Temporal change in the gut community of rats fed high amylose cornstarch is driven by endogenous urea rather than strictly on carbohydrate availability.

AIM: To examine change in the gut community of rats fed high amylose maize starch (HAMS). METHODS AND RESULTS: Rats were fed AIN93G diets containing HAMS (5% resistant starch type 2) or alphacell (control). HAMS increased faecal short-chain fatty acid output, faecal propionate and total bacteria output but reduced gut pH and blood urea concentrations compared with rats ingesting the control diet. Feeding HAMS resulted in a gut community dominated by four phylotypes homologous with Ruminococcus bromii, Bacteroides uniformis and with yet to be cultivated organisms aligning into the Family Porphyromonadaceae. Enrichment of phylotypes aligning within the Bacteroidetes occurred primarily in the caecum, whereas those homologous with R. bromii were found primarily in the faeces. HAMS altered community structure such that the phylum Bacteroidetes represented the dominant gut lineage and progressively reduced faecal community phylotype richness over the duration of feeding. CONCLUSIONS: Feeding HAMS resulted in a caecal and faecal community dominated by organisms that require ammonia as a primary nitrogen source. Gut ammonia derived from endogenous urea represents an important factor contributing to caecal community composition in addition to the ability to utilize HAMS. Increases in faecal propionate, rather than butyrate as is often observed following resistant starch feeding, reflected a gut community dominated by the Bacteroidetes. SIGNIFICANCE: Diet-mediated change is often viewed strictly in terms of available carbohydrate. Here, we have shown that ammonia derived from endogenous urea is an important factor contributing to gut community composition and structure in rats fed this substrate.

Continuous feeding of antimicrobial growth promoters to commercial swine during the growing/finishing phase does not modify faecal community erythromycin resistance or community structure.

AIMS: To investigate the effect of continuous feeding of antimicrobial growth promoters (tylosin or virginiamycin) on the swine faecal community. METHODS AND RESULTS: The study consisted of two separate on-farm feeding trials. Swine were fed rations containing tylosin (44 or 88 mg kg(-1) of feed) or virginiamycin (11 or 22 mg kg(-1) of feed) continuously over the growing/finishing phases. The temporal impact of continuous antimicrobial feeding on the faecal community was assessed and compared to nondosed control animals through anaerobic cultivation, the analysis of community 16S rRNA gene libraries and faecal volatile fatty acid content. Feeding either antimicrobial had no detectable effect on the faecal community. CONCLUSIONS: Erythromycin methylase genes encoding resistance to the macrolide-lincosamide-streptogramin B (MLS(B) ) antimicrobials are present at a high level within the faecal community of intensively raised swine. Continuous antimicrobial feeding over the entire growing/finishing phase had no effect on community erm-methylase gene copy numbers or faecal community structure. SIGNIFICANCE AND IMPACT OF THE STUDY: Antimicrobial growth promoters are believed to function by altering gut bacterial communities. However, widespread MLS(B) resistance within the faecal community of intensively raised swine likely negates any potential effects that these antimicrobials might have on altering the faecal community. These findings suggest that if AGP-mediated alterations to gut communities are an important mechanism for growth promotion, it is unlikely that these would be associated with the colonic community.

Understanding the acid tolerance response of bifidobacteria.

AIMS: To investigate the effect of pH on the viability and the acid tolerance response (ATR) of bifidobacteria. METHODS AND RESULTS: The impact of low pH on the viability of five species of bifidobacteria was examined under conditions of strict anaerobiosis. Although differences in the ability to resist the lethal effects of low pH were apparent among the species, cell viability could be improved by the provision of fermentable substrate during an acidic pH stress or through the use of stationary phase cells. While a stationary phase ATR was found to occur in two species of bifidobacteria, there was no adaptive response in exponential phase cells. Proteomic analysis of exponential phase Bifidobacterium longum subjected to a mild acid pre-exposure (pH 4.5, 2 h) prior to an acid challenge revealed a substantial loss in the total number of cellular proteins. In contrast, proteomic analysis of stationary phase cells revealed an increased abundance of proteins associated with the general stress response as well as the beta-subunit of the F(0)F(1)-ATPase, known to be important in bifidobacteria acid tolerance. CONCLUSION: Neither Bif. longum or Bifidobacterium breve possesses an inducible exponential phase ATR. SIGNIFICANCE AND IMPACT OF THE STUDY: These findings provide further insights into the impact of pH on the viability of bifidobacteria and may partially explain the loss in viability associated with their storage in acid foods.

