Regulation of beta-glucosidase in Bacteroides ruminicola by a different mechanism: growth rate-dependent derepression

Bacteroides ruminicola B(1)4, a predominant ruminal and cecal bacterium, was grown in batch and continuous cultures, and beta-glucosidase activity was measured by following the hydrolysis of p-nitrophenyl-beta-glucopyranoside. Specific activity was high when the bacterium was grown in batch cultures containing cellobiose, mannose, or lactose (greater than 286 U/g of protein). Activity was reduced approximately 90% when the organism was grown on glucose, sucrose, fructose, maltose, or arabinose. The specific activity of cells fermenting glucose was initially low but increased as glucose was depleted. When glucose was added to cultures growing on cellobiose, beta-glucosidase synthesis ceased immediately. Catabolite repression by glucose was not accompanied by diauxic growth and was not relieved by cyclic AMP. Since glucose-grown cultures eventually exhibited high beta-glucosidase activity, cellobiose was not needed as an inducer. Catabolite repression explained beta-glucosidase activity of batch cultures and high-dilution-rate chemostats where glucose accumulated, but it could not account for activity at slow dilution rates. Maximal beta-glucosidase activity was observed at a dilution rate of approximately 0.35 h-1, and cellobiose-limited chemostats showed a 15-fold decrease in activity as the dilution rate declined. An eightfold decline was observed in glucose-limited chemostats. Since inducer availability was not a confounding factor in glucose-limited chemostats, the growth rate-dependent derepression could not be explained by other mechanisms.     

Effect of phenolic monomers on the growth and beta-glucosidase activity of Bacteroides ruminicola and on the carboxymethylcellulase, beta-glucosidase, and xylanase activities of Bacteroides succinogenes.

trans-p-Coumaric acid inhibited the growth of Bacteroides ruminicola on both cellobiose and glucose, while trans-ferulic acid and vanillin retarded growth. The phenolic monomers varied in their potential to inhibit the Bacteroides succinogenes beta-glucosidase, carboxymethylcellulase, and xylanase, with p-coumaric acid being the most inhibitory. The B. ruminicola beta-glucosidase was inhibited less than 10% by all three compounds.     

Effect of phenolic monomers on the growth and beta-glucosidase activity of Bacteroides ruminicola and on the carboxymethylcellulase, beta-glucosidase, and xylanase activities of Bacteroides succinogenes

trans-p-Coumaric acid inhibited the growth of Bacteroides ruminicola on both cellobiose and glucose, while trans-ferulic acid and vanillin retarded growth. The phenolic monomers varied in their potential to inhibit the Bacteroides succinogenes beta-glucosidase, carboxymethylcellulase, and xylanase, with p-coumaric acid being the most inhibitory. The B. ruminicola beta-glucosidase was inhibited less than 10% by all three compounds.     

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.     

Specificity of the heme requirement for growth of Bacteroides ruminicola

Caldwell, D. R. (U.S. Department of Agriculture, Beltsville, Md.), D. C. White, M. P. Bryant, and R. N. Doetsch. Specificity of the heme requirement for growth of Bacteroides ruminicola. J. Bacteriol. 90:1645-1654. 1965.-Previous studies suggested that most strains of Bacteroides ruminicola subsp. ruminicola require heme for growth. Present studies with heme-requiring strain 23 showed that protoheme was replaced by various porphyrins, uroporphyrinogen, coproporphyrinogen, certain iron-free metalloporphyrins, hemes, and certain heme-proteins containing readily removable hemes. Strain 23 utilized a wider range of tetrapyrroles than hemin-requiring bacteria previously studied. Inactive compounds included porphyrin biosynthesis intermediates preceding the tetrapyrrole stage and related compounds; uroporphyrin, chlorophyll, pheophytin, phycoerythrin, bilirubin, pyrrole, FeSO(4) with or without chelating agents; and representative ferrichrome compounds. Strain 23, two other strains representing predominant biotypes of B. ruminicola subsp. ruminicola, and one closely related strain grew in media containing heme-free protoporphyrin, mesoporphyrin, hematoporphyrin, or deuteroporphyrin, apparently inserting iron into several nonvinyl porphyrins. Porphobilinogen and porphyrin synthesis, apparently via the commonly known heme synthesis pathway, occurred during growth of heme-independent B. ruminicola subsp. brevis strain GA33 in a tetrapyrrole-free medium containing delta-aminolevulinic acid, but delta-aminolevulinic acid metabolism to porphobilinogen or porphyrins could not be detected in cells of heme-requiring strain 23 grown in the same medium with hemin added. Growth of strain 23 with uroporphyrinogen, coproporphyrinogen, or protoporphyrin IX replacing hemin suggests that part of the commonly known heme-biosynthesis pathway is present in this strain, but nutritional and metabolic evidence indicates that some or all of the enzymes synthesizing the tetrapyrrole nucleus from linear molecules are lacking or inactive.     

