María Dolores Molina-Sánchez

Excision of the Sinorhizobium meliloti group II intron RmInt1 as circles in vivo.

Excision of group II introns as circles has been described only for a few eukaryotic introns and little is known about the mechanisms involved, the relevance or consequences of the process. We report that splicing of the bacterial group II intron RmInt1 in vivo leads to the formation of both intron lariat and intron RNA circles. We determined that besides being required for the intron splicing reaction, the maturase domain of the intron-encoded protein also controls the balance between lariat and RNA intron circle production. Furthermore, comparison with in vitro self-splicing products indicates that in vivo, the intron-encoded protein appears to promote the use of a correct EBS1/IBS1 intron-exon interaction as well as cleavage at, or next to, the expected 3′ splice site. These findings provide new insights on the mechanism of excision of group II introns as circles.

Structural features in the C‐terminal region of the Sinorhizobium meliloti RmInt1 group II intron‐encoded protein contribute to its maturase and intron DNA‐insertion function

Group II introns are both catalytic RNAs and mobile retroelements that move through a process catalyzed by a RNP complex consisting of an intron‐encoded protein and the spliced intron lariat RNA. Group II intron‐encoded proteins are multifunctional and contain an N‐terminal reverse transcriptase domain, followed by a putative RNA‐binding domain (domain X) associated with RNA splicing or maturase activity and a C‐terminal DNA binding/DNA endonuclease region. The intron‐encoded protein encoded by the mobile group II intron RmInt1, which lacks the DNA binding/DNA endonuclease region, has only a short C‐terminal extension (C‐tail) after a typical domain X, apparently unrelated to the C‐terminal regions of other group II intron‐encoded proteins. Multiple sequence alignments identified features of the C‐terminal portion of the RmInt1 intron‐encoded protein that are conserved throughout evolution in the bacterial ORF class D, suggesting a group‐specific functionally important protein region. The functional importance of these features was demonstrated by analyses of deletions and mutations affecting conserved amino acid residues. We found that the C‐tail of the RmInt1 intron‐encoded protein contributes to the maturase function of this reverse transcriptase protein. Furthermore, within the C‐terminal region, we identified, in a predicted α‐helical region and downstream, conserved residues that are specifically required for the insertion of the intron into DNA targets in the orientation that would make it possible to use the nascent leading strand as a primer. These findings suggest that these group II intron intron‐encoded proteins may have adapted to function in mobility by different mechanisms to make use of either leading or lagging‐oriented targets in the absence of an endonuclease domain.

Host Factors Influencing the Retrohoming Pathway of Group II Intron RmInt1, Which Has an Intron-Encoded Protein Naturally Devoid of Endonuclease Activity.

Bacterial group II introns are self-splicing catalytic RNAs and mobile retroelements that have an open reading frame encoding an intron-encoded protein (IEP) with reverse transcriptase (RT) and RNA splicing or maturase activity. Some IEPs carry a DNA endonuclease (En) domain, which is required to cleave the bottom strand downstream from the intron-insertion site for target DNA-primed reverse transcription (TPRT) of the inserted intron RNA. Host factors complete the insertion of the intron. By contrast, the major retrohoming pathway of introns with IEPs naturally lacking endonuclease activity, like the Sinorhizobium meliloti intron RmInt1, is thought to involve insertion of the intron RNA into the template for lagging strand DNA synthesis ahead of the replication fork, with possible use of the nascent strand to prime reverse transcription of the intron RNA. The host factors influencing the retrohoming pathway of such introns have not yet been described. Here, we identify key candidates likely to be involved in early and late steps of RmInt1 retrohoming. Some of these host factors are common to En+ group II intron retrohoming, but some have different functions. Our results also suggest that the retrohoming process of RmInt1 may be less dependent on the intracellular free Mg2+ concentration than those of other group II introns.

The underlying process of early ecological and genetic differentiation in a facultative mutualistic Sinorhizobium meliloti population

The question of how genotypic and ecological units arise and spread in natural microbial populations remains controversial in the field of evolutionary biology. Here, we investigated the early stages of ecological and genetic differentiation in a highly clonal sympatric Sinorhizobium meliloti population. Whole-genome sequencing revealed that a large DNA region of the symbiotic plasmid pSymB was replaced in some isolates with a similar synteny block carrying densely clustered SNPs and displaying gene acquisition and loss. Two different versions of this genomic island of differentiation (GID) generated by multiple genetic exchanges over time appear to have arisen recently, through recombination in a particular clade within this population. In addition, these isolates display resistance to phages from the same geographic region, probably due to the modification of surface components by the acquired genes. Our results suggest that an underlying process of early ecological and genetic differentiation in S. meliloti is primarily triggered by acquisition of genes that confer resistance to soil phages within particular large genomic DNA regions prone to recombination.

