Downloaded from www.microbiologyresearch.org by IP: 54.70.40.11 On: Tue, 04 Dec 2018 17:42:37 The S.ma.I2 class C group II intron inserts at integron attC sites Cecilia Quiroga, 1,2 Paul H. Roy 2,3 and Daniela Centro ´n 1 Correspondence Daniela Centro ´n dcentron@gmail.com 1 Departamento de Microbiologı ´a, Inmunologı ´a y Parasitologı ´a, Facultad de Medicina, Universidad de Buenos Aires, Buenos Aires, Argentina 2 Centre de Recherche en Infectiologie, Universite ´ Laval, Que ´ bec, Canada 3 De ´ partement de Biochimie et de Microbiologie, Universite ´ Laval, Que ´ bec, Canada Received 18 July 2007 Revised 3 January 2008 Accepted 13 January 2008 We previously found the class C S.ma.I2 group II (GII) intron in Serratia marcescens SCH909 inserted into the variable region of a class 1 integron within the attC site of the ant(299)-Ia gene cassette. Here, we demonstrate that this ant(299)-Ia :: S.ma.I2 gene cassette is a recombinationally active element despite the presence of the S.ma.I2 intron. In addition, S.ma.I2 is an active GII intron capable of performing self-splicing and invading specific target sites. Intron homing to a DNA target site is RecA-independent and recognizes the intron binding site (IBS)1 and IBS3 regions, formed by the 59 TTGTT 39 consensus sequence located within the inverse core site of attC integrons. Our results also indicate that the process for S.ma.I2 intron mobilization involves a secondary structure provided by the folding of the complete attC site. Moreover, phylogenetic analysis of the class C GII introns showed a clear divergent clade formed by introns that insert within specific sites usually associated with lateral gene transfer. INTRODUCTION Group II (GII) introns are mobile genetic units that have recently been found to be widespread in bacterial genomes and are mostly inserted within other mobile elements, such as conjugative plasmids, transposons, integron-associated gene cassettes, and insertion sequences (Pyle, 2000; Dai & Zimmerly, 2002). They possess an intron- encoded protein (IEP) with several activities that include a maturase, a reverse transcriptase and a site-specific DNA endonuclease (Endo) (Kennell et al., 1993; Zimmerly et al., 1995a; Matsuura et al., 1997). GII introns mediate their dissem- ination by the well-characterized target DNA primed reverse transcription (TPRT; Zimmerly et al., 1995b; Cousineau et al., 2000), which involves an RNA–DNA pairing. This pairing is achieved through two regions: the exon binding sites (EBSs) in the intron RNA, and the intron binding sites (IBSs) in the targeted DNA (Lambowitz & Belfort, 1993; Michel & Ferat, 1995). These regions vary in sequence and length based upon the GII intron classification (Dai & Zimmerly, 2002). Based on their IEP sequences, they have been classified into chloroplast-like classes, mitochondrial-like classes and bacterial classes A–E (Zimmerly et al., 2001). Their RNAs and IEPs have largely coevolved; thus, it is possible to follow the evolution of the ribozyme by understanding the evolution of the reverse transcriptase (Toor et al., 2001). It has been proposed that the class C introns are direct descendants of a common ancestor of GII introns (Rest & Mindell, 2003). The class C GII introns, which have a novel RNA secondary structure, lack the Endo domain of the IEP (Endo 2 ), have a shorter EBS–IBS pairing, and are located downstream of transcriptional terminators (Granlund et al., 2001; Dai & Zimmerly, 2002). While Endo + introns use the TPRT mechanism, introns lacking the Endo activity use the host replication machinery to invade novel target sites (Jime ´nez-Zurdo et al., 2003; Zhong & Lambowitz, 2003). RNA–DNA pairing in most GII introns involves EBS regions 1, 2 and 3, which constitute an 11–12 nt motif (Matsuura et al., 1997; Mohr et al., 2000); in contrast, the class C GII introns lack the EBS2 region, resulting in a shorter RNA–DNA pairing (Toor et al., 2001). Previously, we reported the presence of a class C group II intron identified as S.ma.I2 (Figs 1 and 2a) in a class 1 integron in the multiresistant Serratia marcescens strain SCH909 (Sm909) (Centro ´n & Roy, 2002). The S.ma.I2 GII intron is inserted within the attC site of the ant(299)-Ia gene cassette, in the opposite orientation to the cassette ORF (Fig. 2a). Cassettes are mobile elements typically composed of a promoterless structural gene and a recombination site known as a 59-base element or attC Abbreviations: CS, consensus sequence; EBS, exon binding site; Endo, endonuclease; GII, group II; IBS, intron binding site; IEP, intron-encoded protein; wt, wild-type. A supplementary table showing the GII intron and E1 sequences used in this study, and two supplementary figures showing the structures used for intron self-splicing and the secondary structures of the target sites corresponding to class C GII introns, are available with the online version of this paper. Microbiology (2008), 154, 1341–1353 DOI 10.1099/mic.0.2007/016360-0 2007/016360 G 2008 SGM Printed in Great Britain 1341