Abstract
BackgroundIntegrons are genetic elements capable of the acquisition, rearrangement and expression of genes contained in gene cassettes. Gene cassettes generally consist of a promoterless gene associated with a recombination site known as a 59-base element (59-be). Multiple insertion events can lead to the assembly of large integron-associated cassette arrays. The most striking examples are found in Vibrio, where such cassette arrays are widespread and can range from 30 kb to 150 kb. Besides those found in completely sequenced genomes, no such array has yet been recovered in its entirety. We describe an approach to systematically isolate, sequence and annotate large integron gene cassette arrays from bacterial strains.ResultsThe complete Vibrio sp. DAT722 integron cassette array was determined through the streamlined approach described here. To place it in an evolutionary context, we compare the DAT722 array to known vibrio arrays and performed phylogenetic analyses for all of its components (integrase, 59-be sites, gene cassette encoded genes). It differs extensively in terms of genomic context as well as gene cassette content and organization. The phylogenetic tree of the 59-be sites collectively found in the Vibrio gene cassette pool suggests frequent transfer of cassettes within and between Vibrio species, with slower transfer rates between more phylogenetically distant relatives. We also identify multiple cases where non-integron chromosomal genes seem to have been assembled into gene cassettes and others where cassettes have been inserted into chromosomal locations outside integrons.ConclusionOur systematic approach greatly facilitates the isolation and annotation of large integrons gene cassette arrays. Comparative analysis of the Vibrio sp. DAT722 integron obtained through this approach to those found in other vibrios confirms the role of this genetic element in promoting lateral gene transfer and suggests a high rate of gene gain/loss relative to most other loci on vibrio chromosomes. We identify a relationship between the phylogenetic distance separating two species and the rate at which they exchange gene cassettes, interactions between the non-mobile portion of bacterial genomes and the vibrio gene cassette pool as well as intragenomic translocation events of integrons in vibrios.
Highlights
Integrons are genetic elements capable of the acquisition, rearrangement and expression of genes contained in gene cassettes
By making genomic libraries and screening individual clones by cassette PCR or with PCR primers targeting the intI gene, we can recover large inserts that comprise exclusively, or in large part, of contiguous arrays of mobile gene cassettes. We demonstrate this principle on an environmental strain of a Vibrio sp. from an aquaculture facility in Darwin, Australia
We show that using a fosmid vector to construct a genomic library (480 clones), we can recover the whole 82 kb integron array in four PCR screening rounds, the first of which identifies a clone bearing the intI
Summary
Integrons are genetic elements capable of the acquisition, rearrangement and expression of genes contained in gene cassettes. Multiple insertion events can lead to the assembly of large integron-associated cassette arrays. The targets for SSR are gene cassettes [3] These are independently mobilizable units of DNA, usually composed of a single gene bound by a recombination site, designated a 59-base element (59-be) or alternatively attC. The DNA integrase catalysing SSR, IntI, is encoded by the integron This integrase recognises two families of recombination sites. The first of these is the cassette associated 59-be [4] and the second is a site contained within the integron designated attI. Integrons are normally associated with arrays comprising multiple cassettes, which in some species of Vibrio can number well in excess of a hundred [7]. The integron and gene cassettes comprise both a gene capture and gene expression system
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