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Applied and Environmental Microbiology, May 2005, p. 2347-2354, Vol. 71, No. 5
0099-2240/05/$08.00+0     doi:10.1128/AEM.71.5.2347-2354.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Construction, Analysis, and ß-Glucanase Screening of a Bacterial Artificial Chromosome Library from the Large-Bowel Microbiota of Mice{dagger}

Jens Walter,1 Marco Mangold,1 and Gerald W. Tannock1,2*

Department of Microbiology and Immunology, University of Otago, Dunedin, New Zealand,1 Agricultural, Food and Nutritional Science, University of Alberta, Edmonton, Alberta, Canada2

Received 27 September 2004/ Accepted 1 December 2004


   ABSTRACT
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
A metagenomic (community genomic) library consisting of 5,760 bacterial artificial chromosome clones was prepared in Escherichia coli DH10B from DNA extracted from the large-bowel microbiota of BALB/c mice. DNA inserts detected in 61 randomly chosen clones averaged 55 kbp (range, 8 to 150 kbp) in size. A functional screen of the library for ß-glucanase activity was conducted using lichenin agar plates and Congo red solution. Three clones with ß-glucanase activity were detected. The inserts of these three clones were sequenced and annotated. Open reading frames (ORF) that encoded putative proteins with identity to glucanolytic enzymes (lichenases and laminarinases) were detected by reference to databases. Other putative genes were detected, some of which might have a role in environmental sensing, nutrient acquisition, or coaggregation. The insert DNA from two clones probably originated from uncultivated bacteria because the ORF had low sequence identity with database entries, but the genes associated with the remaining clone resembled sequences reported in Bacteroides species.


   INTRODUCTION
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The microbiota of the distal digestive tract of monogastric animals is comprised mainly of obligately anaerobic bacteria, many phylotypes of which have not yet been cultivated under laboratory conditions (reviewed in reference 39). The largest bacterial community in terms of quantity and biodiversity is located in the cecum and colon (the large bowel). The carbon and energy requirements of the large-bowel microbiota are met from two sources: complex carbohydrates, proteins and fats that have escaped digestion by host processes in the small bowel, and the components of host secretions (reviewed in references 21 and 40). Important in these respects are plant polysaccharides such as cellulose, arabinoxylans, resistant starch, and glucans derived from the diet of the host, and large, heavily glycosylated glycoproteins (mucins) that are the principle components of mucus which is produced abundantly by goblet cells in the intestinal mucosa (13, 26). The composition of the large-bowel microbiota of monogastric animals such as humans, mice, chickens, and pigs is now well known through the application of nucleic acid-based methods of analysis (18, 19, 30, 38). Yet these largely phylogenetic studies, based on hypervariable nucleotide base sequences of 16S rRNA genes, while demonstrating that in the case of the murine microbiota, for example, 75% of 16S rRNA gene sequences were novel, have provided little information about the processes that shape and sustain the large bowel ecosystem.

Metagenomics is a facet of synecology in which a microbial community is studied in terms of its collective genomes, rather than focusing on the diversity of species and their individual genomes (28). The metagenomic approach entails the cloning and sequencing of large fragments of community genomic DNA that have been extracted directly from the ecosystem of choice. This abrogates the problem of noncultivability of the majority of the community inhabitants. The cloned DNA fragments are large enough to encode operons and therefore might result in the expression, by a surrogate bacterial host, of several enzymes that could catalyze a relatively complex metabolic process, including the synthesis of secondary metabolites (34). Bacterial artificial chromosome (BAC) vectors are especially suitable in the preparation of metagenomic clone libraries because they stably maintain large DNA inserts (greater than 100 kbp) in Escherichia coli (3). The replication of the BAC vector in the E. coli host is strictly controlled so that one to two copies of the cloned DNA are present per cell. Metagenomic libraries derived from microbial community genomes can be screened for heterologous phenotypic traits that include enzymes and other proteins that are essential to the functioning of the ecosystem (27). Hence they provide a means of assessing details of community biochemistry and genetics and of accessing nature's bounty in the form of bacterial enzymes and other bioactive substances.

