Biochemical analysis of a highly specific, pH stable xylanase gene identified from a bovine rumen-derived metagenomic library.

A metagenomic library was generated using microbial DNA extracted from the rumen contents of a grass hay-fed dairy cow using a bacterial artificial chromosome-based vector system. Functional screening of the library identified a gene encoding a potent glycoside hydrolase, xyn10N18, localised within a xylanolytic gene cluster consisting of four open-reading frames (ORFs). The ORF, xyn10N18, encodes an endo-ß-1,4-xylanase with a glycosyl hydrolase family 10 (GH10) catalytic domain, adopts a canonical α8/ß8-fold and possesses conserved catalytic glutamate residues typical of GH10 xylanases. Xyn10N18 exhibits optimal catalytic activity at 35 °C and pH 6.5 and was highly stable to pH changes retaining at least 85 % relative catalytic activity over a broad pH range (4.0-12.0). It retained 25 % of its relative activity at both low (4 °C) and high (55 °C) temperatures, however the stability of the enzyme rapidly decreased at temperatures of >40 °C. The specific activity of Xyn10N18 is enhanced by the divalent cations Mn(2+) and Co(2+) and is dramatically reduced by Hg(2+) and Cu(2+). Interestingly, EDTA had little effect on specific activity indicating that divalent cations do not function mechanistically. The enzyme was highly specific for xylan containing substrates and showed no catalytic activity against cellulose. Analysis of the hydrolysis products indicated that Xyn10N18 was an endoxylanase. Through a combination of structural modelling and in vitro enzyme characterisation this study provides an understanding of the mechanism and the substrate specificity of this enzyme serving as a starting point for directed evolution of Xyn10N18 and subsequent downstream use in industry.

Physical and genetic characterization of an outer-membrane protein (OmpM1) containing an N-terminal S-layer-like homology domain from the phylogenetically Gram-positive gut anaerobe Mitsuokella multacida.

Thin sectioning and freeze-fracture-etch of the ovine ruminal isolate Mitsuokella multacida strain 46/5(2) revealed a Gram-negative envelope ultra-structure consisting of a peptidoglycan wall overlaid by an outer membrane. Sodium-dodecyl-sulfate-polyacrylamide gel electrophoretic (SDS-PAGE) analysis of whole cells, cell envelopes and Triton X-100 extracted envelopes in combination with thin-section and N-terminal sequence analyses demonstrated that the outer membrane contained two major proteins (45 and 43 kDa) sharing identical N-termini (A-A-N-P-F-S-D-V-P-A-D-H-W-A-Y-D). A gene encoding a protein with a predicted N-terminus identical to those of the 43 and 45 kDa outer-membrane proteins was cloned. The 1290 bp open reading frame encoded a 430 amino acid polypeptide with a predicted molecular mass of 47,492 Da. Cleavage of a predicted 23 amino acid leader sequence would yield a protein with a molecular mass of 45,232 Da. Mass spectroscopic analysis confirmed that the cloned gene (ompM1) encoded the 45 kDa outer-membrane protein. The N-terminus of the mature OmpM1 protein (residues 24-70) shared homology with surface-layer homology (SLH) domains found in a wide variety of regularly structured surface-layers (S-layers). However, the outer-membrane locale, resistance to denaturation by SDS and high temperatures and the finding that the C-terminal residue was a phenylalanine suggested that ompM1 encoded a porin. Threading analysis in combination with the identification of membrane spanning domains indicated that the C-terminal region of OmpM1 (residues 250-430) likely forms a 16-strand beta-barrel and appears to be related to the unusual N-terminal SLH-domain-containing beta-barrel-porins previously described in the cyanobacterium Synechococcus PCC6301.

Functional symmetry of the beta-galactoside carrier in Escherichia coli.

Cytoplasmic membrane vesicles with either normal or inverted orientation were prepared from Escherichia coli. The lactose transport activity of these vesicle preparations was compared. The parameters measured were net efflux, counterflux, and K+/valinomycin-induced active uptake of lactose. With membrane vesicles derived from both wild-type and cytochrome-deficient strains the right-side-out and inverted membrane preparations showed similar rates of lactose flux in all assays. According to these criteria, the activity of the beta-galactoside transport protein is inherently symmetrical. One major difference was observed between the native and inverted vesicle preparations: the inverted vesicles had approximately twice the specific activity of native vesicles in the counterflux and K+/valinomycin-induced uptake assays. This difference can be largely ascribed to the presence in the normal vesicle preparation of vesicles with a high passive permeability to lactose. Such vesicles are apparently absent from the inverted vesicle preparations.

