Publications by authors named "Nicolás Toro"

62 Publications

Prokaryotic reverse transcriptases: from retroelements to specialized defense systems.

FEMS Microbiol Rev 2021 May 13. Epub 2021 May 13.

Structure, Dynamics and Function of Rhizobacterial Genomes, Grupo de Ecología Genética de la Rizosfera, Department of Soil Microbiology and Symbiotic Systems, Estación Experimental del Zaidín, Consejo Superior de Investigaciones Científicas, C/ Profesor Albareda 1, 18008 Granada, Spain.

Reverse transcriptases (RTs) catalyze the polymerization of DNA from an RNA template. These enzymes were first discovered in RNA tumor viruses in 1970, but it was not until 1989 that they were found in prokaryotes as a key component of retrons. Apart from RTs encoded by the "selfish" mobile retroelements known as group II introns, prokaryotic RTs are extraordinarily diverse, but their function has remained elusive. However, recent studies have revealed that different lineages of prokaryotic RTs, including retrons, those associated with CRISPR-Cas systems, Abi-like RTs, and other yet uncharacterized RTs, are key components of different lines of defense against phages and other mobile genetic elements. Prokaryotic RTs participate in various antiviral strategies, including abortive infection (Abi), in which the infected cell is induced to commit suicide to protect the host population, adaptive immunity, in which a memory of previous infection is used to build an efficient defense, and other as yet unidentified mechanisms. These prokaryotic enzymes are attracting considerable attention, both for use in cutting-edge technologies, such as genome editing, and as an emerging research topic. In this review, we discuss what is known about prokaryotic RTs, and the exciting evidence for their domestication from retroelements to create specialized defense systems.
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http://dx.doi.org/10.1093/femsre/fuab025DOI Listing
May 2021

Systematic prediction of genes functionally associated with bacterial retrons and classification of the encoded tripartite systems.

Nucleic Acids Res 2020 12;48(22):12632-12647

Structure, Dynamics and Function of Rhizobacterial Genomes, Grupo de Ecología Genética de la Rizosfera, Department of Soil Microbiology and Symbiotic Systems, Estación Experimental del Zaidín, Consejo Superior de Investigaciones Científicas, C/ Profesor Albareda 1, 18008 Granada, Spain.

Bacterial retrons consist of a reverse transcriptase (RT) and a contiguous non-coding RNA (ncRNA) gene. One third of annotated retrons carry additional open reading frames (ORFs), the contribution and significance of which in retron biology remains to be determined. In this study we developed a computational pipeline for the systematic prediction of genes specifically associated with retron RTs based on a previously reported large dataset representative of the diversity of prokaryotic RTs. We found that retrons generally comprise a tripartite system composed of the ncRNA, the RT and an additional protein or RT-fused domain with diverse enzymatic functions. These retron systems are highly modular, and their components have coevolved to different extents. Based on the additional module, we classified retrons into 13 types, some of which include additional variants. Our findings provide a basis for future studies on the biological function of retrons and for expanding their biotechnological applications.
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http://dx.doi.org/10.1093/nar/gkaa1149DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7736814PMC
December 2020

Complete Genome Sequence of Sinorhizobium meliloti Strain AK21, a Salt-Tolerant Isolate from the Aral Sea Region.

Microbiol Resour Announc 2020 Jan 9;9(2). Epub 2020 Jan 9.

Structure, Dynamics, and Function of Rhizobacterial Genomes Research Group, Department of Soil Microbiology and Symbiotic Systems, Estación Experimental del Zaidín, Consejo Superior de Investigaciones Científicas (CSIC), Granada, Spain.

We report here the complete genome sequence of the salt-tolerant strain AK21, isolated from nodules of L. subsp. inhabiting the northern Aral Sea Region. This genome (7.36 Mb) consists of a chromosome and four accessory plasmids, two of which are the symbiotic megaplasmids pSymA and pSymB.
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http://dx.doi.org/10.1128/MRA.01432-19DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6952668PMC
January 2020

Recruitment of Reverse Transcriptase-Cas1 Fusion Proteins by Type VI-A CRISPR-Cas Systems.

Front Microbiol 2019 13;10:2160. Epub 2019 Sep 13.