In-feed administered sub-therapeutic chlortetracycline alters community composition and structure but not the abundance of community resistance determinants in the fecal flora of the rat.

The impact of continuous sub-therapeutic chlortetracycline on community structure, composition and abundance of tetracycline resistance genes in the rat fecal community was investigated. Rats were fed a standard diet containing chlortetracycline at 15 microg g(-1) diet for 28 days, followed by 30 microg g(-1) diet to completion of the study on day-56. These levels are similar to those administered to swine during the grow-out phase. Sub-therapeutic chlortetracycline affected the fecal community as determined through change in the cultivable anaerobic community and through molecular-based analyses including denaturing gradient gel electrophoresis profiles of the variable 2-3 region community 16S rRNA genes over time and through comparative sequence analysis of 16S rRNA gene community libraries. Significant decreases in fecal phylotype diversity occurred in response to sub-therapeutic chlortetracycline, although total bacterial output remained constant over the entire feeding trial. Chlortetracycline at 15 microg g(-1) diet resulted in significant change in community composition, but only modest change to the fecal community structure in terms of the distribution of individual phylotypes among the major fecal lineages. Chlortetracycline at 30 microg g(-1) diet significantly altered the distribution of phylotypes among the major fecal lineages shifting the overall community such that Gram-negative phylotypes aligning within the phylum Bacteroidetes became the dominant lineage (>60% of total community). While chlortetracycline impacted both fecal community structure and composition, there was no significant effect on the abundance of community tetracycline resistance genes [tet(Q), tet(W), tet(O)] or on the emergence of a new putative tetracycline resistance gene identified within the fecal community. While sub-therapeutic chlortetracycline provides sufficient selective pressure to significantly alter the fecal community, the primary outcome appears to be the development of a community which may have a higher inherent tolerance to sub-therapeutic levels of chlortetracycline rather than an overgrowth of the tetracycline resistant bacteria already present within the community.

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.

Dietary fructooligosaccharides alter the cultivable faecal population of rats but do not stimulate the growth of intestinal bifidobacteria.

The effect of fructans on the cultivable faecal community of Bio Breeding rats fed diets containing 5% (m/v) food-grade fructooligosaccharide (FOS) was investigated. Culturing of faecal material using chicory inulin as the sole carbohydrate source revealed the presence of a greater diversity of inulin-utilizing bacterial species in FOS-fed rats as compared with the control rats, although both contained species which effectively utilized inulin. The majority of cultivable inulin-utilizing species fell within the Clostridium coccoides group and Clostridium leptum subgroup, some of which were related to previously cultured butyrate-producing bacteria from the intestines of various animals. The impact of FOS on the growth of the indigenous bifidobacteria community and three inulin-utilizing isolates was assessed using real-time polymerase chain reaction. While dietary FOS was found to stimulate the growth of all three inulin-utilizing isolates, no growth stimulation of the indigenous bifidobacteria community occurred over the duration of the feeding trial.

Proteomic and microscopic analysis of biofilms formed by Listeria monocytogenes 568.

Biofilm formation may be important in the colonization of the food-processing environment by the food-borne pathogen Listeria monocytogenes. Listeria monocytogenes 568 formed adherent multicellular layers on a variety of test surfaces following growth at 37 degrees C with multiple transfers of the test surface into fresh medium. Microscopic examination of these adherent layers suggest that the cells were surrounded by extracellular material. The presence of a carbohydrate containing extracellular polymeric matrix was confirmed by labelling hydrated adherent layers with fluorescein-conjugated concanavalin A, indicating that these adherent layers are biofilms. To gain insight into the physiological state of cells in these biofilms, the proteomes from biofilm- and planktonic-grown cells from the same cultures were compared using 2-dimensional polyacrylamide gel electrophoresis. Nineteen proteins, which exhibited higher levels of expression in biofilm-grown cells, were successfully identified from the 2-D gels using a combination of MALDI-TOF and MS/MS. Proteins that were found to be more highly expressed in biofilm-grown cells were involved in stress response, envelope and protein synthesis, biosynthesis, energy generation, and regulatory functions. In biofilm-grown cells, many proteins in the pH range 4-6 ran as multiple spots arranged horizontally across the 2-D gels.

Flagellin from Listeria monocytogenes is glycosylated with beta-O-linked N-acetylglucosamine.

