Survey of chimeric IStron elements in bacterial genomes: multiple molecular symbioses between group I intron ribozymes and DNA transposons.

IStrons are chimeric genetic elements composed of a group I intron associated with an insertion sequence (IS). The group I intron is a catalytic RNA providing the IStron with self-splicing ability, which renders IStron insertions harmless to the host genome. The IS element is a DNA transposon conferring mobility, and thus allowing the IStron to spread in genomes. IStrons are therefore a striking example of a molecular symbiosis between unrelated genetic elements endowed with different functions. In this study, we have conducted the first comprehensive survey of IStrons in sequenced genomes that provides insights into the distribution, diversity, origin and evolution of IStrons. We show that IStrons have a restricted phylogenetic distribution limited to two bacterial phyla, the Firmicutes and the Fusobacteria. Nevertheless, diverse IStrons representing two major groups targeting different insertion site motifs were identified. This taken with the finding that while the intron components of all IStrons belong to the same structural class, they are fused to different IS families, indicates that multiple intron-IS symbioses have occurred during evolution. In addition, introns and IS elements related to those that were at the origin of IStrons were also identified.

A conserved 3' extension in unusual group II introns is important for efficient second-step splicing.

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.

Group II intron in Bacillus cereus has an unusual 3' extension and splices 56 nucleotides downstream of the predicted site.

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.

Diversity, mobility, and structural and functional evolution of group II introns carrying an unusual 3' extension.

BACKGROUND: Group II introns are widespread genetic elements endowed with a dual functionality. They are catalytic RNAs (ribozymes) that are able of self-splicing and they are also mobile retroelements that can invade genomic DNA. The group II intron RNA secondary structure is typically made up of six domains. However, a number of unusual group II introns carrying a unique extension of 53-56 nucleotides at the 3' end have been identified previously in bacteria of the Bacillus cereus group. METHODS: In the present study, we conducted combined sequence comparisons and phylogenetic analyses of introns, host gene, plasmid and chromosome of host strains in order to gain insights into mobility, dispersal, and evolution of the unusual introns and their extension. We also performed in vitro mutational and kinetic experiments to investigate possible functional features related to the extension. RESULTS: We report the identification of novel copies of group II introns carrying a 3' extension including the first two copies in bacteria not belonging to the B. cereus group, Bacillus pseudofirmus OF4 and Bacillus sp. 2_A_57_CT2, an uncharacterized species phylogenetically close to B. firmus. Interestingly, the B. pseudofirmus intron has a longer extension of 70 bases. From sequence comparisons and phylogenetic analyses, several possible separate events of mobility involving the atypical introns could be identified, including both retrohoming and retrotransposition events. In addition, identical extensions were found in introns that otherwise exhibit little sequence conservation in the rest of their structures, with the exception of the conserved and catalytically critical domains V and VI, suggesting either separate acquisition of the extra segment by different group II introns or a strong selection pressure acting on the extension. Furthermore, we show by in vitro splicing experiments that the 3' extension affects the splicing properties differently in introns belonging to separate evolutionary branches. CONCLUSIONS: Altogether this study provides additional insights into the structural and functional evolution of unusual introns harboring a 3' extension and lends further evidence that these introns are mobile with their extension.

Structural and functional evolution of group II intron ribozymes: insights from unusual elements carrying a 3' extension.

Group II introns are large RNA elements that interrupt genes. They are self-splicing ribozymes that catalyze their own excision and mobile retroelements that can invade new genomic DNA sites. While group II introns typically consist of six structural domains, a number of elements containing an unusual 3' extension of 53-56 nucleotides have recently been identified. Bioinformatic and functional analyses of these introns have revealed that they belong to two evolutionary subgroups and that the 3' extension has a differential effect on the splicing reactions for introns of the two subgroups, a functional difference that may be related to structural differences between the introns. In addition, there is phylogenetic evidence that some introns are mobile with their extension. The unusual introns have provided dramatic examples of the structural and functional evolution of group II ribozymes that have been able to accommodate an extra segment into their compact structure while maintaining functionality.

Exploring the evolution of the Bacillus cereus group repeat element bcr1 by comparative genome analysis of closely related strains.

bcr1 is a chromosomal approximately 155 bp repeated element found uniquely and ubiquitously in the Bacillus cereus group of Gram-positive bacteria; it exhibits several features characteristic of mobile elements, including a variable distribution pattern between strains. Here, highly similar bcr1 elements in non-conserved genomic loci are identified in a set of closely related B. cereus and Bacillus thuringiensis strains near the Bacillus anthracis phylogenetic cluster. It is also shown that bcr1 may be present on small RNA transcripts in the 100-400 bp size range. In silico folding of bcr1 at the RNA level indicated that transcripts may form a double-hairpin-like structure predicted to have high structural stability. A functional role of bcr1 at the RNA level is supported by multiple cases of G-U base-pairing, and compensatory mutations maintaining structural stability of the RNA fold. In silico folding at the DNA level produced similar predicted structures, with the potential to form a cruciform structure at open DNA complexes. The predicted structural stability was greater for bcr1 elements showing high sequence identities to bcr1 elements in non-conserved chromosomal loci in other strains, relative to other bcr1 copies. bcr1 mobility could thus be dependent on the formation of a stable DNA or RNA intermediate. Furthermore, bcr1 elements potentially encoding structurally stable and less stable transcripts were phylogenetically intermixed, indicating that loss of bcr1 mobility may have occurred multiple times during evolution. Repeated elements with similar features in other bacteria have been shown to provide functions such as mRNA stabilization, transcription termination and/or promoter function. Similarly, bcr1 may constitute a mobile element which occasionally gains a function when it enters an appropriate chromosomal locus.

Unusual group II introns in bacteria of the Bacillus cereus group.

A combination of sequence and structure analysis and reverse transcriptase PCR experiments was used to characterize the group II introns in the complete genomes of two strains of the pathogen Bacillus cereus. While B. cereus ATCC 14579 harbors a single intron element in the chromosome, B. cereus ATCC 10987 contains three introns in the chromosome and four in its 208-kb pBc10987 plasmid. The most striking finding is the presence in B. cereus ATCC 10987 of an intron [B.c.I2(a)] located on the reverse strand of a gene encoding a putative cell surface protein which appears to be correlated to strains of clinical origin. Because of the opposite orientation of B.c.I2(a), the gene is disrupted. Even more striking is that B.c.I2(a) splices out of an RNA transcript corresponding to the opposite DNA strand. All other intragenic introns studied here are inserted in the same orientation as their host genes and splice out of the mRNA in vivo, setting the flanking exons in frame. Noticeably, B.c.I3 in B. cereus ATCC 10987 represents the first example of a group II intron entirely included within a conserved replication gene, namely, the alpha subunit of DNA polymerase III. Another striking finding is that the observed 3' splice site of B.c.I4 occurs 56 bp after the predicted end of the intron. This apparently unusual splicing mechanism may be related to structural irregularities in the 3' terminus. Finally, we also show that the intergenic introns of B. cereus ATCC 10987 are transcribed with their upstream genes and do splice in vivo.

The bcr1 DNA repeat element is specific to the Bacillus cereus group and exhibits mobile element characteristics.

Bacillus cereus strains ATCC 10987 and ATCC 14579 harbor an approximately 155-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.