CONICET | Buscador de Institutos y Recursos Humanos

Identification of group II introns in the marine organism Shewanella Bacterial group II introns are widely distributed ribozymes capable of self-splicing. These elements have the ability to move to new regions in a genome through an RNA intermediate by a retrotransposition event. Previously, we found that the class C- group II intron S.ma.I2 isolated from the clinical isolate Serratia marcescens shares a common ancestor with its homologs encoded in the genus Shewanella. Shewanella is a versatile organism of interest for its biotechnological application as biofuel cells and bioremediation, and for its recent relevance as an opportunistic pathogen. Shewanella bacteria thrive in marine niches but it can be also isolated from soft tissue samples. Our work focuses in the study of different class C group II introns among Shewanella isolates and the characterization of their genomic contexts. We searched for S.ma.I2 homologues in 10 Shewanella clinical and 5 marine isolates by PCR using specific primers. Three out of 15 isolates harboured a class C group II intron, corresponding to 1 clinical (S. putrefaciens) and 2 marine isolates (Shewanella spp. and S. vesiculosa). Nucleotide sequence analysis showed that these introns were highly identical to S.ma.I2 (> 95%). Comparative analysis of Shewanella genomes using Artemis showed that out 24 complete projects 5 harboured a class C intron in their chromosomes. These introns were found in the boundaries of different transposases. We looked for these genes in our 3 intron-bearing Shewanella isolates by PCR; however none of our class C introns were associated to them. In order to identify their insertion sites we did an inverse PCR using specific primers. Our results showed that they were inserted in different intergenic region in the chromosome. Sequence analysis of the insertion site using the MFOLD software confirms that alike S.ma.I2 group II introns, these ribozymes also insert downstream of DNA secondary structures. The strong sequence identity between S.ma.I2 group II intron and Shewanella introns observed by experimental and in-silico analysis as well as their insertion downstream of DNA secondary structure supports the hypothesis of a marine origin for this type of ribozymes. Furthermore, we have obtained the complete sequence of the genome of a Shewanella isolate and we are currently in the process of annotating all the features. Once the annotation is complete we will be able to identify additional group II introns that may be encoded and we will start searching for small RNAs using an in silico approach. In the future, we will validate this data by doing the RNAseq analysis of this organism, and we will increase our efforts in the identification of other known and unknown of non-coding RNAs.