We investigated the potential use of metagenomics in the study of the large bowel ecosystem of mice. These animals were chosen as experimental subjects for three reasons: the unique phylogenetic composition of their large-bowel microbiota has been described previously (30); the impact of members of the microbiota on the physiology of the murine host is well studied, even to the molecular details in some cases (11, 12); and the interactions that occur between the gut microbiota and the murine host impinge on biomedical research in which laboratory mice fill an essential role (10). Despite the importance of the gut microbiota in the development of host physiology, the metabolic pathway structure of this community is unknown. We do not know the composition of the consortia that are responsible for the metabolism of complex carbohydrate and glycoprotein substrates available to the gut bacteria.

This article describes the construction of a BAC library using DNA extracted directly from the large-bowel microbiota of BALB/c mice. The library, consisting of 5,760 clones with an average insert size of 55 kbp, was screened for the production of enzymes that degrade lichenin, a 1,3-1,4-ß-glucan. Three clones with this property were detected, sequenced, and annotated. These results demonstrate that metagenomics could provide an important tool in investigations of the gut microbiota from both microbial ecological and biotechnological perspectives.


   MATERIALS AND METHODS
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Bacterial strains, plasmids, media, and growth conditions.
E. coli DH10B was grown in Luria-Bertani (LB) medium at 37°C with agitation. E. coli DH10B cultures containing the BAC vector pIndigoBAC-5 (Epicentre, WI) were grown in LB medium containing 12.5 µg of chloramphenicol per ml.

DNA extraction from large-bowel contents of mice.
The use of mice was approved by the Animal Ethics Committee of the University of Otago (approval number 63/01). All centrifugations were performed at 4°C. Combined cecal and colon contents from seven BALB/c mice were diluted 1:4 in ice-cold phosphate-buffered saline (PBS; pH 7.4), vortexed vigorously, and then centrifuged for 3 min at 9,000 x g. The pellet was washed three times in the same volume of PBS. The pellet was suspended in the initial volume of PBS, and the cell suspension was centrifuged at 150 x g for 3 min to remove debris. The supernatant containing the bacterial cells was centrifuged again at 150 x g for 5 min. The supernatant was collected and then centrifuged at 9,000 x g for 3 min to recover the bacterial cells. The bacteria were washed three times with 1 vol (initial volume of PBS) of STE buffer (0.2 M NaCl, 100 mM EDTA, 10 mM Tris; pH 8.0) and resuspended in 0.125 vol of STE buffer. The cell suspension was mixed with the same volume of 1.5% low-melting-point agarose (FMC Bioproducts, ME) in STE buffer (autoclaved and cooled to 40°C), mixed, and used to make agarose plugs (1- by 9- by 20-mm molds; Bio-Rad Laboratories, CA). The plugs were stored at 4°C for 1 h and then incubated overnight in 2 ml of lysis buffer (STE buffer containing 0.2% sodium deoxycholate, 1% sodium lauryl sarcosine, 5 mg of lysozyme per ml, and 80 µg of mutanolysin per ml) at 37°C in a gently shaking water bath. This DNA preparative method protects high-molecular-mass nucleic acid from shearing. The agarose plugs were then transferred to 2 ml of ESP buffer (1 mg of proteinase K per ml, 1% sodium lauryl sarcosine, 50 mM Tris, 0.5 M EDTA; pH 8.0) and incubated for 24 h at 55°C. The plugs were transferred to fresh ESP buffer and incubated for another 24 h. The plugs were then transferred to 5 ml of 1 mM phenylmethylsulfonyl fluoride solution in TE50 buffer (50 mM EDTA, 10 mM Tris; pH 8.0) and left for 2 h at room temperature. The plugs were washed four times in 5 ml of ice-cold TE50 buffer and were then stored in this buffer.

Preparation, size selection, and concentration of DNA.
Agarose plugs were treated ("pre-electrophoresed") as described by Osoegawa et al. (22). Briefly, the agarose plugs were transferred to 20 ml of 0.5x TBE buffer (31) and held on ice for 3 to 4 h. The plugs were then inserted into a preparative slot in a pulsed-field electrophoretic gel (PFGE with 1% pulsed field certified agarose; Bio-Rad). PFGE was conducted using 0.5x TBE and a Bio-Rad CHEF apparatus for 10 h (14°C) at 4 V/cm, an angle of 120°, and a switch time of 5 s. After this treatment, the plugs were removed from the slot and stored in TE50 buffer.