Partial purification and characterisation of Bfi57I and Bfi89I, restriction endonucleases from different strains of Butyrivibrio fibrisolvens.

Two class-II restriction endonucleases (ENases), Bfi57I and Bfi89I, were partially purified from Butyrivibrio fibrisolvens OB157 and OB189, respectively. Bfi57I (isoschizomer Sau3AI) had the DNA recognition/cleavage sequence 5'-/GATC-3'; it is not inhibited by Dam methylation, but is partially inhibited by M.BamHI methylation. Bfi89I (isoschizomer EaeI) had the recognition/cleavage sequence 5'-Y/GGCCR-3'; unlike the EaeI isoschizomer it is not fully inhibited by M.HaeIII methylation.

Phylogenetic analysis of methanogens from the bovine rumen.

BACKGROUND: Interest in methanogens from ruminants has resulted from the role of methane in global warming and from the fact that cattle typically lose 6 % of ingested energy as methane. Several species of methanogens have been isolated from ruminants. However they are difficult to culture, few have been consistently found in high numbers, and it is likely that major species of rumen methanogens are yet to be identified. RESULTS: Total DNA from clarified bovine rumen fluid was amplified using primers specific for Archaeal 16S rRNA gene sequences (rDNA). Phylogenetic analysis of 41 rDNA sequences identified three clusters of methanogens. The largest cluster contained two distinct subclusters with rDNA sequences similar to Methanobrevibacter ruminantium 16S rDNA. A second cluster contained sequences related to 16S rDNA from Methanosphaera stadtmanae, an organism not previously described in the rumen. The third cluster contained rDNA sequences that may form a novel group of rumen methanogens. CONCLUSIONS: The current set of 16S rRNA hybridization probes targeting methanogenic Archaea does not cover the phylogenetic diversity present in the rumen and possibly other gastro-intestinal tract environments. New probes and quantitative PCR assays are needed to determine the distribution of the newly identified methanogen clusters in rumen microbial communities.

Development of a competitive polymerase chain reaction assay for the ruminal bacterium Butyrivibrio fibrisolvens OB156 and its use for tracking an OB156-derived recombinant.

A competitive polymerase chain reaction assay targeting the 16S rDNA was developed for quantitating the rumen bacterium Butyrivibrio fibrisolvens OB156. A competitor DNA, serving as an internal control in the competitive polymerase chain reaction reaction, was constructed by polymerase chain reaction using a looped oligo longer than the normal primer. Coamplification of the target DNA with known amounts of the competitor DNA allowed quantitation of the target DNA in both pure culture and mixed culture systems, where minimum quantifiable level of OB156 was 1.7x10(2) and 5.6x10(4) cells, respectively. When an erythromycin-resistant recombinant derived from OB156 was inoculated into a rumen fluid culture, its numbers decreased with time. The rate of decrease measured by the competitive polymerase chain reaction assay was much slower than the rate determined by culture enumeration using erythromycin selection. The competitive polymerase chain reaction assay also showed 48 h persistence of the recombinant at 10(4) ml(-1) even after disappearance of culturable recombinant, suggesting maintenance of the target DNA from uncultivable cells. In an in vivo tracking trial, the recombinant became undetectable within 72 h with either assay, indicating rapid hydrolysis and/or outflow of the cells from the rumen.

Analysis of the sequence of a new cryptic plasmid, pRJF2, from a rumen bacterium of the genus Butyrivibrio: comparison with other Butyrivibrio plasmids and application in the development of cloning vector.

A small cryptic plasmid, pRJF2, from Butyrivibrio fibrisolvens strain OB157 was isolated and sequenced. The plasmid is similar in organisation to the previously sequenced Butyrivibrio plasmid, pRJF1, with two open reading frames, ORF1 and ORF2, flanking a region tentatively identified as the replication origin, and a region of unknown function defined by terminal 79 bp invert repeats. The sequences of ORF1, ORF2, and the presumptive replication origin are highly conserved. The sequence between the 79, bp invert repeats is not, and is therefore presumed to be of lesser functional significance, although the 5' and 3' termini are still highly conserved. The functional importance for plasmid replication of these regions was tested by constructing potential shuttle vectors, each lacking one or more of the regions of interest. When the region between the invert repeats was deleted and replaced by the erythromycin resistance gene from pAM beta 1 together with pUC18, to produce the 7.9 kb chimaeric plasmid pYK4, the construct was successfully transformed into E. coli and B. fibrisolvens by electroporation, and was stably maintained in both hosts. Both ORF1 and ORF2 were required for successful transformation of B. fibrisolvens.

