A possible role for group II introns in the formation of mobile gene cassettes in integrons

CECILIA QUIROGA; DANIELA CENTRÓN; PAUL H. ROY

Santa Fe, New Mexico, USA.

Congreso; Keystone Symposia. Transposon and other recombination elements.; 2002

Integrons are elements that use an integron integrase, a tyrosine recombinase, to incorporate gene cassettes into a specific attI site.  Cassettes usually consist of a single, promoterless gene accompanied by an attC site, a palindromic sequence of 60-140 bp.  The integrase integrates and excises cassettes by attI x attC and attC x attC recombination. In plasmid-borne integrons, where cassettes are mostly antibiotic resistance genes, analysis indicates that the structural genes and their attC sites have evolved separately before being assembled. A major unanswered question is how structural genes become associated with attC sites to form cassettes. We have found a group II intron, Smtr, inserted at the structural gene-attC site junction in a class 1 integron from a Serratia marcescens plasmid.  Related bacterial class C group II introns occur at structural gene-attC site junctions in Pseudomonas, Acinetobacter, and Nitrosomonas. We are investigating how group II introns may capture attC sites (possibly by retrohoming to the left end of attC sites, which resembles the target site of the L. lactis intron) and structural genes (possibly by the less specific mechanism of ectopic insertion) and recombine to assemble them.  Relative to the integron, the intron Smtr is “upside down” in that the attC site is exon 1 and the structural gene exon 2. A regulatory region present within the intron controls the expression of the intron-encoded protein (IEP) (a reverse transcriptase-maturase-endonuclease). We have cloned this region into a promoter-probe vector. The consensus sequence for the IEP promoter has been identified by the primer extension method. Surprisingly, cloning of the regulatory region revealed the existence of a second, stronger promoter directed outwardly toward exon 1, resulting in a strong expression of the downstream cassette.