For partial digestion of the DNA, the agarose plugs were transferred to 10 ml of TE buffer (10 mM Tris, 1 mM EDTA; pH 8.0) containing 50 mM NaCl and stored overnight on ice. The buffer was replaced by 10 ml of restriction buffer base (RBB; 50 mM NaCl, 10 mM MgCl, 10 mM Tris; pH 8.0), and the plugs were held for 3 h on ice. Agarose plugs were cut into six pieces (approximately 4 by 6 mm), and each piece was placed in 100 µl of restriction buffer (RBB containing 100 µg of bovine serum albumin per ml and 4 mM spermidine) and held for 30 min on ice. The appropriate amount of HindIII (determined in preliminary experiments using different concentrations of the enzyme) was added, and the samples were placed for 1 h on ice and then incubated at 37°C for 30 min. The reaction was stopped by adding 20 µl of 0.5 M EDTA (pH 8.0) and cooling on ice. After 30 min, the buffer was removed and 1 ml of ice-cold TE50 buffer was added and placed on ice for a further 30 min. This step was repeated twice. The plugs were stored at 4°C in TE50 buffer. DNA was size selected by two rounds of PFGE in 0.5x TBE buffer. The first electrophoretic run was performed for 18 h (14°C) at 6 V/cm, an angle of 120°, and a switch time of 0.1 to 50 s. A gel slice containing DNA of 100 to 150 kbp (recognized by reference to New England Biolabs low-range PFGE marker) was cut from the gel, embedded in a fresh agarose gel, and electrophoresed a second time for 20 h (14°C) at 6 V/cm, an angle of 120°, and a switch time of 15 s. To recover the DNA, the gel slice containing the selected 100- to 150-kbp DNA was electroeluted as described by Strong et al. (37) but with 0.5x TBE buffer. To remove borate ions (which inhibit T4 DNA ligase), the dialysis bags containing the gel slice were dialyzed three times against 500 ml of TE buffer for at least 2 h. The DNA was concentrated approximately 10-fold by drop dialysis against 30% (wt/vol) polyethylene glycol 8000 in 0.5x TE using a membrane (VSWP02500, 0.025-µm pores, white VSWP, 25 mm; Millipore, Ireland) floating on the polyethylene glycol solution (22).

Library construction.
The size-selected DNA was cloned in pIndigoBAC-5 (HindIII cloning-ready; Epicentre) using T4 DNA ligase (Roche Diagnostics, Germany). Vector DNA (1 µl) was mixed with 84 µl of insert DNA and heated at 55°C for 5 min. After cooling to room temperature for 10 min, 5 µl (5U) of T4 DNA ligase and 10 µl of 10x ligase buffer were added and the solution was incubated for 18 h at 14°C. Ligation reactions were heat inactivated (65°C for 15 min) and desalted on agarose cones (1). Five µl of the DNA solution was used to electrotransform E. coli DH10B as described by Sheng et al. (35), and the bacteria were plated on LB agar containing 12.5 µg of chloramphenicol per ml plus X-Gal (5-bromo-4-chloro-3-indolyl-ß-D-galactopyranoside) and IPTG (isopropyl-ß-D-thiogalactopyranoside) (31). White and pale blue colonies (5,760) were picked and stored in 60 96-well microtiter plates as described by Zimmer and Verrinder Gibbins (47). Four electrotransformations were sufficient to derive the complete library.

Determination of cloned insert sizes.
BAC DNA was extracted from E. coli clones using an alkaline lysis method described by Osoegawa et al. (22). BAC plasmids were digested with NotI (Roche), and PFGE was performed for 15 h (12°C) at 6 V/cm, an angle of 120°, and a switch time of 5 to 15 s.

Screening the library for ß-glucanase genes.
The clone library was replicated on LB agar plates containing 0.05% lichenin (Sigma, MO) using a 48-pin replicator (Nunc, Denmark) and incubated aerobically at 37°C for 3 days. Each plate was flooded with 1 ml of Congo red solution (0.1% Congo red in 20% ethanol). Clones that were surrounded by a yellow halo were picked from the original library and retested prior to BAC plasmid extraction. Purified plasmids were used to electrotransform E. coli DH10B, and the ß-glucanase phenotype was again checked to ensure that the cloned DNA was responsible for the enzymatic activity.