Glycan staining of purified flagellin from Listeria monocytogenes serotypes 1/2a, 1/2b, 1/2c, and 4b suggested that the flagellin protein from this organism is glycosylated. Mass spectrometry analysis demonstrated that the flagellin protein of L. monocytogenes is posttranslationally modified with O-linked N-acetylglucosamine (GlcNAc) at up to six sites/monomer. The sites of glycosylation are all located in the central, surface-exposed region of the protein monomer. Immunoblotting with a monoclonal antibody specific for beta-O-linked GlcNAc confirmed that the linkage was in the beta configuration, this residue being a posttranslational modification commonly observed in eukaryote nuclear and cytoplasmic proteins.

Culture-independent phylogenetic analysis of the faecal flora of the rat.

The dominant faecal flora of the rat was determined using randomly cloned 16S rDNA comparative sequence analysis. A total of 109 near full-length 16S rDNA clones were sequenced, representing 69 unique 16S rRNA phylotypes or operational taxonomic units (OTUs). Estimates of species richness indicated that approximately 338 species were present in the faeces, suggesting that only 20% of species were identified. Only two of 39 Gram-negative clones aligned with previously cultured species, the remainder fell into a separate lineage within the Bacteroides-Cytophaga phylum. Several clones within this new group were related to 16S rDNA sequences previously identified from mouse faeces. Lactobacilli were the most abundant Gram-positive species, representing 23% of the total clones but only 7% of OTUs. The remaining Gram-positive clones were distributed among the Clostridium coccoides group (9%), the Clostridium leptum subgroup (18%), and throughout the low GC Gram-positive bacteria (13%). The majority of OTUs (63/69 or 91%) were less than 97% homologous to previously cultured bacteria. Faecal samples were also cultured using a variety of anaerobic media. With the exception of the lactobacilli, the cultured isolates demonstrated low species diversity and poorly reflected the population, as defined through comparative sequence analysis.

Butyrivibriocin AR10, a new cyclic bacteriocin produced by the ruminal anaerobe Butyrivibrio fibrisolvens AR10: characterization of the gene and peptide.

The gene (bviA) encoding the ruminal bacteriocin butyrivibriocin AR10 was cloned from an EcoRI library by using an oligonucleotide probe based on a partial peptide sequence of the previously isolated peptide. The gene encoded an 80 amino acid prebacteriocin that demonstrated significant identity with the cyclic bacteriocin gassericin A. Negative ion time of flight mass spectroscopic analysis (ESI/MS) indicated a mass of 5981.5 Da for the isolated bacteriocin, a molecular mass that could not be generated by removal of a leader peptide alone. However, an N- to C-terminal cyclization of the predicted mature bacteriocin resulted in a peptide that conformed to the determined mass and charge characteristics. Northern blotting confirmed that expression of bviA mirrored the production of the bacteriocin in both liquid and solid media.

Adsorption, attachment and biofilm formation among isolates of Listeria monocytogenes using model conditions.

AIMS: To determine whether isolates of Listeria monocytogenes differ in their ability to adsorb and form biofilms on a food-grade stainless steel surface. METHODS AND RESULTS: Strains were assessed for their ability to adsorb to a test surface over a short time period. Although some differences in numbers of bound cells were found among the strains, there were no correlations between the degree of adsorption and either the serotype or source of the strain. The ability of each strain to form a biofilm when grown with the test surface was also assessed. With the exception of a single strain, all strains adhered as single cells and did not form biofilms. Significant differences in adherence levels were found among strains. Strains demonstrating enhanced attachment produced extracellular fibrils, whereas those which adhered poorly did not. A single strain formed a biofilm consisting of adhered single cells and aggregates of cells. CONCLUSIONS: Significant differences were found in the ability of various L. monocytogenes strains to attach to a test surface. In monoculture, the majority of strains did not form biofilms. SIGNIFICANCE AND IMPACT OF THE STUDY: Differences in attachment and biofilm formation among strains provide a basis to study these characteristics in L. monocytogenes.

Identification of a new plasmid-encoded sec-dependent bacteriocin produced by Listeria innocua 743.

