Construction, analysis, and beta-glucanase screening of a bacterial artificial chromosome library from the large-bowel microbiota of mice.
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 beta-glucanase activity was conducted using lichenin agar plates and Congo red solution. Three clones with beta-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.
Walter
Jens
J
Department of Microbiology and Immunology, University of Otago, PO Box 56, Dunedin, New Zealand.
Mangold
Marco
M
Tannock
Gerald W
GW
D000595:Amino Acid Sequence; D000818:Animals; D022202:Chromosomes, Artificial, Bacterial; D015698:Genomic Library; D006026:Glycoside Hydrolases / Q000235:genetics*; D007420:Intestine, Large / Q000382:microbiology*; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D016366:Open Reading Frames; D010802:Phylogeny
D006026:Glycoside Hydrolases; C028020:licheninase