Cloned insert sequences and annotation.
Sequencing of BAC plasmids was performed by Macrogen, Korea. Open reading frames (ORF) were detected using the GeneMark.hmm program (20) and the ORF search tool provided by the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/gorf/gorf.html). Homology searches were carried out against the GenBank database using the BLASTX and BLASTP algorithms (http://www.ncbi.nlm.nih.gov/BLAST). The start of each ORF was confirmed manually by the identification of putative ribosomal binding sites and, in some cases, the presence of a signal peptide (5). The InterPro database was utilized for protein analysis (http://www.ebi.ac.uk/InterProScan/). Sequences were aligned using the Clustal method of Megalign software of the GeneJockey program (BioSoft, United Kingdom) or with the ClustalX (1.83) program (41). Phylogenetic protein analysis was performed by parsimony using PAUP 4.0 (Sinauer Associates, MA).

Nucleotide sequence accession numbers.
The nucleotide sequences of genes reported in this paper have been deposited in the GenBank database (AY766184, AY766185, AY766186).


   RESULTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Preparation of a BAC library of the large-bowel microbiota.
A BAC library was prepared from combined cecal and colonic contents from BALB/c mice. The library consisted of 5,760 clones stored in 60 microtiter plates at –80°C. Restriction endonuclease analysis (NotI) of 61 randomly chosen clones showed that 95% of the clones contained inserts between 8 and 150 kbp in size, with an average of 55 kbp (examples are shown in Fig. 1). Each BAC restriction pattern was unique, indicating that a variety of different DNA fragments had been cloned. Several of the clones had multiple DNA fragments in NotI digests, which indicated that DNA from phylotypes with a high mol% G+C content had been cloned. Assuming a bacterial genome equivalent to be 3 Mbp, we have covered an estimated 100 bacterial genomes in our library.



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  		FIG. 1. PFGE of randomly selected BAC clones digested with NotI. Lanes: 1 through 16, random clones; P, pIndigoBAC-5; M, low-range PFGE marker (New England Biolabs).

 
ß-Glucanase-secreting clones.
The library was screened for the expression and secretion of ß-glucanases that degraded lichenin. Three clones (14A, 31B, and 54B) were detected. Clones 14A and 31B had more activity than 54B (larger zones on lichenin agar plates) (Fig. 2). BAC were purified from these clones and were used to electrotransform E. coli DH10B. The transformants had ß-glucanase activity, which confirmed that this phenotype was encoded by the BAC insert. Restriction endonuclease analysis showed that the three clones contained different-sized inserts with different restriction patterns.



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  		FIG. 2. Detection of ß-glucanase activity on a lichenin agar plate that had been flooded with Congo red solution. E. coli DH10B containing pIndigoBAC-5 (vector-only control) is also shown.

 
Sequence analysis of clones 14A, 31B, and 54B.
The cloned inserts were sequenced and annotated to discover the genomic information that they contained. The inserts were 9.6 kbp (14A), 42.7 kbp (31B), and 39.9 kbp (54B) in size, and sequence analysis revealed that each insert was distinctive and contained several ORF (Fig. 3).



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  		FIG. 3. Physicalmaps of BAC clone inserts. Arrows indicate the location and direction of transcription of the ORF. Shading of arrows indicates gene products with related functions. Further details of putative protein functions and GenBank accession numbers are given in the supplemental material (Tables S1 to S3).

 
The 14A sequence (average mol% G+C content of 42) contained seven ORF (Fig. 3) of which two were genes (genes 2 and 3) encoding proteins (Bgl14A1 and Bgl14A2) with similarity to known ß-glucanases (Table 1). The closest matches (amino acid sequence identities of about 50%) for five out of the seven genes, including the two ß-glucanases, were database entries from various Clostridium species.