Structure, Dynamics and Function of Rhizobacterial Genomes, Department of Soil Microbiology and Symbiotic Systems, Estación Experimental del Zaidín, Consejo Superior de Investigaciones Científicas, Granada, Spain.

Type VI CRISPR-Cas systems contain a single effector nuclease (Cas13) that exclusively targets single-stranded RNA. It remains unknown how these systems acquire spacers. It has been suggested that type VI systems with adaptation modules can acquire spacers from RNA bacteriophages, but sequence similarities suggest that spacers may provide immunity to DNA phages. We searched databases for Cas13 proteins with linked RTs. We identified two different type VI-A systems with adaptation modules including an RT-Cas1 fusion and Cas2 proteins. Phylogenetic reconstruction analyses revealed that these adaptation modules were recruited by different effector Cas13a proteins, possibly from RT-associated type III-D systems within the bacterial classes Alphaproteobacteria and Clostridia. These type VI-A systems are predicted to acquire spacers from RNA molecules, paving the way for future studies investigating their role in bacterial adaptive immunity and biotechnological applications.
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http://dx.doi.org/10.3389/fmicb.2019.02160DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6753606PMC
September 2019

Spacer acquisition from RNA mediated by a natural reverse transcriptase-Cas1 fusion protein associated with a type III-D CRISPR-Cas system in Vibrio vulnificus.

Nucleic Acids Res 2019 11;47(19):10202-10211

Structure, Dynamics and Function of Rhizobacterial Genomes, Grupo de Ecología Genética de la Rizosfera, Department of Soil Microbiology and Symbiotic Systems, Estación Experimental del Zaidín, Consejo Superior de Investigaciones Científicas, C/ Profesor Albareda 1, 18008 Granada, Spain.

The association of reverse transcriptases (RTs) with CRISPR-Cas system has recently attracted interest because the RT activity appears to facilitate the RT-dependent acquisition of spacers from RNA molecules. However, our understanding of this spacer acquisition process remains limited. We characterized the in vivo acquisition of spacers mediated by an RT-Cas1 fusion protein linked to a type III-D system from Vibrio vulnificus strain YJ016, and showed that the adaptation module, consisting of the RT-Cas1 fusion, two different Cas2 proteins (A and B) and one of the two CRISPR arrays, was completely functional in a heterologous host. We found that mutations of the active site of the RT domain significantly decreased the acquisition of new spacers and showed that this RT-Cas1-associated adaptation module was able to incorporate spacers from RNA molecules into the CRISPR array. We demonstrated that the two Cas2 proteins of the adaptation module were required for spacer acquisition. Furthermore, we found that several sequence-specific features were required for the acquisition and integration of spacers derived from any region of the genome, with no bias along the 5'and 3'ends of coding sequences. This study provides new insight into the RT-Cas1 fusion protein-mediated acquisition of spacers from RNA molecules.
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http://dx.doi.org/10.1093/nar/gkz746DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6821258PMC
November 2019

Multiple origins of reverse transcriptases linked to CRISPR-Cas systems.

RNA Biol 2019 10 11;16(10):1486-1493. Epub 2019 Jul 11.

Structure, Dynamics and Function of Rhizobacterial Genomes (Grupo de Ecología Genética de la Rizosfera), Department of Soil Microbiology and Symbiotic Systems, Estación Experimental del Zaidín, Consejo Superior de Investigaciones Científicas , Granada , Spain.

Prokaryotic genomes harbour a plethora of uncharacterized reverse transcriptases (RTs). RTs phylogenetically related to those encoded by group-II introns have been found associated with type III CRISPR-Cas systems, adjacent or fused at the C-terminus to Cas1. It is thought that these RTs may have a relevant function in the CRISPR immune response mediating spacer acquisition from RNA molecules. The origin and relationships of these RTs and the ways in which the various protein domains evolved remain matters of debate. We carried out a large survey of annotated RTs in databases (198,760 sequences) and constructed a large dataset of unique representative sequences (9,141). The combined phylogenetic reconstruction and identification of the RTs and their various protein domains in the vicinity of CRISPR adaptation and effector modules revealed three different origins for these RTs, consistent with their emergence on multiple occasions: a larger group that have evolved from group-II intron RTs, and two minor lineages that may have arisen more recently from Retron/retron-like sequences and Abi-P2 RTs, the latter associated with type I-C systems. We also identified a particular group of RTs associated with CRISPR- loci in clade 12, fused C-terminally to an archaeo-eukaryotic primase (AEP), a protein domain (AE-Prim_S_like) forming a particular family within the AEP proper clade. Together, these data provide new insight into the evolution of CRISPR-Cas/RT systems.
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http://dx.doi.org/10.1080/15476286.2019.1639310DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6779382PMC
October 2019

A group II intron-encoded protein interacts with the cellular replicative machinery through the β-sliding clamp.

