Potential Role of Group IIC-attCIntrons in Integron Cassette Formation

Abstract

Integrons are natural expression vectors in which gene cassettes are integrated downstream of a promoter region by a site-specific recombinase. Gene cassettes usually consist of a single gene followed by a recombination site designated attC. A major unanswered question is how a gene becomes associated with an attC site. Here, we investigate the potential role of a specific lineage of group IIC introns, named group IIC-attC, in cassette formation. Group IIC-attC introns preferentially target attC while retaining the ability to target transcriptional terminators. We show using a PCR-based mobility assay with Escherichia coli that the S.ma.I2 intron from the genome of a clinical isolate of Serratia marcescens can target both attC site and putative terminator motifs of resistance genes. Quantitative results showed that S.ma.I2 is more efficient in targeting various attC sequences than three group IIC-attC introns (54 to 64% sequence identity) from the genomes of environmental isolates. We also show that purified group IIC-attC intron-encoded reverse transcriptases have both RNA-dependent and DNA-dependent DNA polymerase activities in vitro. These data permit us to suggest a new model for gene cassette formation, in which a group IIC-attC intron targets separately a transcriptional terminator adjoining a gene and an isolated attC, joins the gene and the attC by homologous recombination, and then splices and reverse transcribes a gene-attC RNA template, leading to the formation of a cassette.Integrons and gene cassettes are considered important genetic elements in the evolution of multiresistance plasmids and transposons in gram-negative bacteria (39). Integrons can be categorized as mobile or chromosomal depending on their genomic location (i.e., on plasmids or on chromosomes). Mobile integrons and their cassettes are known to have a role in the dissemination of antibiotic resistance genes, whereas the majority of chromosomal integron cassettes include open reading frames (ORFs), most of whose products have no known function (21). Integrons are natural expression vectors composed of an integrase gene (intI) followed by a cassette promoter region and an integrase-specific recombination site (called an attI site) into which new cassettes are integrated (12). Gene cassettes usually consist of one promoterless gene associated with a distinct integrase-specific recombination site (called an attC site) that is located downstream of the gene. attC sites found in mobile integron cassettes exhibit little sequence similarity but contain a characteristic central palindromic sequence (11). attC sites can vary considerably in length (57 to 141 bp), and their sequence similarities are restricted primarily to the boundaries, which correspond to two pairs of conserved inverted repeats, 1L-2L and 2R-1R (40). Usually, a unique attC site is associated with one gene and is named by reference to the gene (e.g., the aminoglycoside resistance gene aadA1 and the attC aadA1 site). In contrast, attC sequences from chromosomal integrons with l...

The Integron: Adaptation On Demand

“…This is performed by a Group IIC-attC intron, a type of intron with affinity for palindromic sequences including attC sites, as well as retrotranscriptase activity (133). Unfortunately, direct evidence supporting this model remains scarce, and these elements have seldom been found within integrons (133,134). Although the rationale for the RNA origin of cassettes is interesting, some arguments against this theory are also solid.…”

“…In this model, the transcriptional terminator of the gene of interest is fused to an attC site through homologous recombination and further retrotranscribed into a DNA cassette. This is performed by a Group IIC-attC intron, a type of intron with affinity for palindromic sequences including attC sites, as well as retrotranscriptase activity (133). Unfortunately, direct evidence supporting this model remains scarce, and these elements have seldom been found within integrons (133,134).…”

“…Still, some major questions remain unanswered, such as the de novo creation of cassettes. In cassettes, the general absence of promoters, together with the paucity of pseudogenes or noncoding sequence, has been interpreted to be representative of an RNA origin (133). In this model, the transcriptional terminator of the gene of interest is fused to an attC site through homologous recombination and further retrotranscribed into a DNA cassette.…”

Group IIC Intron with an Unusual Target of Integration in Enterobacter cloacae

“…1B). The hypothesis for the role of group IIC-attC introns in gene cassette formation would not include the type of integration found for this novel group IIC intron (16). Analysis of these flanking exon sequences of the E.cl.GOC intron from the mobility assays showed that this intron recognizes a degenerate consensus homing site, 5=-RRYG T/TRR, which is at the 5= end of the 1R of most attC sites (top strand) (Fig.…”

“…It has been shown that group II introns that cannot carry out site-specific second-strand cleavage due to the lack of an En domain are still mobile, but the residual mobility shows a pronounced strand bias and dependence on replication (20,52). Recently, a potential role for group IIC-attC introns in gene cassette formation has been suggested (16), in which a group IIC-attC intron separately targets a transcriptional terminator adjoining a gene and an isolated attC, joins the gene and the attC by homologous recombination, and then splices and reverse transcribes a gene-attC RNA template, leading to the formation of the cassette.…”