Design, expression, and initial characterization of MB1, a de novo protein enriched in essential amino acids.

Using recently emerging protein folding principles we have designed a protein enriched in the essential amino acids methionine, threonine, lysine and leucine. Our preliminary study of consensus residues (based on charge, hydrophobicity and volume) of natural alpha-helical bundle proteins indicated that the residues M, T, K, and L could be inserted in an alpha-helical bundle structure. We therefore attempted to create a stable de novo protein, highly enriched in these essential amino acids, that would adopt the alpha-helical bundle fold. The design process was an iterative one. The consensus residues (based on the properties profile) for bundle helices were found considering the four helices taken together, helices I to IV individually, or only their N- and C-termini. Using these data, the helices in our de novo protein were designed by inserting the residues M, T, K and L as often as possible at positions where their volume, hydrophobicity and charge match the consensus found in natural bundle helices. Short sequences of strong turn formers were used to join the helices and adjust the predicted p1 to 7.7, while a number of local and global factors were used to refine our design. Further, the sequence was checked to eliminate various known protease targets in E. coli. The sequence of our de novo protein, MB1, is: MAT-EDMTDMMTTLFKTMQLLTK-SEPTA-MDEATKTATTMKNHLQNLMQK-TKNKE DMTDMATTYFKTMQLLTK-TEPSA-MDEATKTATTMKNHLQNLMQK-GVA+ ++ , where dashes separate long helices from short, turn forming linkers. A gene coding for this protein was assembled from synthetic oligonucleotides, then fused to the maltose binding protein gene under the control of a tac promoter. The fusion protein was expressed in E. coli, purified and cleaved to yield maltose binding protein and our de novo protein, MB1. MB1 was found to be helical, to have the expected molecular weight (11 kDa) and the expected content (57%) of the essential amino acids M, T, K and L.

Evidence of increased diversity of methanogenic archaea with plant extract supplementation.

This study evaluated the effects of selected essential oils on archaeal communities using the ovine rumen model. Forty weaned Canadian Arcott ewes, fed with barley-based diet, were allotted to one of three essential oil supplementation treatments or a control (10 ewes per treatment) for 13 weeks. The treatments were cinnamaldehyde, garlic oil, juniper berry oil, and a control with no additive. Rumen content was sampled after slaughter and grouped by treatment by combining subsamples from each animal. DNA was extracted from the pooled samples and analyzed for methanogenic archaea using quantitative polymerase chain reaction, denaturing gradient gel electrophoresis, cloning, and sequencing. Our results suggest that the total copy number of archaeal 16S rRNA was not significantly affected by the treatments. The phylogenetic analysis indicated a trend toward an increased diversity of methanogenic archaea related to Methanosphaera stadtmanae, Methanobrevibacter smithii, and some uncultured groups with cinnamaldehyde, garlic, and juniper berry oil supplementation. The trends in the diversity of methanogenic archaea observed with the essential oil supplementation may have resulted from changes in associated protozoal species. Supplementation of ruminant diets with essential oils may alter the diversity of rumen methanogens without affecting the methanogenic capacity of the rumen.

Production of enterolysin A by rumen Enterococcus faecalis strain and occurrence of enlA homologues among ruminal Gram-positive cocci.

AIMS: Purification and partial characterization of an extracellular bacteriocin produced by the ruminal isolate Enterococcus faecalis II/1 and determine the frequency of occurrence of enterolysin A structural gene within the ruminal cocci. METHODS AND RESULTS: Bacteriocin produced by E. faecalis II/1 was purified to homogeneity. Purified bacteriocin exhibited a single band on sodium dodecylsulphate polyacrylamide gel electrophoresis with an apparent molecular weight of about 35 kDa. The amino acid sequence of the first 30 amino acids of purified bacteriocin was identical with the enterolysin A sequence. The DNA sequence of the nearly complete E. faecalis II/1 bacteriocin structural gene was identical to the enterolysin A gene sequence, confirming that this bacteriocin is identical to enterolysin A, a cell wall-degrading bacteriocin from E. faecalis LMG 2333. Enterolysin A structural genes were detected in approximately one-sixth of the Gram-positive ruminal cocci examined by PCR using primers targeting the enterolysin A structural gene. CONCLUSIONS: Bacteriocin produced by E. faecalis II/1 is identical to enterolysin A. Enterolysin A structural gene homologues are frequently encountered in rumen enterococcal and streptococcal bacterial strains. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first evidence of a large heat-labile bacteriocin produced by rumen E. faecalis strain, enlarging the number and types of known anti-bacterial proteins produced by rumen bacteria.

