Construction, Analysis, and β-Glucanase Screening of a Bacterial Artificial Chromosome Library from the Large-Bowel Microbiota of Mice

Abstract

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 ␤-glucanase activity was conducted using lichenin agar plates and Congo red solution. Three clones with ␤-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.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 fra...

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“…Bacteria are known for hydrolyzing these biomolecules by excreting (1) lichenases: Clostridium acetobutylicum; C. thermocellum, Bacillus spp., Bacillus macerens, B. circulans, B. brevis, R. flavefaciens; (2) lumiarinases: Rhodothermus marinus; C. thermocellum, Thermotoga neopolitana, T. maritima; (3) lichenin, 1,3-1,4-b glucan (b glucanases): Clostridium spp., Bacteriodes sp. [184].…”

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