Phylogeny-function analysis of (meta)genomic libraries: screening for expression of ribosomal RNA genes by large-insert library fluorescent in situ hybridization (LIL-FISH).
We assessed the utility of fluorescent in situ hybridization (FISH) in the screening of clone libraries of (meta)genomic or environmental DNA for the presence and expression of bacterial ribosomal RNA (rRNA) genes. To establish proof-of-principle, we constructed a fosmid-based library in Escherichia coli of large-sized genomic DNA fragments of the mycophagous soil bacterium Collimonas fungivorans, and hybridized 768 library clones with the Collimonas-specific fluorescent probe CTE998-1015. Critical to the success of this approach (which we refer to as large-insert library FISH or LIL-FISH) was the ability to induce fosmid copy number, the exponential growth status of library clones in the FISH assay and the use of a simple pooling strategy to reduce the number of hybridizations. Twelve out of 768 E. coli clones were suspected to harbour and express Collimonas 16S rRNA genes based on their hybridization to CTE998-1015. This was confirmed by the finding that all 12 clones were also identified in an independent polymerase chain reaction-based screening of the same 768 clones using a primer set for the specific detection of Collimonas 16S ribosomal DNA (rDNA). Fosmids isolated from these clones were grouped by restriction analysis into two distinct contigs, confirming that C. fungivorans harbours at least two 16S rRNA genes. For one contig, representing 1-2% of the genome, the nucleotide sequence was determined, providing us with a narrow but informative view of Collimonas genome structure and content.
Leveau
Johan H J
JH
Netherlands Institute of Ecology (NIOO-KNAW), Centre for Terrestrial Ecology, Boterhoeksestraat 48, 6666 GA Heteren, the Netherlands. j.leveau@nioo.knaw.nl
Gerards
Saskia
S
de Boer
Wietse
W
van Veen
Johannes A
JA
D001483:Base Sequence; D015139:Blotting, Southern; D004777:Environment; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015723:Gene Library; D016680:Genome, Bacterial; D017404:In Situ Hybridization, Fluorescence / Q000379:methods*; D008969:Molecular Sequence Data; D042802:Oxalobacteraceae / Q000235:genetics* / Q000378:metabolism; D010802:Phylogeny; D016133:Polymerase Chain Reaction / Q000379:methods; D012336:RNA, Ribosomal, 16S / Q000235:genetics / Q000378:metabolism*; D015183:Restriction Mapping; D017422:Sequence Analysis, DNA
D012336:RNA, Ribosomal, 16S