Integrase-directed recovery of functional genes from genomic libraries

Large population sizes, rapid growth and 3.8 billion years of evolution firmly establish microorganisms as a major source of the planet's biological and genetic diversity. However, up to 99% of the micro- organisms in a given environment cannot be cul- tured. Culture-independent methods that directly access the genetic potential of an environmental sample can unveil new proteins with diverse func- tions, but the sequencing of random DNA can generate enormous amounts of extraneous data. Integrons are recombination systems that accumu- late open reading frames (gene cassettes), many of which code for functional proteins with enormous adaptive potential. Some integrons harbor hundreds of gene cassettes and evidence suggests that the gene cassette pool may be limitless in size. Accessing this genetic pool has been hampered since sequence-based techniques, such as hybrid- ization or PCR, often recover only partial genes or a small subset of those present in the sample. Here, a three-plasmid genetic strategy for the sequence-independent recovery of gene cassettes from genomic libraries is described and its use by retrieving functional gene cassettes from the chro- mosomal integron of Vibrio vulnificus ATCC 27562 is demonstrated. By manipulating the natural activity of integrons, we can gain access to the caches of functional genes amassed by these structures.