcr) gene. pRDB5 was mobilized out of B. uniformis by a self-transmissible Bacteroides chromosomal element designated Tcr Emr 12256. pRDB5 replicated in Escherichia coli as well as in Bacteroides spp. and was also mobilized from E. coli to B. uniformis by using IncP plasmid R751. However, direct transfer from E. coli to P. ruminicola B(1)4 was not detected. Thus, to introduce cloned DNA into P. ruminicola B(1)4, it was necessary first to mobilize the plasmid from E. coli to B. uniformis and then to mobilize the plasmid from B. uniformis to P. ruminicola B(1)4.     

Effects of potassium ion concentrations on the antimicrobial activities of ionophores against ruminal anaerobes.

The antimicrobial activities of monensin and lasalocid against representative strains of ruminal bacteria were evaluated in medium containing three different concentrations of potassium (1.3, 7.9, or 23.3 mM). The growth of Eubacterium ruminantium was inhibited by low concentrations of ionophores (less than or equal to 0.16 mg/liter), while the strain of Streptococcus bovis tested was resistant to high concentrations of ionophores (40 mg/liter) at all potassium concentrations tested. The MICs of the ionophores for strains of Bacteroides succinogenes, Butyrivibrio fibrisolvens, Ruminococcus albus, and Ruminococcus flavefaciens and for one strain of Bacteroides ruminicola increased with increasing potassium concentrations in the medium. High concentrations of ionophores (40 mg/liter) decreased the maximum cell yields or increased the lag times or both in cultures of one strain of Bacteroides ruminicola and two strains of Selenomonas ruminantium but did not completely inhibit the growth of these organisms. Increased potassium concentrations in the medium (from 7.9 to 23.3 mM) decreased the lag times or increased the cell yields or both when these three strains were grown in ionophore-containing medium, while the activities of lasalocid and monensin against these organisms were enhanced in the medium containing low potassium concentrations (1.3 mM). The data from this study suggest that extracellular potassium concentrations may influence the antimicrobial activities of ionophores in the rumen.     

Conjugal transfer of a shuttle vector from the human colonic anaerobe Bacteroides uniformis to the ruminal anaerobe Prevotella (Bacteroides) ruminicola B(1)4.

Prevotella ruminicola (formerly Bacteroides ruminicola) is an anaerobic, gram-negative, polysaccharide-degrading bacterium which is found in the rumina of cattle. Since P. ruminicola is thought to make an important contribution to digestion of plant material in rumina, the ability to alter this strain genetically might help improve the efficiency of rumen fermentation. However, previously there has been no way to introduce foreign DNA into P. ruminicola strains. In this study we transferred a shuttle vector, pRDB5, from the colonic species Bacteroides uniformis to P. ruminicola B(1)4. The transfer frequency was 10(-6) to 10(-7) per recipient. pRDB5 contains sequences from pBR328, a cryptic colonic Bacteroides plasmid pB8-51, and a colonic Bacteroides tetracycline resistance (Tcr) gene. pRDB5 was mobilized out of B. uniformis by a self-transmissible Bacteroides chromosomal element designated Tcr Emr 12256. pRDB5 replicated in Escherichia coli as well as in Bacteroides spp. and was also mobilized from E. coli to B. uniformis by using IncP plasmid R751. However, direct transfer from E. coli to P. ruminicola B(1)4 was not detected. Thus, to introduce cloned DNA into P. ruminicola B(1)4, it was necessary first to mobilize the plasmid from E. coli to B. uniformis and then to mobilize the plasmid from B. uniformis to P. ruminicola B(1)4.     

Cyclic adenosine 3'',5''-monophosphate levels and activities of adenylate cyclase and cyclic adenosine 3'',5''-monophosphate phosphodiesterase in Pseudomonas and Bacteroides.