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  		TABLE 1. Predicted genes in BAC clones

 
The 31B sequence (average mol% G+C content of 45.5) contained 29 ORF (Fig. 3). One gene (gene 5) encoded a putative ß-glucanase (Bgl31B). The majority of ORF-encoded proteins had high sequence similarity to putative proteins of Bacteroides thetaiotaomicron, with 10 proteins showing more than 80% amino acid sequence identity. However, a Bacteroides homologue could not be found for the putative ß-glucanase. The 31B sequence encoded several additional enzymes (Table 1) involved in carbohydrate hydrolysis such as ß-glucosidase (gene 9), {alpha}-glucosidase (gene 13), and {alpha}-amylase (gene20).

The 54B sequence (average mol% G+C content of 46.5) contained 32 ORF (Fig. 3). One gene (gene 31) encoded a putative ß-glucanase (Bgl54B). As shown in Table 1, the 54B ORF showed low similarities to entries in the database. Genes 6, 7, 10, and 13, located between nucleotides 7700 and 17100, encoded proteins likely involved in the mobilization of genetic elements and horizontal gene transfer. The insert of clone 54B also contained genes 23 (pad54B) and 24 (hbp54B) that encoded large surface proteins with signal peptides. These proteins resembled molecules involved in adherence or coaggregation. Pad54B had highest similarity to a hydrocephalus-inducing protein of Mus musculus that has been suggested to interact with the cytoskeleton (7). In addition, InterPro analysis of Pad54B revealed the presence of a filamin/ABP280 repeat. This domain is also present in the actin-binding cytoskeleton protein filamin. A PapD-like domain was also detected in the sequence of Pad54B. The PapD-like superfamily of periplasmic chaperones directs the assembly of over 30 diverse adhesive surface organelles that mediate the attachment of many different pathogenic bacteria to tissues. PapD, a prototype chaperone, is necessary for the assembly of P pili which contain the adhesin PapG that mediates the attachment of uropathogenic E. coli to kidney cells (2, 32). These observations suggest that proteins important in bacteria-eucaryotic cell interactions were encoded by the genomes of the gut bacteria. Hbp54B showed similarities to a hemin-binding protein of Porphyromonas gingivalis that has been shown to be involved in bacterial coaggregation (36). Thus, Pad54B and Hbp54B resembled proteins that have been associated with adherence or coaggregation and, if they have this function in the gut, could be important colonization determinants.

Comparative sequence analysis of clones 31B and 54B showed that the ß-glucanases were arranged in gene clusters that might be involved in the regulation of ß-glucanase gene expression. Genes lbs31B and lbs54B, predicted to encode a periplasmic ligand-binding sensor, were present in close proximity, and in the same orientation, to bgl31b and bgl54b. The Lbs proteins were 35% similar to each other, with the highest similarity occurring at the C-terminal end. InterPro analysis revealed that this region contained an effector domain of bipartite response regulators (helix-turn-helix motif). Bipartite response regulator proteins are involved in a two-component signal transduction system that detects and responds to environmental changes. Lbs31B and Lbs54B might therefore be involved in the regulation of the ß-glucanase genes. Additionally, Nbt31B and Nbt54B (this sequence was truncated) had high sequence similarity (65%) to each other and low similarity to putative outer membrane proteins involved in nutrient binding. Nbt31B contained a TonB-dependent receptor domain (pfam00593). In E. coli, TonB interacts with outer membrane receptor proteins that carry out the high-affinity binding and energy-dependent uptake of specific substrates (such as iron siderophores or vitamin B12) into the periplasm (24). A receptor like this has not been described for the uptake of ß-glucans, but the presence of a novel protein in association with ß-glucanases in two unique BAC clones suggested that Nbt31B and Nbt54B might have this function.

Characteristics and phylogeny of ß-glucanases.
Four ORF were detected that encoded proteins with similarities to known ß-glucanases that degrade lichenin (bgl14A1, bgl14A2, bgl31B, bgl54B) (Table 1). Amino acid sequence comparisons indicated that all four enzymes belonged to glycoside hydrolase family 16 (pfam00722). Three of the protein sequences contained a signal peptide, but Bgl14A2 did not. Bgl14A2 contained a CenC-like carbohydrate binding domain (pfam02018) (Fig. 4). When compared to the amino acid sequences of known ß-glucanases, the overall identities of the catalytic domain were relatively low (40 to 64%) but Bgl31B and Bgl54B were 75% identical (48% over the complete amino acid sequence). Signature motifs and catalytic residues were revealed by alignment of the catalytic domain sequences. As shown in Fig. 5, the catalytic domain of Bgl14A1 resembled that of lichenases, while the sequences of Bgl14A2, Bgl31B, and Bgl54B showed insertions in the active site that were characteristic of laminarinases (17). Both lichenases and laminarinases degrade lichenin (17, 48).