Nucleic Acids Res 2019 08;47(14):7605-7617

Structure, Dynamics and Function of Rhizobacterial Genomes (Grupo de Ecología Genética de la Rizosfera), Department of Soil Microbiology and Symbiotic Systems, Estación Experimental del Zaidín, Consejo Superior de Investigaciones Científicas, C/Profesor Albareda 1, 18008 Granada, Spain.

Group II introns are self-splicing mobile genetic retroelements. The spliced intron RNA and the intron-encoded protein (IEP) form ribonucleoprotein particles (RNPs) that recognize and invade specific DNA target sites. The IEP is a reverse transcriptase/maturase that may bear a C-terminal endonuclease domain enabling the RNP to cleave the target DNA strand to prime reverse transcription. However, some mobile introns, such as RmInt1, lack the En domain but nevertheless retrohome efficiently to transient single-stranded DNA target sites at a DNA replication fork. Their mobility is associated with host DNA replication, and they use the nascent lagging strand as a primer for reverse transcription. We searched for proteins that interact with RmInt1 RNPs and direct these RNPs to the DNA replication fork. Co-immunoprecipitation assays suggested that DnaN (the β-sliding clamp), a component of DNA polymerase III, interacts with the protein component of the RmInt1 RNP. Pulldown assays, far-western blots and biolayer interferometry supported this interaction. Peptide binding assays also identified a putative DnaN-interacting motif in the RmInt1 IEP structurally conserved in group II intron IEPs. Our results suggest that intron RNP interacts with the β-sliding clamp of the DNA replication machinery, favouring reverse splicing into the transient ssDNA at DNA replication forks.
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http://dx.doi.org/10.1093/nar/gkz468DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6698660PMC
August 2019

DNA cleavage and reverse splicing of ribonucleoprotein particles reconstituted with linear RmInt1 RNA.

RNA Biol 2019 07 14;16(7):930-939. Epub 2019 Apr 14.

a Structure, Dynamics and Function of Rhizobacterial Genomes, Grupo de Ecología Genética de la Rizosfera, Department of Soil Microbiology and Symbiotic Systems , Estación Experimental del Zaidín, Consejo Superior de Investigaciones Científicas , Granada , Spain.

The RmInt1 group II intron is an efficient self-splicing mobile retroelement that catalyzes its own excision as lariat, linear and circular molecules. , the RmInt1 lariat and the reverse transcriptase (IEP) it encodes form a ribonucleoprotein particle (RNP) that recognizes the DNA target for site-specific full intron insertion via a two-step reverse splicing reaction. RNPs containing linear group II intron RNA are generally thought to be unable to complete the reverse splicing reaction. Here, we show that reconstituted RNPs containing linear RmInt1 ΔORF RNA can mediate the cleavage of single-stranded DNA substrates in a very precise manner with the attachment of the intron RNA to the 3´exon as the first step of a reverse splicing reaction. Notably, we also observe molecules in which the 5´exon is linked to the RmInt1 RNA, suggesting the completion of the reverse splicing reaction, albeit rather low and inefficiently. That process depends on DNA target recognition and can be successful completed by RmInt1 RNPs with linear RNA displaying 5´ modifications.
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http://dx.doi.org/10.1080/15476286.2019.1601379DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6546360PMC
July 2019

Metabarcoding reveals that rhizospheric microbiota of Quercus pyrenaica is composed by a relatively small number of bacterial taxa highly abundant.

Sci Rep 2019 02 8;9(1):1695. Epub 2019 Feb 8.

Departamento de Microbiología del Suelo y Sistemas Simbióticos, Estación Experimental del Zaidín, CSIC, calle Profesor Albareda 1, 18008, Granada, Spain.