Plasmid-associated transfer of tetracycline resistance in Bacteroides ruminicola.

Tetracycline resistance was transferred at frequencies between 10(-7) and 10(-6) per recipient cell in anaerobic matings between two strains of the strictly anaerobic rumen bacterium Bacteroides ruminicola. The donor strain, 223/M2/7, was a multiple-plasmid-bearing tetracycline-resistant strain from the ovine rumen, and the recipient, F101, was a rifampin-resistant mutant of B14, a bovine strain belonging to B. ruminicola subsp. brevis. Resistance transfer could occur in the presence of DNase, but not in dummy mating mixtures in which filtrate from a donor culture replaced donor cells. Acquisition of tetracycline resistance by the recipient was accompanied by the appearance of a 19.5-kilobase pair plasmid (designated pRRI4) which was homologous with a plasmid of similar size and restriction pattern present in the donor strain. A transconjugant (F115) carrying pRRI4 was also able to act as a donor of tetracycline resistance and plasmid DNA in matings with another recipient. Derivatives of F115 that had spontaneously lost tetracycline resistance lacked detectable plasmid DNA. It is concluded that pRRI4 mediated the transfer of tetracycline resistance. Transfer of resistance was not detectably enhanced by pregrowth of the donor in medium containing tetracycline. Transfer of tetracycline resistance was not detected from 223/M2/7 to a strain, 23 belonging to B. ruminicola subsp. ruminicola.     

Fermentation of Peptides by Bacteroides ruminicola B14

The maximum growth rate of Bacteroides ruminicola B₁4 was significantly improved when either Trypticase or acetate and C₄-C₅ fatty acids were added to defined medium containing macrominerals, microminerals, vitamins, hemin, cysteine hydrochloride, and glucose. The organism was unable to grow with peptides as the sole energy source, but growth yields from glucose were significantly improved when Trypticase was added to batch cultures containing basal medium, acetate, and C₄-C₅ volatile fatty acids. During periods of rapid growth, very little peptide was deaminated to ammonia, but after growth ceased there was a linear increase in ammonia. Fifteen grams of Trypticase per liter resulted in maximum ammonia production. In glucose-limited chemostats, ammonia production from peptides was inversely proportional to the dilution rate, and 87% of the variation in ammonia production could be explained by retention time in the culture vessel. Chemostats receiving Trypticase had higher theoretical maximum growth yields and lower maintenance energy expenditures than similar cultures not receiving peptide. Cells from the Trypticase cultures contained more carbohydrate, and this difference was most evident at rapid dilution rates. When corrections were made for cell composition and the amount of peptides that were fermented, it appeared that peptide carbon skeletons could be used for maintenance energy. B. ruminicola B₁4 was unable to grow on peptides alone because it was unable to utilize peptides at a fast enough rate to meet its maintenance requirement.     

Heterologous expression of the Bacteroides ruminicola xylanase gene in Bacteroides fragilis and Bacteroides uniformis

A cloned xylanase gene from the ruminal bacterium Bacteroides ruminicola 23 was transferred by conjugation into the colonic species Bacteroides fragilis and Bacteroides uniformis by using the Escherichia coli-Bacteroides shuttle vector pVAL-1. The cloned gene was expressed in both species, and xylanase specific activity in crude extracts was found to be at least 1400-fold greater than that found in the B. ruminicola strain. Analysis of crude extract proteins from the recombinant B. fragilis by SDS-PAGE demonstrated a new 60,000 molecular weight protein. The xylanase activity expressed in both E. coli and B. fragilis was capable of degrading xylan to xylooligosaccharides in vitro. This is the first demonstration that colonic Bacteroides species can express a gene from a ruminal Bacteroides species.     

Properties of ionophore-resistant Bacteroides ruminicola enriched by cultivation in the presence of tetronasin.

Bacteroides ruminicola M384 was grown in the presence of increasing concentrations of tetronasin, an ionophore that has been developed as a feed additive for ruminants. The resulting culture, B. ruminicola M384/TnR, was then maintained in medium containing 0.1 microgram tetronasin/ml. Growth of the parent strain was eliminated by the addition of 0.1 micrograms tetronasin/ml, but the growth rate of B. ruminicola M384/TnR, which grew more slowly than the parent strain, was unaffected by adding tetronasin. Bacteroides ruminicola M384/TnR retained its resistance to tetronasin even after repeated subculture in the absence of the ionophore, suggesting that a mutation had occurred. The absence of plasmids in individual colonies of B. ruminicola M384/TnR implied that the mutation was chromosomal. Bacteroides ruminicola M384/TnR was also more resistant to the ionophores monensin and lasalocid and, to a lesser degree, to the antibiotic avoparcin than B. ruminicola M384. Binding of [14C]tetronasin to B. ruminicola M384/TnR was lower than binding of the ionophore to the parent stain, and this difference was eliminated by washing cells with EDTA. The peptidolytic activity of B. ruminicola M384 towards triphenylalanine (Mr = 460) was unaffected in B. ruminicola M384/TnR, but the rate of breakdown tetraphenylalanine (Mr = 607) was decreased. This difference was also abolished by EDTA. It was concluded that growth of B. ruminicola in the presence of tetronasin resulted in a mutation affecting the permeability of the cell envelope, such that permeation of tetronasin and molecules of a similar size (Mr = 628) was decreased.     