A modified Gilman assay was used to determine the concentrations of cyclic adenosine 3',5'-monophosphate (cAMP) in rapidly filtered cells and in the culture filtrates of Pseudomonas aeruginosa, Escherichia coli K-12, and Bacteroides fragilis. In P. aeruginosa cultures, levels of cAMP in the filtrate increased with the culture absorbance (3.5 to 19.8 X 10(-9) M) but did not vary significantly with the carbon source used to support growth. Intracellular concentrations (0.8 to 3.2 X 10(-5) M) were substantially higher and did not vary appreciably during growth or with carbon source. Sodium cAMP (5 mM) failed to reverse the catabolite repression of inducible glucose-6-phosphate dehydrogenase (EC 1.1.1.49) synthesis caused by the addition of 10 mM succinate. Exogenous cAMP also had no discernible effect on the catabolite repression control of inducible mannitol dehydrogenase (EC 1.1.1.67). P. aeruginosa was found to contain both soluble cAMP phosphodiesterase (EC 3.1.4.17) and membrane-associated adenylate cyclase (EC 4.6.1.1) activity, and these were compared to the activities detected in crude extracts of E. coli. B. fragilis crude cell extracts contain neither of these enzyme activities, and little or no cAMP was detected in cells or culture filtrates of this anaerobic bacterium.     

Comparison of the post-antibiotic effect (PAE) induced by ceftizoxime, ceftriaxone, cefoxitin, ampicillin-sulbactam, and ticarcillin-clavulanate against selected isolates of Bacteroides fragilis and B. thetaiotaomicron

The exposure of bacteria to various groups of antimicrobials at different concentrations can produce damage to the bacteria that persists even after removal of the antimicrobial agent. The post antibiotic effect (PAE) of beta-lactams on aerobic gram-negative bacilli is relatively short (<1 h), however, little information is available regarding anaerobic gram-negative bacilli. We studied the PAE of ceftizoxime, ampicillin-sulbactam, ticarcillin-clavulanate, cefoxitin, and ceftriaxone against strains of Bacteroides fragilis and B. thetaiotaomicron at antimicrobial concentrations 4x, 8x, and 16x the MIC values using colony count determinations of treated and untreated cultures. Against B. fragilis H931, ceftizoxime-induced PAE values were 2 h, 3 h 24 min, and 11 h 36 min at 4xMIC, 8xMIC, and 16xMIC while for the B. thetaiotaomicron isolates PAEs ranged from 2 h 27 min to 6 h 12 min at the same concentrations. Cefoxitin PAE values were 3 h 6 min and 2 h 18 min for the clinical isolates at 16xMIC and 3 h 24 min and 1 h 12 min against the laboratory strains at 16xMIC respectively, and for ceftriaxone 1 h 12 min and 5 h 12 min, respectively, for the B. thetaiotaomicron D933 and B. fragilis H931 strains at 16xMIC. With ampicillin-sulbactam, the longest PAE values were observed at 16xMIC with all the test isolates of B. fragilis and B. thetaiotaomicron. PAE values induced by ticarcillin-clavulanate overall were the shortest for the two clinical isolates. These studies indicate substantial PAE values for beta-lactams against selected anaerobes which may be an important factor in the dosing regimen of these test agents.     

Some effects of uncouplers and inhibitors on growth and electron transport in rumen bacteria.

Uncouplers and inhibitors of electron transport affected growth and electron transport of rumen bacteria in various ways. Selenomonas ruminantium was not affected by inhibitor and uncoupler concentrations which affected growth and electron transport of Bacteroides ruminicola, B. succinogenes, and Butyrivibrio fibrisolvens. Inhibitors, when active, led to accumulation of reduced electron carriers before the site of action, but differences were found among organisms in the site of action of these inhibitors. Uncouplers reduced the glucose molar growth yields (Ygluc) of B. ruminicola, B. succinogenes, and B. fibrisolvens compared with those obtained without uncouplers. The extent of Ygluc reduction accompanying inhibitor exposure reflected electron transport chain structure. S. ruminantium appeared to obtain its adenosine 5'-triphosphate from substrate-level processes only. The other organisms studied appeared to obtain adenosine 5'-triphosphate both from substrate-level processes and from electron transport but differed in the amount of adenosine 5'-triphosphate obtained from glucose catabolism and in the proportions of adenosine 5'-triphosphate obtained from substrate-level reactions and electron transport.