Group II introns in the Bacillus cereus group with unusual splicing properties
Appears in the following Collection
- Farmasøytisk institutt 
AbstractMobile genetic elements have had, and still have an impact on the evolution of the genomes providing means for adaptation and structural organization. These elements are one of the major driving forces for the general evolution of all life forms. For the organisms and their genomes these elements are essential for development and adaptation to different environments. The Bacillus cereus group of bacteria includes the related species B. cereus (sensu stricto), B. thuringiensis, B. weihenstephanensis, B. mycoides, B. pseudomycoides, and B.anthracis. These bacteria are very closely related at the genomic level, both in terms of gene content and synteny. Nevertheless, they show different phenotypic characteristics and pathogenic properties, and are altogether found worldwide in diverse habitats. Several of the major phenotypic characteristics of the members of the B. cereus group are determined by the different plasmids they have acquired. Besides these vehicles of genetic information, mobile genetic elements like transposons and group II introns do also induce some of the genetic and phenotypic variation and could therefore influence the dynamic behavior of the B. cereus group of bacteria.
Introns, or intervening sequences (IVS), are elements interrupting the sequence of genes. These are present in precursor mRNA and are removed by a process called splicing. The group II introns are a type of mobile retrotransposons that can also perform self-splicing. Group II introns are classified according to features of their RNA structure and the sequence of their intron-encoded reverse-transcriptase protein. The typical structure is made of six RNA domains (I-VI), which are involved in a network of tertiary interactions that fold the ribozyme into its catalytically active structure. Self-splicing proceeds in two steps via branching or hydrolysis pathways, releasing a lariat or linear intron, respectively In vivo the active intron need its own intron-encoded protein for splicing as well as for mobility.
The work presented in this Thesis starts with the classification and functional characterization of a total of eight group II introns present in the genomes of two strains of B. cereus,ATCC 14579 and ATCC 10987. The splice boundaries were as expected except for the B.c.I4 intron of B. cereus ATCC 10987, which spliced 56 nucleotides downstream of the predicted 3’ splice site. This extraordinary intron was then investigated in more detail. We showed that the extra 56-bp 3’ segment is an integral part of the intron RNA molecule downstream of domain VI, while splicing through branching still occurred at the expected site. B.c.I4 represented therefore a unique arrangement never seen before, and our studies imply that the intron must have adapted to splice with the 3’ extension. RNA secondary structure predictions suggest that the 56-bp segment folds into two stable stem-loop structures.
We later identified four new group II introns, B.th.I5, B.th.I6(a and b), and B.th.I7 from B. thuringiensis BGSC 4D1 that harbor a 3’ extension similar to that of B.c.I4. This showed that the presence of a 3’ extension was more common that previously thought and that B.c.I4 was not an isolated case. Surprisingly, these introns do not form a single evolutionary lineage even though the structure and sequence of the extensions are highly conserved. Furthermore, our in vitro splicing studies demonstrated that the larger of the two stems in the 3’ extension is important for an efficient second-step splicing with the extension. Though the initial studies showed that the whole extension of B.c.I4 was not essential for splicing, later studies suggested that it has an effect on the balance between splicing via hydrolyis and splicing via branching. Most remarkably, analysis of B.th.I6 revealed that this intron does not appear to be able to perform an efficient second splicing step when the extension is removed as opposed to B.c.I4. This difference may come from evolutionary divergence that is accompanied by differences in specific (sub)domains of the secondary structure.
We have further reported five divergent copies of the B.th.I6 group II intron in five B. cereus and B. thuringiensis strains. By using sequence comparisons and phylogenetic analysis of the host gene of these introns from 43 different B. cereus group strains, we could infer several separate events of mobility, thus strongly indicating that the B.th.I6 intron is mobile with the 3’ extension. Altogether, the results presented here indicate that the 3’ extension can be regarded as a functional domain VII that does contribute to the splicing properties, when present as an integral part of the intron. In addition to illustrating the adaptability and flexibility of group II introns, the study of these unusual introns has shed light on the structural and functional evolution of group II ribozymes in general.
List of papers
Paper 1 "Unusual group II introns in bacteria of the Bacillus cereus group." Tourasse NJ; Stabell, FB; Reiter L; Kolstø AB. J Bacteriol 187 : 5437-5461, 2005.
Paper 2 "Group II intron in Bacillus cereus has an unusual 3' extension and splices 56 nucleotides downstream of the predicted site." Stabell FB, Tourasse NJ, Ravnum S, Kolsto AB. Nucleic Acids Res 35 : 1612-1623, 2007.
Paper 3 "A conserved 3’ extension in unusual group II introns is important for the second splicing step." Stabell FB, Tourasse NJ, Kolstø AB. Nucleic Acids Res 37 : 3202-3214, 2009.
Paper 4 "Group II introns carrying an unusual 3’ extension show different splicing properties." Stabell FB, Tourasse NJ, Kolstø AB. Submitted
Paper 5 "Dispersal of a group II intron carrying an unusual 3’ extension in bacteria of the Bacillus cereus group." Tourasse NJ§*, Stabell FB§, Kolstø AB. Submitted