Enhancement of gene detection frequencies by combining DNA-based stable-isotope probing with the construction of metagenomic DNA libraries

DNA-based stable-isotope probing (SIP) using C-13-labeled growth substrates as bait is a powerful tool for the selective DNA isolation from microorganisms that are actively involved in consuming these substrates. To enhance the detection frequency of target genes in screens for new natural products, we have combined for the first time DNA-based SIP with the construction of metagenomic libraries. To isolate genes encoding coenzyme B-12-dependent glycerol dehydratases an enrichment of glycerol-fermenting microorganisms from a sediment sample of the Wadden Sea was performed by using glycerol-C-13(3) as sole carbon source. Subsequently, the C-13-labeled DNA was separated from the naturally abundant C-12-DNA by density centrifugation, and used for library generation. Screening of the constructed libraries for the target genes revealed that the gene detection frequencies employing DNA-based SIP for enrichment of genomes harboring dehydratase genes were 2.1- to 3.8-fold higher than those recorded by using a traditional step with unlabeled glycerol for enrichment.