Listeria innocua 743 produces an inhibitory activity demonstrating broad-spectrum inhibition of Listeria monocytogenes isolates. Gel-electrophoretic analysis of culture supernatants indicated that two inhibitors with different molecular weights were produced by this strain. Insertion of Tn917 into a 2.9 Kb plasmid (pHC743) generated mutants with either an impaired ability or a loss in ability to produce one of the inhibitors. Sequence analysis of the transposon insertion regions revealed the presence of two continuous open reading frames, the first encoding a new pediocin-like bacteriocin (lisA) and the second encoding a protein homologous with genes involved in immunity toward other bacteriocins (lisB). Translation of the bacteriocin gene (lisA) initiates from a noncanonical start codon and encodes a 71-amino-acid prebacteriocin which lacked the double glycine leader peptidase processing site common in other type II bacteriocins. Alignment of the sequence with the processed N termini of related bacteriocins suggests that the mature bacteriocin consists of 43 amino acids, with a predicted molecular mass of 4,484 Da. Mutants containing insertions into lisA were sensitive to the inhibitor, indicating that lisAB forms a single operon and that lisB represents the immunity protein. Cloning of an amplicon containing the lisAB operon into Escherichia coli resulted in expression and export of the bacteriocin. This finding confirms that the phenotype is dependent on the structural and immunity gene only and that export of this bacteriocin is sec dependent. This is the first confirmation of bacteriocin production in a Listeria spp., and it is of interest that this bacteriocin is closely related to the pediocin family of bacteriocins produced by lactic acid bacteria.

Construction of a promoter-rescue plasmid for Butyrivibrio fibrisolvens and its use in characterization of a flagellin promoter.

The Butyrivibrio fibrisolvens/Escherichia coli shuttle vector pBHerm has been modified to produce a plasmid (pBHE) that can be used for the identification and characterization of promoters in B. fibrisolvens. pBHE allows the insertion of a test promoter immediately upstream of a promoterless erythromycin resistance gene (ermAM). The efficacy of the pBHE plasmid in isolating and characterizing promoters was tested by inserting the flagellin gene (flaA) promoter from B. fibrisolvens OR77. Transcription of the ermAM gene from the flaA promoter was significantly higher than that observed when the ermAM gene was under the control of its own promoter. The flagelling gene of OR77 appears to be transcribed from two different promoters that produce transcripts initiating approximately 130 bp apart. Two mutant flaA promoter constructs, containing mutations in the -10 and -35 regions of either of the two putative promoter regions, showed drastic alterations in both the origin and amounts of the two transcripts produced. Mutations in either promoter affected transcription from both promoters, indicating that both regions contribute to gene expression.

Characterization of a major envelope protein from the rumen anaerobe Selenomonas ruminantium OB268.

Cell envelopes from the Gram-negative staining but phylogenetically Gram-positive rumen anaerobe Selenomonas ruminantium OB268 contained a major 42 kDa heat modifiable protein. A similarly sized protein was present in the envelopes of Selenomonas ruminantium D1 and Selenomonas infelix. Sodium dodecyl sulfate polyacrylamide gel electrophoresis of Triton X-100 extracted cell envelopes from S. ruminantium OB268 showed that they consisted primarily of the 42 kDa protein. Polyclonal antisera produced against these envelopes cross-reacted only with the 42 kDa major envelope proteins in both S. ruminantium D1 and S. infelix, indicating a conservation of antigenic structure among each of the major envelope proteins. The N-terminus of the 42 kDa S. ruminantium OB268 envelope protein shared significant homology with the S-layer (surface) protein from Thermus thermophilus, as well as additional envelope proteins containing the cell surface binding region known as a surface layer-like homologous (SLH) domain. Thin section analysis of Triton X-100 extracted envelopes demonstrated the presence of an outer bilayer over-laying the cell wall, and a regularly ordered array was visible following freeze-fracture etching through this bilayer. These findings suggest that the regularly ordered array may be composed of the 42 kDa major envelope protein. The 42 kDa protein has similarities with regularly ordered outer membrane proteins (rOMP) reported in certain Gram-negative and ancient eubacteria.

Evidence for production of a new lantibiotic (butyrivibriocin OR79A) by the ruminal anaerobe Butyrivibrio fibrisolvens OR79: characterization of the structural gene encoding butyrivibriocin OR79A.

