Introduction of the Bacteroides ruminicola xylanase gene into the Bacteroides thetaiotaomicron chromosome for production of xylanase activity.

The xylanase gene from the ruminal bacterium Bacteroides ruminicola 23 is highly expressed in colonic Bacteroides species when carried on plasmid pVAL-RX. In order to stabilize xylanase expression in the absence of antibiotic selection, the xylanase gene was introduced into the chromosome of Bacteroides thetaiotaomicron 5482 by using suicide vector pVAL-7. Xylanase activity in the resulting strain, B. thetaiotaomicron BTX, was about 30% of that observed in B. thetaiotaomicron 5482 containing the xylanase gene on pVAL-RX. The data obtained from continuous culture experiments using antibiotic-free medium showed that expression of xylanase activity in strain BTX was extremely stable, with no demonstrated loss of the inserted xylanase gene over 60 generations, with dilution rates from 0.42 to 0.03 h-1. In contrast, the plasmid-borne xylanase gene was almost completely lost by 60 generations in the absence of antibiotic selection. Incubation of strain BTX with oatspelt xylan resulted in the degradation of more than 40% of the xylan to soluble xylooligomers. The stability of xylanase expression in B. thetaiotaomicron BTX suggests that this microorganism might be suitable for introduction into the rumen and increased xylan degradation.     

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.     

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.     

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.     

Incorporation of leucine into phospholipids of Bacteroides thetaiotaomicron.

L-[4,5-3H]- or L-[U-14C]leucine was incorporated by Bacteroides thetaiotaomicron into acid-precipitable material even when the bacteria were treated with concentrations of tetracycline high enough to prevent growth. Similar results were obtained when L-[2,3,4-3H]valine or L-[4,5-3H]isoleucine was used instead of leucine. In bacteria which had been treated with tetracycline, the acid-precipitable label was not solubilized by treatment with protease, lysozyme, or deoxyribonuclease. However, virtually all of the label was extractable with chloroform-methanol, indicating that the label had been incorporated into membrane lipids. Since L-[1-14C]leucine was not incorporated into lipids, leucine was probably decarboxylated before incorporation. When a chloroform extract from bacteria which had been labeled with both [32P]phosphate and [3H]leucine was resolved into component phospholipids by two-dimensional thin-layer chromatography, 3H was incorporated into all of the phospholipids. When these phospholipids were deacylated, the 3H from leucine was associated with released fatty acids rather than with the head groups. Thus, it appears that B. thetaiotaomicron can utilize leucine and similar amino acids not only by incorporating them into protein but also by incorporating portions of these amino acids into membrane phospholipids.     

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.     

Distribution of xylanase genes and enzymes among strains of Prevotella (Bacteroides) ruminicola from the rumen

The distribution of two xylanase genes was examined by Southern hybridization among 26 strains of the rumen anaerobic bacterium Prevotella (Bacteroides) ruminicola. Hybridization with a xylanase/endoglucanase gene from the type strain 23 was found in six strains while hybridization with a xylanase gene from strain D31d was found in 14 strains. Sequences related to both genes were present, on different restriction fragments, in six strains, whereas no hybridization to either gene was detected in five other strains capable of hydrolysing xylan, or in seven strains that showed little or no xylanase activity. Zymogram analyses of seven xylanolytic strains of P. ruminicola demonstrated interstrain variation in the apparent molecular masses of the major xylanases and carboxymethylcellulases that could be renatured following SDS polyacrylamide gel electrophoresis.     

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.     

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.     

Transposon-induced norfloxacin-sensitive mutants of Bacteroides thetaiotaomicron

To elucidate the mechanism of norfloxacin (a fluoroquinolone) resistance of Bacteroides thetaiotaomicron, a member of the B. fragilis group, we isolated transposon-induced mutants sensitive to this agent using Tn4351. Four norfloxacin-sensitive mutants showed reduced levels of resistance, at least, to ethidium bromide. Cloning and sequencing of three chromosomal fragments adjacent to Tn4351 from the mutants revealed that two partial open reading frames (orfs) were disrupted by a transposon. Amino acid sequences of partial orf products had strong homologies to those of Escherichia coli RecB and B. ovatus transketolase. Two mutants carried a recB homolog inserted by Tn4351 together with R751 (cointegration) and by itself (simple transposition) at the amino- and carboxyl-terminal portions, respectively. Since mutations in recB produce E. coli cells sensitive to DNA-damaging treatments by quinolones, it is concluded that decreases of the minimum inhibitory concentrations (MICs) of the agents for B. thetaiotaomicron resulted from disruption of the recB homolog. Another mutant carried a transketolase gene inserted by Tn4351. There is no reasonable explanation why disruption of the transketolase gene caused a decrease of the MIC of norfloxacin for this organism, although Streptococcus pneumoniae RecP related to DNA recombination was reported to be transketolase.     

