Group II introns are genetic retroelements capable of selfsplicing and mobility that are widespread in prokaryotes. Originally discovered in organelles of fungi, plants, and lower eukaryotes ϳ20 years ago (33), they were first found in bacteria ϳ10 years later (15) and lately have been identified in archaea of the Methanosarcina genus (11, 55). About 25% of the completely sequenced microbial genomes, covering a diverse range of bacterial phyla, contain one or more introns (either full length or fragmented). A compilation of bacterial group II introns showed that these elements are often inserted in intergenic regions, and when located inside genes, they are rarely found within highly conserved or housekeeping genes (10). Usually, bacterial group II introns are located on mobile DNA elements such as plasmids, insertion elements, transposons, or pathogenicity islands, which could account for their spread among bacteria (4, 10).Group II introns typically consist of a catalytic RNA containing an internal protein-encoding open reading frame (ORF), although many ORF-less introns exist in eukaryotic organelles. Functional introns are able to excise out of RNA transcripts (self-splicing), and insert (reverse splice) into identical intronless DNA sites (process called homing) or into novel (ectopic) genomic locations but at a much lower frequency (retrotransposition). The homing process is highly site specific and occurs at target regions spanning ϳ30 bp around the insertion point (18, 51). Selection of splice sites is determined by base pairings between three motifs in the intron RNA (exon-binding sequences EBS1, EBS2, and EBS3 or ␦) and the complementary sequences in the flanking exons (intron-binding sequences IBS1, IBS2, and IBS3 or ␦Ј, respectively), and these pairings are required for both splicing and reverse splicing (insertion). The splicing reactions are intrinsically catalyzed by the RNA part, but the intronic protein participates in vivo in both the splicing and insertion events (see references 5 and 55 for reviews). Because of the similar in vitro splicing mechanism, including formation of a lariat structure, group II introns are thought to be the ancestors of the nuclear spliceosomal introns of eukaryotes. The secondary structure of the group II RNA is made up of six domains linked by tertiary interactions, where domain V is the presumed catalytic core and domain VI contains a bulged A that is the branching point of the lariat (34,35,42,54). The structure is used to divide the group II introns into subclasses (54). The intron-encoded protein (IEP), located in domain IV, is a multifunctional protein that can have three functional domains: a reverse transcriptase (RT) domain for synthesis of DNA strand upon insertion, a maturase (X) domain involved in splicing, and an endonuclease (En or Zn) domain for target DNA cleavage, although the latter region is lacking in most IEPs. In between the X and En domains is a DNA-binding (D) region (3,5,50,57).For many published bacterial genome sequences, the IEPs are annot...

All group II introns known to date fold into six functional domains. However, we recently identified an intron in Bacillus cereus ATCC 10987, B.c.I4, that splices 56 nt downstream of the expected 3′ splice site in vivo (Tourasse et al. 2005, J. Bacteriol., 187, 5437–5451). In this study, we confirmed by ribonuclease protection assay that the 56-bp segment is part of the intron RNA molecule, and computational prediction suggests that it might form a stable stem-loop structure downstream of domain VI. The splicing of B.c.I4 was further investigated both in vivo and in vitro. Lariat formation proceeded primarily by branching at the ordinary bulged adenosine in domain VI without affecting the fidelity of splicing. In addition, the splicing efficiency of the wild-type intron was better than that of a mutant construct deleted of the 56-bp 3′ extension. These results indicate that the intron has apparently adapted to the extra segment, possibly through conformational adjustments. The extraordinary group II intron B.c.I4 harboring an unprecedented extra 3′ segment constitutes a dramatic example of the flexibility and adaptability of group II introns.

The B.c.I4 group II intron from Bacillus cereus ATCC 10987 harbors an unusual 3′ extension. Here, we report the discovery of four additional group II introns with a similar 3′ extension in Bacillus thuringiensis kurstaki 4D1 that splice at analogous positions 53/56 nt downstream of domain VI in vivo. Phylogenetic analyses revealed that the introns are only 47–61% identical to each other. Strikingly, they do not form a single evolutionary lineage even though they belong to the same Bacterial B class. The extension of these introns is predicted to form a conserved two-stem–loop structure. Mutational analysis in vitro showed that the smaller stem S1 is not critical for self-splicing, whereas the larger stem S2 is important for efficient exon ligation and lariat release in presence of the extension. This study clearly demonstrates that previously reported B.c.I4 is not a single example of a specialized intron, but forms a new functional class with an unusual mode that ensures proper positioning of the 3′ splice site.

Bacillus cereus strains ATCC 10987 and ATCC 14579 harbor a ϳ155-bp repeated element, bcr1, which is conserved in B. cereus, B. anthracis, B. thuringiensis, and B. mycoides but not in B. subtilis and B. licheniformis. In this study, we show by Southern blot hybridizations that bcr1 is present in all 54 B. cereus group strains tested but absent in 11 Bacillus strains outside the group, suggesting that bcr1 may be specific and ubiquitous to the B. cereus group. By comparative analysis of the complete genome sequences of B. cereus ATCC 10987, B. cereus ATCC 14579, and B. anthracis Ames, we show that bcr1 is exclusively present in the chromosome but absent from large plasmids carried by these strains and that the numbers of full-length bcr1 repeats for these strains are 79, 54, and 12, respectively. Numerous copies of partial bcr1 elements are also present in the three genomes (91, 128, and 53, respectively). Furthermore, the genomic localization of bcr1 is not conserved between strains with respect to chromosomal position or organization of gene neighbors, as only six full-length bcr1 loci are common to at least two of the three strains. However, the intergenic sequence surrounding a specific bcr1 repeat in one of the three strains is generally strongly conserved in the other two, even in loci where bcr1 is found exclusively in one strain. This finding indicates that bcr1 either has evolved by differential deletion from a very high number of repeats in a common ancestor to the B. cereus group or is moving around the chromosome. The identification of bcr1 repeats interrupting genes in B. cereus ATCC 10987 and ATCC 14579 and the presence of a flanking TTTAT motif in each end show that bcr1 exhibits features characteristic of a mobile element.

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