The ruminal anaerobe Butyrivibrio fibrisolvens OR79 produces a bacteriocin-like activity demonstrating a very broad spectrum of activity. An inhibitor was isolated from spent culture fluid by a combination of ammonium sulfate and acidic precipitations, reverse-phase chromatography, and high-resolution gel filtration. N-terminal analysis of the isolated inhibitor yielded a 15-amino-acid sequence (G-N/Q-G/P-V-I-L-X-I-X-H-E-X-S-M-N). Two different amino acid residues were detected in the second and third positions from the N terminus, indicating the presence of two distinct peptides. A gene with significant homology to one combination of the determined N-terminal sequence was cloned, and expression of the gene was confirmed by Northern blotting. The gene (bvi79A) encoded a prepeptide of 47 amino acids and a mature peptide, butyrivibriocin OR79A, of 25 amino acids. Significant sequence homology was found between this peptide and previously reported lantibiotics containing the double-glycine leader peptidase processing site. Immediately downstream of bvi79A was a second, partial open reading frame encoding a peptide with significant homology to proteins which are believed to be involved in the synthesis of lanthionine residues. These findings indicate that the isolated inhibitory peptides represent new lantibiotics. Results from both total and N-terminal amino acid sequencing indicated that the second peptide was identical to butyrivibriocin OR79A except for amino acid substitutions in positions 2 and 3 of the mature lantibiotic. Only a single coding region was detected when restriction enzyme digests of total DNA were probed either with an oligonucleotide based on the 5' region of bvi79A or with degenerate oligonucleotides based on the predicted sequence of the second peptide.

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.

Are ruminal bacteria armed with bacteriocins?

The production of toxic compounds or antibiotics is a common component of intermicrobial competitive interactions, and many of these toxins have been adopted and adapted for the control of microbial populations. One class of these toxins, the bacteriocins, is a heterogeneous group of proteinaceous antibiotics that often display a high degree of target specificity, although many have a very wide spectrum of activity. To date, only limited information is available concerning the occurrence of bacteriocins among ruminal isolates or the sensitivity of ruminal microorganisms to exogenous bacteriocins. A survey of 50 strains of Butyrivibrio spp. isolated from a variety of sources (sheep, deer, and cattle) for bacteriocin production indicated a high incidence of bacteriocin-like activity (50%). Many of these inhibitory compounds appear to have a broad spectrum of activity, which suggests that bacteriocins may have a significant impact on both the competitive fitness of individual microbial strains within the rumen and on the overall structure of the microbial population within the rumen. Selected bacteriocins from lactic acid bacteria also were shown to have activity against Butyrivibrio spp. and may have application in ruminant systems. Bacteriocins may provide an alternative group of antibiotics for the manipulation of ruminal microbial populations. Bacteriocins have significant advantages over other antibiotics in target specificity, susceptibility to proteolytic digestion, possibility of genetic transfer and manipulation, and, in the case of some bacteriocins derived from lactic acid bacteria, a long history of safe use.

Contractile-tailed bacteriophages adsorb to Escherichia coli O128ab lipopolysaccharide that is altered by large plasmids to provide receptors and lipopolysaccharide heterogeneity within the serogroup.

The verotoxigenic Escherichia coli strain H.I.8 (originally O128:B12, now not typeable) contained a ColB+M plasmid and two morphologically identical temperate bacteriophages (H18A and H18B). Both phages were O128ab specific, using the lipopolysaccharide O side chains of susceptible clinical isolates as receptors. SDS polyacrylamide gel electrophoresis with silver staining of O128ab lipopolysaccharide revealed four distinct types of ladder with different interband spacings. No specificity was found between ladder type and sensitivity to either phage. One of the numerous large plasmids present in O128ab isolates was found to modify the structure of the lipopolysaccharide O side chains to provide phage receptors.

Molecular analysis of archael flagellins: similarity to the type IV pilin-transport superfamily widespread in bacteria.

Ultrastructural, biochemical and genetic evidence has shown that the flagella and flagellin proteins from members of the archaea are distinct from their bacterial counterparts. The most important evidence is the sequence dissimilarity between archael and bacterial flagellins. We report here similarity between archael flagellins and members of the bacterial type IV pilin-transport superfamily. In addition to sequence similarity, the archael flagellins and the type IV pilin-transport superfamily share an unusual signal sequence cleavage site and may have functional parallels. This relationship has important implications for the assembly and biogenesis of archael flagella.

A general method of isolating high molecular weight DNA from methanogenic archaea (archaebacteria).

High molecular weight DNA was readily isolated from all methanogens treated, as well as from thermophilic anaerobic eubacteria, by grinding cells frozen in liquid N2, prior to lysis with SDS. DNA can subsequently be purified by the usual phenol-chloroform extractions. The procedure yields DNA readily cut by restriction enzymes and suitable for oligonucleotide probing, as well as for mole percent G + C content determination by thermal denaturation. The method routinely yields DNA of high molecular weight and is an improvement over DNA isolation methods for many methanogens, which often involve an initial breakage of the cells in a French pressure cell.