Potential role of group IIC-attC introns 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.

FIG. 1.

Schematic diagrams of group IIC-attC introns and their surrounding sequences (A) in mobile integrons, (B) in chromosomal integron cassette arrays, and (C) in attC sites without any nearby integrase homolog. The gray arrows indicate open reading frames; the cross-hatched arrows indicate intron ORFs; and the gray boxes indicate attC sites. The information for E. cloacae was obtained from reference (asterisk).

FIG. 2.

(A) Two-plasmid mobility assay with E. coli. E₁ and E₂, exon sequences; Plac, lac promoter; Ap^R and Cm^R, ampicillin and chloramphenicol resistance genes, respectively; pMB1 and p15A, plasmid replication origins. “Intron*.for” indicates one of four intron-specific primers, NeI1.for, GsI2.for, SbI1.for, or SmaI2.for (Table 2). (B) In vivo mobility of group IIC-attC introns into various attC site sequences. Agarose gels contained the PCR products obtained using PACYC-5′ and one of the four intron-specific primers. These primers amplify the 3′ intron-exon integration junction and are specific for homing into the bottom strand of the cloned attC sites. +, positive PCR result for integration; −, negative PCR result for integration. Secondary structures of the bottom strand sequence (homing strand) of all attC sites tested are shown in Fig. S2 in the supplemental material.

FIG. 3.

S.ma.I2 intron mobility into putative transcriptional terminators (Term sites) of various resistance genes. (A) Agarose gels containing the PCR products obtained using primer pair “a” or “b” in order to detect insertion of S.ma.I2 into putative IBS1-IBS3 motifs in bottom strand (bs) or top strand (ts) sequences, respectively. (B) S.ma.I2 homing site for each Term site based on the sequencing results. The asterisks indicate the intron insertion sites between the four-base IBS1 motif and the one-base IBS3 motif (underlined). Lowercase letters indicate the gene stop codons. Vienna outputs (above each sequence) indicate base pairing from a potential stem-loop structure. A period indicates sequence identity with the wild type. Secondary structures of the homing strand are shown in Fig. S4 in the supplemental material. WT, wild type; mut., mutant.

FIG. 4.

RT activity of purified group IIC-attC IEPs. Quantitative RNA-dependent (RT) and DNA-dependent activities of purified MBP-IEPs were assayed using poly(rA)-oligo(dT)_12-18 and activated calf thymus DNA substrates, respectively. Assays were performed in triplicate, and the error bars indicate standard deviations. Purified MBP was used as a negative control. The commercial Superscript III (SSCIII) and Biotools DNA polymerase enzymes were used as positive controls for the RT and DNA-dependent DNA polymerase (pol.) activities, respectively.

FIG. 5.

Model for integron cassette neoformation mediated by a group IIC-attC intron, adapted from a previously suggested mechanism (4). The proposed model involves two independent homing events placing one intron copy immediately downstream of a gene (ORF) and another intron copy in an isolated attC site (step 1). The next step involves recombination, possibly RecA mediated, between the two introns (step 2). The next event involves splicing of the intron (step 3), followed by reverse transcription using an unknown primer and the gene-attC RNA as the template (step 4). The final step involves integration of the cassette into the integron by an integrase (step 5).