Maintenance of Laboratory strains of obligately anaerobic rumen bacteria.

Cultures of rumen bacteria can be stored at -20 degrees C for at least 2 years in a liquid medium containing 20% glycerol. Thawing, sampling, and refreezing do not significantly affect viability.

Isolation of Plasmid DNA from Butyrivibrio fibrisolvens.

A procedure based on successive precipitation of cell lysates with sodium dodecyl sulfate-NaCl and polyethylene glycol 6000 was developed which allows the isolation of plasmid DNA from Butyrivibrio fibrisolvens. A survey of B. fibrisolvens strains isolated from the bovine rumen showed that plasmids are a common feature of this species.

Isolation and properties of a (1,3)-beta-D-glucanase from Ruminococcus flavefaciens.

A (1,3)-beta-D-glucanase [(1,3)-beta-D-glucan-3-glucanohydrolase] from Ruminococcus flavefaciens grown on milled filter paper was purified 3,700-fold (19% yield) and appeared as a single major protein and activity band upon polyacrylamide gel electrophoresis. The enzyme did not hydrolyze 1,6-beta linkages (pustulan) or 1,3-beta linkages in glucans with frequent 1,6-beta-linkage branch points (scleroglucan). Curdlan and carboxymethylpachyman were hydrolyzed at 50% the rate of laminarin. The enzyme had a Km of 0.37 mg of laminarin per ml, a pH optimum of 6.8, and a temperature optimum of 55 degrees C and was stable to heating at 40 degrees C for 60 min. The molecular mass of the enzyme was estimated to be 26 kDa by gel filtration and 25 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme was completely inhibited by 1 mM Hg2+, Cu2+, and KMnO4, 75% by 1 mM Ag2+, and Ni2+, and 50% by 1 mM Mn2+ and Fe3+. In a 2-h incubation with laminaridextrins (seven to nine glucose units) or curdlan and excess enzyme, the major products were glucose (30 to 37%), laminaribiose (17 to 23%), laminaritriose (18 to 28%), laminaritetraose (13 to 21%), and small amounts of large laminarioligosaccharides. With laminarihexaose and laminaripentaose, the products were equal quantities of laminaribiose and glucose (30%) and laminaritetraose and laminaritriose (18 to 21%). Laminaribiose or laminaritriose were not hydrolyzed, indicating a requirement for at least four contiguous 1,3-beta-linked glucose units for enzyme activity. The enzyme appeared to have the properties of both an exo- and an endoglucanase.(ABSTRACT TRUNCATED AT 250 WORDS)

Changes in oxidation reduction potentials and volatile fatty acid production by rumen bacteria when methane synthesis is inhibited.

Rumen inoculum was cultured in specially designed fermenters that allowed simultaneous measurement of pH, oxidation-reduction potentials, and gas production. The cultures were maintained at pH 6.8 by addition of 1 M NaHCO3 and continuous infusion of artificial saliva. Gas flow was maintained at 20.0 ml/min with a stream of O2-free N2. Monensin at 7.0 micrograms/ml inhibited CH4 production 49% below control concentrations. The sodium salt of 2-bromoethanesulfonic acid added at an initial concentration of 5 x 10(-5) M inhibited CH4 production by 86% and increased H2 production from less than .5 mumol/min in the control to 24.5 mumol/min in the inhibited fermenter. The redox potentials in the control fermenter remained above -.20 V and did not change with the addition of monensin. Bromoethanesulfonic acid rapidly decreased the redox potential in the fermenter to -.33 V. Volatile fatty acid production was not significantly altered by the addition of 2-bromoethanesulfonic acid. The addition of monensin gave the expected decrease in acetate:propionate ratios, decreased acetate and butyrate production, and increased valerate (but not propionate) production.

Isolation and characterization of a bacteriocin (Butyrivibriocin AR10) from the ruminal anaerobe Butyrivibrio fibrisolvens AR10: evidence in support of the widespread occurrence of bacteriocin-like activity among ruminal isolates of B. fibrisolvens.