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  		FIG. 4. Diagrammatic representations of ß-glucanases with their functional domains. Catalytic domains are black, and the numbers indicate amino acid positions.

 


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  		FIG. 5. Comparison of amino acid sequences of active-site regions of ß-glucanases. Residues identified as catalytic amino acids in Bacillus macerans lichenase and Rhodothermus marinus laminarinase are indicated by arrows (17). lich, lichenase; lam, laminarinase. Boxes indicate conserved amino acids, and GenBank accession numbers are given on the right-hand side of the diagram.

 
The protein sequences of the four ß-glucanases were compared phylogenetically to known 1,3-1,4-ß-glucanases (lichenases) and 1,3(4)-ß-glucanases (laminarinases). Bowel microbiota ß-glucanases were only distantly related to enzymes of cultivated and noncultivated bacteria (Fig. 6).



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  		FIG. 6. Unrooted dendrogram constructed by parsimony based on the catalytic domains of bacterial ß-glucanases. Bootstrap values for 1,000 trials are shown at major internal nodes. The Bacillus group consists of sequences from B. subtilis (CAA86922and BAA00405, B. amyloliquefaciens (AAO43052and A29091), B. licheniformis (AAQ67340, and three sequences of uncultured soil bacteria (AAK50612 AAK610, AAK50614.

 

   DISCUSSION
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The construction and analysis of metagenomic (microbial community) libraries has provided knowledge of the genetics and biochemistry of noncultivable inhabitants of soil and marine communities (4, 9, 27). We have followed the same technological approach to begin to investigate the metabolic structure of the bowel community and to detect, by a functional screen, enzymes encoded by the genomes of the gut microbiota of mice. Phylogeny of bacterial communities can also be investigated by screening metagenomic libraries for 16S rRNA genes. We did not pursue this option because a catalogue of the murine gut microbiota derived from PCR-amplified 16S rRNA genes has been provided by Salzman et al. (30) and because Béjà et al. (4) have reported that the qualitative phylogenetic representation obtained with a BAC library was in general agreement with previous reports about the recovery of PCR-amplified rRNA genes from a marine community.

As in the case of soil, the digesta contains compounds that copurify with DNA and prevent the acquisition of high-quality nucleic acid required for the preparation of metagenomic libraries (25). Phenol-chloroform treatment, which has been shown to remove contaminants from DNA extracted from feces, is not suitable for the derivation of metagenomic libraries because the DNA is sheared as a result of the procedure. Instead, we found removal of contaminants could be achieved by a preliminary electrophoretic treatment described by Osoegawa et al. (22). The average insert size of our BAC library was 55 kbp, a value that is intermediate between the soil (44.5 kbp) and marine (80 kbp) libraries reported in the literature (4, 27).

In addition to the detection of genes encoding four novel ß-glucanases, sequence analysis of clone inserts revealed other genomic properties of gut bacteria that reflected adaptation to the bowel ecosystem. The ß-glucanase genes of clones 31B and 54B were arranged in similar gene clusters encoding proteins that might be involved in sensing and acquiring substrates and in regulating gene expression when these growth substrates are available. This is consistent with reports of the stringent regulation of pathways concerning the exploitation of complex growth substrates by Bacteroides thetaiotaomicron and Bifidobacterium longum. Analysis of the genome sequences of these bowel inhabitants showed that a considerable proportion of the genome encoded well-regulated pathways for the utilization of complex nutrients. These regulated pathways minimize energy expenditure and enable optimal rates of proliferation to be achieved (33, 44, 46). BAC insert proteins Lbs31B and Lbs54B had a C-terminal effector domain that is found in response regulators, and the proteins showed similarities, admittedly low, to hybrid two-component systems of Bacteroides thetaiotaomicron (20 and 22%, respectively). Hybrid two-component systems were reported to be common in the Bacteroides thetaiotaomicron genome and were linked to genes associated with the acquisition and processing of polysaccharides (45). While the amino acid sequences of the two proteins were very different to that of the hybrid two-component systems of Bacteroides, they had 35% identity to each other, suggesting that they have a similar function in the phylotypes from which they originated.