Digestion of proteoglycan by Bacteroides thetaiotaomicron

It has been shown previously that Bacteroides thetaiotaomicron, a human colonic anaerobe, can utilize the tissue mucopolysaccharide chondroitin sulfate as a source of carbon and energy and that the enzymes involved in this utilization are all cell associated (A. A. Salyers and M. B. O'Brien, J. Bacteriol. 143:772-780, 1980). Since chondroitin sulfate does not generally occur in isolated form in tissue, but rather is bound covalently in proteoglycan, we investigated the extent to which chondroitin sulfate which is bound in such a sterically hindered complex can be utilized by intact bacteria. Intact cells of B. thetaiotaomicron were able to digest chondroitin sulfate in proteoglycan, although at a slightly slower rate than free chondroitin sulfate. Prior digestion of proteoglycan with trypsin to produce small fragments of protein with several chondroitin sulfate chains attached did not increase the rate at which the bound chondroitin sulfate was digested. Accordingly, the slower rate of digestion was probably due to attachment of chondroitin sulfate chains to the protein backbone rather than to steric hindrance by other components of the proteoglycan. When proteoglycan which had been incubated with intact bacteria was treated with sodium borohydride to release the undigested fragments of chondroitin sulfate from the protein backbone, the size and composition of the fragments indicated that intact bacteria were able to digest all but three monosaccharides of the chondroitin sulfate chains. Thus, despite steric hindrance due to attachment of the chondroitin sulfate chains to the protein backbone, digestion of bound chondroitin sulfate by intact bacteria was nearly complete.     

Use of a unique gene sequence as a probe to enumerate a strain of Bacteroides ruminicola introduced into the rumen

Cloned fragments of genomic DNA from the ruminal anaerobe Bacteroides ruminicola subsp. brevis B14 were isolated and used as hybridization probes to identify closely related bacterial species. One DNA fragment unique to strain B14 was tested to determine its sensitivity in detecting homologous sequences among total ruminal microbial DNA. In a DNA titration experiment, the probe was capable of detecting strain B14 sequences in vitro down to 0.1% of the total bacterial DNA present in a hybridization assay. There was no detectable signal for total ruminal bacterial DNA. The specificity of this DNA fragment was exploited to enumerate strain B14 in a fresh mixed suspension of ruminal bacteria in vitro and after inoculation of the strain into the rumen. In vitro strain B14 had a half-life of 9 h. However, following inoculation into the rumen there was a very rapid loss of the strain to below the detectable limit within 3 h. The half-life was less than 30 min. This loss was not due to ruminal dilution or to bacteriophage attack but was possibly the result of a specific bacteriocinlike activity present in the rumen and detectable in fresh ruminal fluid.     

Specificity of a Bacteroides thetaiotaomicron Marker for Human Feces

A bacterial primer set, known to produce a 542-bp amplicon specific for Bacteroides thetaiotaomicron, generated this product in PCR with 1 ng of extracted DNA from 92% of 25 human fecal samples, 100% of 20 sewage samples, and 16% of 31 dog fecal samples. The marker was not detected in 1 ng of fecal DNA from 61 cows, 35 horses, 44 pigs, 24 chickens, 29 turkeys, and 17 geese.     

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.     

Use of a unique gene sequence as a probe to enumerate a strain of Bacteroides ruminicola introduced into the rumen.

Cloned fragments of genomic DNA from the ruminal anaerobe Bacteroides ruminicola subsp. brevis B14 were isolated and used as hybridization probes to identify closely related bacterial species. One DNA fragment unique to strain B14 was tested to determine its sensitivity in detecting homologous sequences among total ruminal microbial DNA. In a DNA titration experiment, the probe was capable of detecting strain B14 sequences in vitro down to 0.1% of the total bacterial DNA present in a hybridization assay. There was no detectable signal for total ruminal bacterial DNA. The specificity of this DNA fragment was exploited to enumerate strain B14 in a fresh mixed suspension of ruminal bacteria in vitro and after inoculation of the strain into the rumen. In vitro strain B14 had a half-life of 9 h. However, following inoculation into the rumen there was a very rapid loss of the strain to below the detectable limit within 3 h. The half-life was less than 30 min. This loss was not due to ruminal dilution or to bacteriophage attack but was possibly the result of a specific bacteriocinlike activity present in the rumen and detectable in fresh ruminal fluid.     

Galactosamine inhibition of protein synthesis in Bacteroides thetaiotaomicron

Galactosamine does not support growth of Bacteroides thetaiotaomicron. Despite this, galactosamine was more effective than utilizable carbohydrates such as glucose in preventing synthesis of the inducible enzymes alpha-glucosidase and chondroitin lyase. Galactosamine also stopped overall protein synthesis. By contrast glucose and other utilizable carbohydrates increased the rate of protein synthesis. Addition of glucose to bacteria which had been treated with galactosamine restored the ability of the bacteria to synthesize protein and to produce inducible enzymes. Moreover, when B. thetaiotaomicron was incubated with [1-14C]galactosamine for 30 min at 37 degrees C, about one-third of the label which was taken up by the cells comigrated with glucosamine-6-phosphate on a thin-layer chromatogram. Thus galactosamine appears to be phosphorylated by the bacteria. After 2 h incubation of the bacteria with [1-14C]galactosamine, there was a significant increase in the amount of label which could be extracted from acidified extracellular fluid with diethyl ether. This indicates that galactosamine can be metabolized to the level of volatile fatty acids. The rate of uptake of galactosamine and the amount of labeled fatty acids produced from galactosamine were both much lower than the values obtained when glucosamine was the substrate. Thus, although some metabolism of galactosamine occurs, the rate is apparently too slow to enable galactosamine to support growth of B. thetaiotaomicron.     

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.