Melojo oak (Quercus pyrenaica Willd.) is a key tree species of Mediterranean forests; however, these forests show an advanced stage of deterioration in the Iberian Peninsula. Plant-associated microorganisms play an essential role improving their host's fitness, hence, a better understanding of oak rhizospheric microbiome, especially of those active members, could be the first step towards microbiome-based approaches for oak-forest improvement. Here we reported, for the first time, the diversity of total (DNA-based) and potentially active (RNA-based) bacterial communities of different melojo-oak forest formations through pyrosequencing of 16S rRNA gene amplicons. We found that potentially active bacterial communities were as rich and diverse as total bacterial communities, but different in terms of relative abundance patterns in some of the studied areas. Both core microbiomes were dominated by a relatively small percentage of OTUs, most of which showed positive correlation between both libraries. However, the uncoupling between abundance (rDNA) and potential activity (rRNA) for some taxa suggests that the most abundant taxa are not always the most active, and that low-abundance OTUs may have a strong influence on oak's rhizospheric ecology. Thus, measurement of rRNA:rDNA ratio could be helpful in identifying major players for the development of bacterial bioinoculants.
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http://dx.doi.org/10.1038/s41598-018-38123-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6368570PMC
February 2019

On the Origin and Evolutionary Relationships of the Reverse Transcriptases Associated With Type III CRISPR-Cas Systems.

Front Microbiol 2018 15;9:1317. Epub 2018 Jun 15.

Structure, Dynamics and Function of Rhizobacterial Genomes, Grupo de Ecología Genética de la Rizosfera, Department of Soil Microbiology and Symbiotic Systems, Estación Experimental del Zaidín, Consejo Superior de Investigaciones Científicas, Granada, Spain.

Reverse transcriptases (RTs) closely related to those encoded by group II introns but lacking the intron RNA structure have been found associated with type III clustered regularly interspaced short palindromic repeats (CRISPR)-Cas systems, a prokaryotic immune system against invading viruses and foreign genetic elements. Two models have been proposed to explain the origin and evolutionary relationships of these RTs: (i) the "single point of origin" model, according to which these RTs originated from a single acquisition event in bacterial, with the various protein domains (RT, RT-Cas1, and Cas6-RT-Cas1 fusions) corresponding to single points in evolution; and (ii) the "various origins" model, according to which, independent acquisition events in different evolutionary episodes led to these fusions. We tested these alternative hypotheses, by analyzing and integrating published datasets of RT sequences associated with CRISPR-Cas systems and inferring phylogenetic trees by maximum likelihood (ML) methods. The RTs studied could be grouped into 13 clades, mostly in bacteria, in which they probably evolved. The various clades appear to form three independent lineages in bacteria and a recent lineage in archaea. Our data show that the Cas6 domain was acquired twice, independently, through RT-Cas1 fusion, in the bacterial lineages. Taken together, there more evidence to support the "various origins" hypothesis.
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http://dx.doi.org/10.3389/fmicb.2018.01317DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6013744PMC
June 2018

Contribution of Mobile Group II Introns to Genome Evolution.

Front Microbiol 2018 4;9:627. Epub 2018 Apr 4.

Structure, Dynamics and Function of Rhizobacterial Genomes (Grupo de Ecología Genética de la Rizosfera), Department of Soil Microbiology and Symbiotic Systems, Consejo Superior de Investigaciones Científicas, Estación Experimental del Zaidín, Granada, Spain.

Mobile group II introns are ribozymes and retroelements that probably originate from bacteria. , the nitrogen-fixing endosymbiont of legumes of genus , 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 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 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.
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http://dx.doi.org/10.3389/fmicb.2018.00627DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5894124PMC
April 2018

The Reverse Transcriptases Associated with CRISPR-Cas Systems.

Sci Rep 2017 08 2;7(1):7089. Epub 2017 Aug 2.

Structure, Dynamics and Function of Rhizobacterial Genomes, Grupo de Ecología Genética de la Rizosfera, Department of Soil Microbiology and Symbiotic Systems, Estación Experimental del Zaidín, Consejo Superior de Investigaciones Científicas, C/Profesor Albareda 1, 18008, Granada, Spain.