Pad54B and Hpd54B show features of bacterial adhesins or proteins that mediate bacterial coaggregation. The colonization of dental surfaces by bacterial cells is often facilitated by coaggregation, a phenomenon in which bacterial adhesins recognize receptors on the cell surface of other types of bacteria (15). Coaggregation, although not yet investigated with respect to the bowel ecosystem, could be important in the formation of bacterial consortia that degrade complex polymers. Nutritional interactions would be enhanced by the close proximity of the adherent bacterial partners.

Genes encoding rRNA were not detected in the clone inserts, so an accurate identification of the bacterial origin of the ß-glucanase genes cannot be given. The closest homologues encoded by the BAC inserts were of bacterial origin, and most of the genes of clone 31B had high homology to Bacteroides genes, suggesting that the insert DNA originated from a member of this genus. Laminarinase activity has been described for Bacteroides isolates from the human colon (29). Putative proteins encoded by clones 14A and 54B lacked close homologues, suggesting that the DNA originated from bacteria that have yet to be described.

1,3-1,4-ß-Glucans are components of plant cell walls, especially of those of the endosperm of cereals such as barley, rye, sorghum, rice, and wheat. The consumption of 1,3-1,4-ß-glucans has been reported to have an immunomodulating and cholesterol-lowering effect in humans and might contribute to the cardioprotective effect of oat fiber. They are therefore of considerable interest in human nutrition (6, 8, 16). 1,3-1,4-ß-Glucans are not hydrolyzed in the small bowels of monogastric animals and thus pass to the large bowel, where they become substrates for bacterial fermentation. The production of extracellular ß-glucanases, such as those that we have detected in our study, catalyze the hydrolysis of these dietary polymers. Doubtless some of the hydrolysis products, of various degrees of polymerization, become available as fermentable substrates to other members of the community, much as occurs in the rumen ecosystem when complex plant polymers are degraded (14, 43). Further investigations to detect the bacterial phylotypes that utilize the hydrolysis products will allow us to begin to construct the community structure (consortium) by which ß-glucans are degraded to organic acids in the large bowel.

The ß-glucanases detected in this study have biotechnological interest. The low nutritional value of barley for poultry is due to the inadequate hydrolysis of 1,3-1,4-ß-glucans in the chicken gut, which results in high viscosity digesta that limit nutrient uptake and decrease the growth rate of the birds. Addition of enzyme preparations containing ß-glucanases, or transgenic malt containing a 1,3-1,4-ß-glucanase, to barley-based diets has been reported to improve digestibility and to reduce the problem of "sticky droppings" (23, 42). ß-Glucanases derived and purified from gut metagenomic clones might be especially suitable for use in animal nutrition because they originate from bacteria highly adapted to life in the bowel.

In our murine study, we have demonstrated the potential of BAC libraries to investigate the genetics and biochemistry of the gut microbiota. Our investigation provides a prototype that could be readily expanded to the gut microbiota of other animals, including humans. Metabolic pathway construction should reveal much about the functioning of gut ecosystems, and biotechnological mining of community genomes might provide valuable returns considering the biodiversity of the gut microbiota associated with animals that have adapted to life in diverse habitats. Gut microbes likely produce a wealth of novel substances, and metagenomics provides a means of appraising and obtaining them for biotechnological and pharmaceutical uses.


   ACKNOWLEDGMENTS
 
The support of the University of Otago Research Committee is gratefully acknowledged.


   FOOTNOTES
 
* Corresponding author. Mailing address: Department of Microbiology and Immunology, University of Otago, PO Box 56, Dunedin, New Zealand. Phone: 64 3 479 7713. Fax: 64 3 479 8540. E-mail: gerald.tannock{at}stonebow.otago.ac.nz. Back

{dagger} Supplemental material for this article may be found at http://aem.asm.org/. Back


   REFERENCES
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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Applied and Environmental Microbiology, May 2005, p. 2347-2354, Vol. 71, No. 5
0099-2240/05/$08.00+0     doi:10.1128/AEM.71.5.2347-2354.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



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