Plasmid-associated transfer of tetracycline resistance in Bacteroides ruminicola

Tetracycline resistance was transferred at frequencies between 10(-7) and 10(-6) per recipient cell in anaerobic matings between two strains of the strictly anaerobic rumen bacterium Bacteroides ruminicola. The donor strain, 223/M2/7, was a multiple-plasmid-bearing tetracycline-resistant strain from the ovine rumen, and the recipient, F101, was a rifampin-resistant mutant of B14, a bovine strain belonging to B. ruminicola subsp. brevis. Resistance transfer could occur in the presence of DNase, but not in dummy mating mixtures in which filtrate from a donor culture replaced donor cells. Acquisition of tetracycline resistance by the recipient was accompanied by the appearance of a 19.5-kilobase pair plasmid (designated pRRI4) which was homologous with a plasmid of similar size and restriction pattern present in the donor strain. A transconjugant (F115) carrying pRRI4 was also able to act as a donor of tetracycline resistance and plasmid DNA in matings with another recipient. Derivatives of F115 that had spontaneously lost tetracycline resistance lacked detectable plasmid DNA. It is concluded that pRRI4 mediated the transfer of tetracycline resistance. Transfer of resistance was not detectably enhanced by pregrowth of the donor in medium containing tetracycline. Transfer of tetracycline resistance was not detected from 223/M2/7 to a strain, 23 belonging to B. ruminicola subsp. ruminicola.     

Fermentation of Peptides by Bacteroides ruminicola B(1)4

The maximum growth rate of Bacteroides ruminicola B(1)4 was significantly improved when either Trypticase or acetate and C(4)-C(5) fatty acids were added to defined medium containing macrominerals, microminerals, vitamins, hemin, cysteine hydrochloride, and glucose. The organism was unable to grow with peptides as the sole energy source, but growth yields from glucose were significantly improved when Trypticase was added to batch cultures containing basal medium, acetate, and C(4)-C(5) volatile fatty acids. During periods of rapid growth, very little peptide was deaminated to ammonia, but after growth ceased there was a linear increase in ammonia. Fifteen grams of Trypticase per liter resulted in maximum ammonia production. In glucose-limited chemostats, ammonia production from peptides was inversely proportional to the dilution rate, and 87% of the variation in ammonia production could be explained by retention time in the culture vessel. Chemostats receiving Trypticase had higher theoretical maximum growth yields and lower maintenance energy expenditures than similar cultures not receiving peptide. Cells from the Trypticase cultures contained more carbohydrate, and this difference was most evident at rapid dilution rates. When corrections were made for cell composition and the amount of peptides that were fermented, it appeared that peptide carbon skeletons could be used for maintenance energy. B. ruminicola B(1)4 was unable to grow on peptides alone because it was unable to utilize peptides at a fast enough rate to meet its maintenance requirement.     

Isolation of an endoglucanase gene from Bacteroides ruminicola subsp. brevis

A gene coding for endo-1, 4-beta-glucanase activity has been isolated from Bacteroides ruminicola subsp. brevis by cloning in Escherichia coli. After restriction mapping of a 6.4 kb insert, a 2.2 kb DNA fragment was sub-cloned in pUC19 to produce the enzymically active clone pJW3. Recloning of the gene fragment in the reverse orientation in pUC18 (clone pJW4) indicated that a gene promoter was present in the cloned fragment and was able to function in E. coli. The clone pJW4 displayed increased activity which was attributed to expression from the lac promoter of pUC18. The enzyme encoded by pJW4 was optimally active at pH 5.5-6.0, and in the temperature range 37-42 degrees C. The preferred substrate was carboxymethylcellulose, but the enzyme displayed 50-60% of maximal activity on both acid-swollen cellulose and soluble xylan. No significant activity was detected on ball-milled filter paper or particulate xylan. Deletion experiments confirmed that both cellulase and xylanase activities were altered to a similar extent by deletion of DNA from the 3' end of the gene, suggesting that both are a function of the same polypeptide product.     

Alternative pathways for biosynthesis of leucine and other amino acids in Bacteroides ruminicola and Bacteroides fragilis.