CRISPR (clustered regularly interspaced short palindromic repeats) and associated proteins (Cas) act as adaptive immune systems in bacteria and archaea. Some CRISPR-Cas systems have been found to be associated with putative reverse transcriptases (RT), and an RT-Cas1 fusion associated with a type III-B system has been shown to acquire RNA spacers in vivo. Nevertheless, the origin and evolutionary relationships of these RTs and associated CRISPR-Cas systems remain largely unknown. We performed a comprehensive phylogenetic analysis of these RTs and associated Cas1 proteins, and classified their CRISPR-Cas modules. These systems were found predominantly in bacteria, and their presence in archaea may be due to a horizontal gene transfer event. These RTs cluster into 12 major clades essentially restricted to particular phyla, suggesting host-dependent functioning. The RTs and associated Cas1 proteins may have largely coevolved. They are, therefore, subject to the same selection pressures, which may have led to coadaptation within particular protein complexes. Furthermore, our results indicate that the association of an RT with a CRISPR-Cas system has occurred on multiple occasions during evolution.
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http://dx.doi.org/10.1038/s41598-017-07828-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5541045PMC
August 2017

The rhizosphere microbiome of burned holm-oak: potential role of the genus Arthrobacter in the recovery of burned soils.

Sci Rep 2017 07 20;7(1):6008. Epub 2017 Jul 20.

Departamento de Microbiología del Suelo y Sistemas Simbióticos, Estación Experimental del Zaidín, Consejo Superior de Investigaciones Científicas, calle Profesor Albareda 1, 18008, Granada, Spain.

After a forest wildfire, the microbial communities have a transient alteration in their composition. The role of the soil microbial community in the recovery of an ecosystem following such an event remains poorly understood. Thus, it is necessary to understand the plant-microbe interactions that occur in burned soils. By high-throughput sequencing, we identified the main bacterial taxa of burnt holm-oak rhizosphere, then we obtained an isolate collection of the most abundant genus and its growth promoting activities were characterised. 16S rRNA amplicon sequencing showed that the genus Arthrobacter comprised more than 21% of the total community. 55 Arthrobacter strains were isolated and characterized using RAPDs and sequencing of the almost complete 16S rRNA gene. Our results indicate that isolated Arthrobacter strains present a very high genetic diversity, and they could play an important ecological role in interaction with the host plant by enhancing aerial growth. Most of the selected strains exhibited a great ability to degrade organic polymers in vitro as well as possibly presenting a direct mechanism for plant growth promotion. All the above data suggests that Arthrobacter can be considered as an excellent PGP rhizobacterium that may play an important role in the recovery of burned holm-oak forests.
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http://dx.doi.org/10.1038/s41598-017-06112-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5519729PMC
July 2017

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

Sci Rep 2017 04 6;7(1):675. Epub 2017 Apr 6.

Departamento de Química Orgánica, Universidad de Sevilla, C/Profesor García González, 1, 41012, Sevilla, Spain.

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.
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http://dx.doi.org/10.1038/s41598-017-00730-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5429615PMC
April 2017

Analysis of rhizobial endosymbionts of Vicia, Lathyrus and Trifolium species used to maintain mountain firewalls in Sierra Nevada National Park (South Spain).

Syst Appl Microbiol 2017 Mar 28;40(2):92-101. Epub 2016 Dec 28.

Departamento de Microbiología del Suelo y Sistemas Simbióticos, Estación Experimental del Zaidín, Consejo Superior de Investigaciones Científicas, Granada, Spain.

Forest fires lead to the annual disappearance of many natural formations that require the creation of firewall areas. They can be maintained by enriching their pastures with attractive plants for grazing livestock, mainly legumes, which have a high protein content and low dependence on N fertilizers due to their ability to establish nitrogen-fixing symbiosis with rhizobia. In this study, the rhizobia isolated from the nodules of six legumes from the genera Vicia, Lathyrus and Trifolium were analysed in a firewall zone established in Lanjarón (Granada) close to the Sierra Nevada National Park (Spain). The results showed a high genetic diversity of the isolated strains that had 3, 16, 14 and 13 different types of rrs, recA, atpD and glnII genes, respectively. All strains were phylogenetically close to the species from the Rhizobium leguminosarum group, although they were not identified as any of them. The isolated strains belonged to the symbiovars viciae and trifolii but high phylogenetic diversity was found within both symbiovars, since there were 16 and 14 nodC gene types, respectively. Some of these strains clustered with strains isolated in other countries and continents, but others formed atpD, recA, glnII and nodC clusters and lineages only found to date in this study.
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http://dx.doi.org/10.1016/j.syapm.2016.11.008DOI Listing
March 2017

Functionality of Reconstituted Group II Intron RmInt1-Derived Ribonucleoprotein Particles.

