Phylogenetic screening of a bacterial, metagenomic library using homing endonuclease restriction and marker insertion.
Metagenomics provides access to the uncultured majority of the microbial world. The approaches employed in this field have, however, had limited success in linking functional genes to the taxonomic or phylogenetic origin of the organism they belong to. Here we present an efficient strategy to recover environmental DNA fragments that contain phylogenetic marker genes from metagenomic libraries. Our method involves the cleavage of 23S ribsosmal RNA (rRNA) genes within pooled library clones by the homing endonuclease I-CeuI followed by the insertion and selection of an antibiotic resistance cassette. This approach was applied to screen a library of 6500 fosmid clones derived from the microbial community associated with the sponge Cymbastela concentrica. Several fosmid clones were recovered after the screen and detailed phylogenetic and taxonomic assignment based on the rRNA gene showed that they belong to previously unknown organisms. In addition, compositional features of these fosmid clones were used to classify and taxonomically assign a dataset of environmental shotgun sequences. Our approach represents a valuable tool for the analysis of rapidly increasing, environmental DNA sequencing information.
Yung
Pui Yi
PY
Centre for Marine Bio-Innovation and School of Biotechnology and Biomolecular Sciences, University of New South Wales, 2035 NSW, Australia.
Burke
Catherine
C
Lewis
Matt
M
Egan
Suhelen
S
Kjelleberg
Staffan
S
Thomas
Torsten
T
D000818:Animals; D001419:Bacteria / Q000145:classification*; D001483:Base Sequence; D004262:DNA Restriction Enzymes; D004706:Endodeoxyribonucleases; D020459:Genes, rRNA; D005819:Genetic Markers; D016680:Genome, Bacterial; D015698:Genomic Library; D056186:Metagenomics / Q000379:methods*; D008969:Molecular Sequence Data; D010802:Phylogeny; D011161:Porifera / Q000382:microbiology; D012338:RNA, Ribosomal, 23S / Q000235:genetics
D005819:Genetic Markers; D012338:RNA, Ribosomal, 23S; D004706:Endodeoxyribonucleases; D004262:DNA Restriction Enzymes; C071132:endodeoxyribonuclease I-CeuI