Forty-nine isolates of Butyrivibrio fibrisolvens and a single isolate of Butyrivibrio crossotus were screened for the production of inhibitors by a deferred plating procedure. Twenty-five isolates produced factors which, to various degrees, inhibited the growth of the other Butyrivibrio isolates. None of the inhibitory activity was due to bacteriophages. The inhibitory products from 18 of the producing strains were sensitive to protease digestion. Differences in the ranges of activity among the Butyrivibrio isolates and protease sensitivity profiles suggest that a number of different inhibitory compounds are produced. These findings suggest that the production of bacteriocin-like inhibitors may be a widespread characteristic throughout the genus Butyrivibrio. The bacteriocin-like activity from one isolate, B. fibrisolvens AR10, was purified and confirmed to reside in a single peptide. Crude bacteriocin extracts were prepared by ammonium sulfate and methanol precipitation of spent culture supernatants, followed by dialysis and high-speed centrifugation. The active component was isolated from the semicrude extract by reverse-phase chromatography. Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis confirmed that the peptide was purified to homogeneity, having an estimated molecular mass of approximately 4,000 Da. The N terminus of the peptide was blocked. A cyanogen bromide cleavage fragment of the native peptide yielded a sequence of 20 amino acids [(M)GIQLAPAXYQDIVNXVAAG]. No homology with previously reported bacteriocins was found. Butyrivibriocin AR10 represents the first bacteriocin isolated from a ruminal anaerobe.

Use of Congo red-polysaccharide interactions in enumeration and characterization of cellulolytic bacteria from the bovine rumen.

The interaction of the direct dye Congo red with intact beta-D-glucans provides the basis for a rapid and sensitive assay system for bacterial strains possessing beta-(1 leads to 4),(1 leads to 3)-D-glucanohydrolase, beta-(1 leads to 4)-D-glucanohydrolase, and beta-(1 leads to 3)-D-glucanohydrolase activities. A close correspondence was observed between cellulolytic activity and beta-(1 leads to 4)-D-glucanohydrolase and beta-(1 leads to 4),(1 leads to 3)-D-glucanohydrolase activities in isolates from the bovine rumen. Many of these isolates also possessed beta-(1 leads to 3)-D-glucanohydrolase activity, and this characteristic may have taxonomic significance.

Cloning and expression of a Bacteroides succinogenes mixed-linkage beta-glucanase (1,3-1,4-beta-D-glucan 4-glucanohydrolase) gene in Escherichia coli.

A pseudorandom genomic library of Bacteroides succinogenes DNA, cloned into pUC8 in Escherichia coli, was screened for beta-glucanase activity on 0.1% lichenan plates. Six high-activity clones, containing identical 5.2-kilobase inserts of B. succinogenes DNA, were obtained. The clones exhibited activity solely on beta-glucan substrates containing beta-(1----3)(1----4) linkages, thus manifesting a specific fibrolytic enzyme previously unrecognized in B. succinogenes. A subclone (pJI10) of the original insert (1.35 kilobases in size) expressed full beta-glucanase activity under control of its own promoter. The expression of beta-glucanase in pJI10 appeared subject to catabolite regulation by glucose. Detailed analysis of enzyme activity in the parental and deleted derivatives, subcloned into pUC18 and pUC19, suggested that the apparent glucose repression was an artifact arising as a consequence of interactions with the lac transcriptional unit in the plasmid vector.

DNA sequence of a Fibrobacter succinogenes mixed-linkage beta-glucanase (1,3-1,4-beta-D-glucan 4-glucanohydrolase) gene.

The DNA sequence of a mixed-linkage beta-glucanase (1,3-1,4-beta-D-glucan 4-glucanohydrolase [EC 3.2.1.73]) gene from Fibrobacter succinogenes cloned in Escherichia coli was determined. The general features of this gene are very similar to the consensus features for other gram-negative bacterial genes. The gene product was processed for export in E. coli. There is a high level of sequence homology between the structure of this glucanase and the structure of a mixed-linkage beta-glucanase from Bacillus subtilis. The nonhomologous region of the amino acid sequence includes a serine-rich region containing five repeats of the sequence Pro-Xxx-Ser-Ser-Ser-Ser-(Ala or Val) which may be functionally related to the serine-rich region observed in Pseudomonas fluorescens cellulase and the serine- and/or threonine-rich regions observed in Cellulomonas fimi endoglucanase and exoglucanase, in Clostridium thermocellum endoglucanases A and B, and in Trichoderma reesei cellobiohydrolase I, cellobiohydrolase II, and endoglucanase I.