Front Mol Biosci 2016 27;3:58. Epub 2016 Sep 27.

Structure, Dynamics and Function of Rhizobacterial Genomes, Department of Soil Microbiology and Symbiotic Systems, Estación Experimental del Zaidín, Consejo Superior de Investigaciones Científicas Granada, Spain.

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. reconstituted ribonucleoprotein complexes from the 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 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 expression system to produce soluble and active RmInt1 IEP and reconstituted RNPs with purified components . 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.
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http://dx.doi.org/10.3389/fmolb.2016.00058DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5037169PMC
September 2016

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

PLoS One 2016 2;11(9):e0162275. Epub 2016 Sep 2.

Structure, Dynamics and Function of Rhizobacterial Genomes, Department of Soil Microbiology and Symbiotic Systems, Estación Experimental del Zaidín, Consejo Superior de Investigaciones Científicas, Calle Profesor Albareda 1, 18008, Granada, Spain.

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.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0162275PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5010178PMC
August 2017

The early events underlying genome evolution in a localized Sinorhizobium meliloti population.

BMC Genomics 2016 08 5;17:556. Epub 2016 Aug 5.

Grupo de Ecología Genética, Estación Experimental del Zaidín, Consejo Superior de Investigaciones Científicas, Calle Profesor Albareda 1, 18008, Granada, Spain.

Background: Population genetic analyses based on genome-wide sequencing data have been carried out for Sinorhizobium medicae and S. meliloti, two closely related bacterial species forming nitrogen-fixing symbioses with plants of the genus Medicago. However, genome coverage was low or the isolates had a broad geographic distribution, making it difficult to interpret the estimated diversity and to unravel the early events underlying population genetic variations and ecological differentiation.

Results: Here, to gain insight into the early genome level variation and diversification within S. meliloti populations, we first used Illumina paired-end reads technology to sequence a new clone of S. meliloti strain GR4, a highly competitive strain for alfalfa nodulation. The Illumina data and the GR4 genome sequence previously obtained with 454 technology were used to generate a high-quality reference genome sequence. We then used Illumina technology to sequence the genomes of 13 S. meliloti isolates representative of the genomic variation within the GR4-type population, obtained from a single field site with a high degree of coverage. The genome sequences obtained were analyzed to determine nucleotide diversity, divergence times, polymorphism and genomic variation. Similar low levels of nucleotide diversity were observed for the chromosome, pSymB and pSymA replicons. The isolates displayed other types of variation, such as indels, recombination events, genomic island excision and the transposition of mobile elements.

Conclusions: Our results suggest that the GR4-type population has experienced a process of demographic expansion and behaves as a stable genotypic cluster of genome-wide similarity, with most of the genome following a clonal pattern of evolution. Although some of genetic variation detected within the GR4-type population is probably due to genetic drift, others might be important in diversification and environmental adaptation.
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http://dx.doi.org/10.1186/s12864-016-2878-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4974801PMC
August 2016

Bacterial Group II Introns: Identification and Mobility Assay.

Methods Mol Biol 2016 ;1400:21-32

Grupo de Ecología Genética de la Rizosfera, Estación Experimental del Zaidín, Consejo Superior de Investigaciones Científicas, Profesor Albareda 1, 18008, Granada, Spain.

Group II introns are large catalytic RNAs and mobile retroelements that encode a reverse transcriptase. Here, we provide methods for their identification in bacterial genomes and further analysis of their splicing and mobility capacities.
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http://dx.doi.org/10.1007/978-1-4939-3372-3_2DOI Listing
December 2016

Bacterial Diversity in the Soda Saline Crater Lake from Isabel Island, Mexico.

Microb Ecol 2016 Jan 21;71(1):68-77. Epub 2015 Sep 21.

Grupo de Ecología Genética, Estación Experimental del Zaidín, Consejo Superior de Investigaciones Científicas, C/ Profesor Albareda 1, 18008, Granada, Spain.