Bacteroides ruminicola is one of several species of anaerobes that are able to reductively carboxylate isovalerate (or isovaleryl-coenzyme A) to synthesize alpha-ketoisocaproate and thus leucine. When isovalerate was not supplied to growing B. ruminicola cultures, carbon from [U-14C]glucose was used for the synthesis of leucine and other cellular amino acids. When unlabeled isovalerate was available, however, utilization of [U-14C]glucose or [2-14C]acetate for leucine synthesis was markedly and specifically reduced. Enzyme assays indicated that the key enzyme of the common isopropylmalate (IPM) pathway for leucine biosynthesis, IPM synthase, was present in B. ruminicola cell extracts. The specific activity of IPM synthase was reduced when leucine was added to the growth medium but was increased by the addition of isoleucine plus valine, whereas the addition of isovalerate had little or no effect. The activity of B. ruminicola IPM synthase was strongly inhibited by leucine, the end product of the pathway. It seems unlikely that the moderate inhibition of the enzyme by isovalerate adequately explains the regulation of carbon flow by isovalerate in growing cultures. Bacteroides fragilis apparently also uses either the isovalerate carboxylation or the IPM pathway for leucine biosynthesis. Furthermore, both of these organisms synthesize isoleucine and phenylalanine, using carbon from 2-methylbutyrate and phenylacetate, respectively, in preference to synthesis of these amino acids de novo from glucose. Thus, it appears that these organisms have the ability to regulate alternative pathways for the biosynthesis of certain amino acids and that pathways involving reductive carboxylations are likely to be favored in their natural habitats.     

Some growth and metabolic characteristics of monensin-sensitive and monensin-resistant strains of Prevotella (Bacteroides) ruminicola.

New strains with enhanced resistance to monensin were developed from Prevotella (Bacteroides) ruminicola subsp. ruminicola 23 and P. ruminicola subsp. brevis GA33 by stepwise exposure to increasing concentrations of monensin. The resulting resistant strains (23MR2 and GA33MR) could initiate growth in concentrations of monensin which were 4 to 40 times greater than those which inhibited the parental strains. Resistant strains also showed enhanced resistance to nigericin and combinations of monensin and nigericin but retained sensitivity to lasalocid. Glucose utilization in cultures of the monensin-sensitive strains (23 and GA33) and one monensin-resistant strain (23MR2) was retarded but not completely inhibited when logarithmic cultures were challenged with monensin (10 mg/liter). Monensin challenge of cultures of the two monensin-sensitive strains (23 and GA33) was characterized by 78 and 51% decreases in protein yield (milligrams of protein per mole of glucose utilized), respectively. Protein yields in cultures of resistant strain 23MR2 were decreased by only 21% following monensin challenge. Cell yields and rates of glucose utilization by resistant strains GA33MR were not decreased by challenge with 10 mg of monensin per liter. Resistant strains produced greater relative proportions of propionate and less acetate than the corresponding sensitive strains. The relative amounts of succinate produced were greater in cultures of strains 23, GA33, and 23MR2 following monensin challenge. However, only minor changes in end product formation were associate with monensin challenge of resistant strain GA33MR. These results suggest that monensin has significant effects on both the growth characteristics and metabolic activities of these predominant, gram-negative ruminal bacteria.     

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.     

Some growth and metabolic characteristics of monensin-sensitive and monensin-resistant strains of Prevotella (Bacteroides) ruminicola.

New strains with enhanced resistance to monensin were developed from Prevotella (Bacteroides) ruminicola subsp. ruminicola 23 and P. ruminicola subsp. brevis GA33 by stepwise exposure to increasing concentrations of monensin. The resulting resistant strains (23MR2 and GA33MR) could initiate growth in concentrations of monensin which were 4 to 40 times greater than those which inhibited the parental strains. Resistant strains also showed enhanced resistance to nigericin and combinations of monensin and nigericin but retained sensitivity to lasalocid. Glucose utilization in cultures of the monensin-sensitive strains (23 and GA33) and one monensin-resistant strain (23MR2) was retarded but not completely inhibited when logarithmic cultures were challenged with monensin (10 mg/liter). Monensin challenge of cultures of the two monensin-sensitive strains (23 and GA33) was characterized by 78 and 51% decreases in protein yield (milligrams of protein per mole of glucose utilized), respectively. Protein yields in cultures of resistant strain 23MR2 were decreased by only 21% following monensin challenge. Cell yields and rates of glucose utilization by resistant strains GA33MR were not decreased by challenge with 10 mg of monensin per liter. Resistant strains produced greater relative proportions of propionate and less acetate than the corresponding sensitive strains. The relative amounts of succinate produced were greater in cultures of strains 23, GA33, and 23MR2 following monensin challenge. However, only minor changes in end product formation were associate with monensin challenge of resistant strain GA33MR. These results suggest that monensin has significant effects on both the growth characteristics and metabolic activities of these predominant, gram-negative ruminal bacteria.