Functionality of In vitro Reconstituted Group II Intron RmInt1-Derived Ribonucleoprotein Particles

The functional unit of mobile group II introns is a ribonucleoprotein particle (RNP) consisting of the intron-encoded protein (IEP) and the excised intron RNA. The IEP has reverse transcriptase activity but also promotes RNA splicing, and the RNA-protein complex triggers site-specific DNA insertion by reverse splicing, in a process called retrohoming. In vitro reconstituted ribonucleoprotein complexes from the Lactococcus lactis group II intron Ll.LtrB, which produce a double strand break, have recently been studied as a means of developing group II intron-based gene targeting methods for higher organisms. The Sinorhizobium meliloti group II intron RmInt1 is an efficient mobile retroelement, the dispersal of which appears to be linked to transient single-stranded DNA during replication. The RmInt1IEP lacks the endonuclease domain (En) and cannot cut the bottom strand to generate the 3′ end to initiate reverse transcription. We used an Escherichia coli expression system to produce soluble and active RmInt1 IEP and reconstituted RNPs with purified components in vitro. The RNPs generated were functional and reverse-spliced into a single-stranded DNA target. This work constitutes the starting point for the use of group II introns lacking DNA endonuclease domain-derived RNPs for highly specific gene targeting methods.

Genomic characterization of Sinorhizobium meliloti AK21, a wild isolate from the Aral Sea Region.

The symbiotic, nitrogen-fixing bacterium Sinorhizobium meliloti has been widely studied due to its ability to improve crop yields through direct interactions with leguminous plants. S. meliloti AK21 is a wild type strain that forms nodules on Medicago plants in saline and drought conditions in the Aral Sea Region. The aim of this work was to establish the genetic similarities and differences between S. meliloti AK21 and the reference strain S. meliloti 1021. Comparative genome hybridization with the model reference strain S. meliloti 1021 yielded 365 variable genes, grouped into 11 regions in the three main replicons in S. meliloti AK21. The most extensive regions of variability were found in the symbiotic plasmid pSymA, which also contained the largest number of orthologous and polymorphic sequences identified by suppression subtractive hybridization. This procedure identified a large number of divergent sequences and others without homology in the databases, the further investigation of which could provide new insight into the alternative metabolic pathways present in S. meliloti AK21. We identified a plasmid replication module from the repABC replicon family, together with plasmid mobilization-related genes (traG and a VirB9-like protein), which suggest that this indigenous isolate harbors an accessory plasmid. Furthermore, the transcriptomic profiles reflected differences in gene content and regulation between S. meliloti AK21 and S. meliloti 1021 (ExpR and PhoB regulons), but provided evidence for an as yet unknown, alternative mechanism involving activation of the cbb3 terminal oxidase. Finally, phenotypic microarrays characterization revealed a greater versatility of substrate use and chemical degradation than for S. meliloti 1021.

Intron Biology, Focusing on Group II Introns, the Ancestors of Spliceosomal Introns

Self-splicing group II introns are large ribozymes and mobile retroelements initially identified in the mitochondrial and chloroplast genomes of lower eukaryotes and plants and subsequently found in bacteria and archaea. Group II introns display structural, functional and mechanistic similarities to eukaryotic pre-mRNA nuclear introns, which may have evolved from mobile group II introns. As in spliceosomal introns, the ribozyme of group II introns excises the intron as a branched, lariat structure, through two sequential transesterification reactions. The movement of group II introns is mediated by a ribonucleoprotein (RNP) complex consisting of the IEP encoded by the ORF and the spliced intron lariat RNA, which remains associated with the IEP. These RNP complexes recognize intron targets through both the IEP and the intron lariat RNA. New possibilities for the use of these introns as biotechnological tools are emerging, due to the small number and flexibility of interactions between IEPs and target sites, through the modification of the intron RNA motifs that recognize DNA target sites by base pairing.

Contribution of mobile group II introns to Sinorhizobium meliloti genome evolution

Mobile group II introns are ribozymes and retroelements that probably originate from bacteria. Sinorhizobium meliloti, the nitrogen-fixing endosymbiont of legumes of genus Medicago, harbors a large number of these retroelements. One of these elements, RmInt1, has been particularly successful at colonizing this multipartite genome. Many studies have improved our understanding of RmInt1 and phylogenetically related group II introns, their mobility mechanisms, spread and dynamics within S. meliloti and closely related species. Although RmInt1 conserves the ancient retroelement behavior, its evolutionary history suggests that this group II intron has played a role in the short- and long-term evolution of the S. meliloti genome. We will discuss its proposed role in genome evolution by controlling the spread and coexistence of potentially harmful mobile genetic elements, by ectopic transposition to different genetic loci as a source of early genomic variation and by generating sequence variation after a very slow degradation process, through intron remnants that may have continued to evolve, contributing to bacterial speciation.