Isabel Lake is a moderate saline soda crater lake located in Isabel Island in the eastern tropical Pacific coast of Mexico. Lake is mainly formed by rainfall and is strongly affected by evaporation and high input of nutrients derived from excretions of a large bird community inhabiting the island. So far, only the island macrobiota has been studied. The knowledge of the prokaryotic biota inhabiting the upper layers of this meromictic lake can give clues for the maintenance of this ecosystem. We assessed the diversity and composition of prokaryotic community in sediments and water of the lake by DGGE profiling, 16S rRNA gene amplicon pyrosequencing, and cultivation techniques. The bacterial community is largely dominated by halophilic and halotolerant microorganisms. Alpha diversity estimations reveal higher value in sediments than in water (P > 0.005). The lake water is dominated by γ-Proteobacteria belonging to four main families where Halomonadaceae presents the highest abundance. Aerobic, phototrophic, and halotolerant prokaryotes such as Cyanobacteria GPIIa, Halomonas, Alcanivorax, Idiomarina, and Cyclobacterium genera are commonly found. However, in sediment samples, Formosa, Muricauda, and Salegentibacter genera corresponding to Flavobacteriaceae family accounted for 15-20 % of the diversity. Heterotrophs like those involved in sulfur cycle, Desulfotignum, Desulfuromonas, Desulfofustis, and Desulfopila, appear to play an important role in sediments. Finally, a collection of aerobic halophilic bacterial isolates was created from these samples; members of the genus Halomonas were predominantly isolated from lake water. This study contributes to state the bacterial diversity present in this particular soda saline crater lake.
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http://dx.doi.org/10.1007/s00248-015-0676-6DOI Listing
January 2016

Localization of a bacterial group II intron-encoded protein in human cells.

Sci Rep 2015 Aug 5;5:12716. Epub 2015 Aug 5.

Grupo de Ecología Genética, Estación Experimental del Zaidín, Consejo Superior de Investigaciones Científicas, Calle Profesor Albareda 1, 18008 Granada, Spain.

Group II introns are mobile retroelements that self-splice from precursor RNAs to form ribonucleoparticles (RNP), which can invade new specific genomic DNA sites. This specificity can be reprogrammed, for insertion into any desired DNA site, making these introns useful tools for bacterial genetic engineering. However, previous studies have suggested that these elements may function inefficiently in eukaryotes. We investigated the subcellular distribution, in cultured human cells, of the protein encoded by the group II intron RmInt1 (IEP) and several mutants. We created fusions with yellow fluorescent protein (YFP) and with a FLAG epitope. We found that the IEP was localized in the nucleus and nucleolus of the cells. Remarkably, it also accumulated at the periphery of the nuclear matrix. We were also able to identify spliced lariat intron RNA, which co-immunoprecipitated with the IEP, suggesting that functional RmInt1 RNPs can be assembled in cultured human cells.
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http://dx.doi.org/10.1038/srep12716DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4525487PMC
August 2015

Inactivation of group II intron RmInt1 in the Sinorhizobium meliloti genome.

Sci Rep 2015 Jul 9;5:12036. Epub 2015 Jul 9.

Grupo de Ecología Genética, Estación Experimental del Zaidín, Consejo Superior de Investigaciones Científicas, Calle Profesor Albareda 1, 18008 Granada, Spain.

Group II introns are self-splicing catalytic RNAs that probably originated in bacteria and act as mobile retroelements. The dispersal and dynamics of group II intron spread within a bacterial genome are thought to follow a selection-driven extinction model. Likewise, various studies on the evolution of group II introns have suggested that they are evolving toward an inactive form by fragmentation, with the loss of the intron 3'-terminus, but with some intron fragments remaining and continuing to evolve in the genome. RmInt1 is a mobile group II intron that is widespread in natural populations of Sinorhizobium meliloti, but some strains of this species have no RmInt1 introns. We studied the splicing ability and mobility of the three full-length RmInt1 copies harbored by S. meliloti 1021, and obtained evidence suggesting that specific mutations may lead to the impairment of intron splicing and retrohoming. Our data suggest that the RmInt1 copies in this strain are undergoing a process of inactivation.
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http://dx.doi.org/10.1038/srep12036DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4496777PMC
July 2015

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

Springerplus 2015 16;4:259. Epub 2015 Jun 16.

Grupo de Ecología Genética, Departamento de Microbiología del Suelo y Sistemas Simbióticos, Estación Experimental del Zaidín, CSIC, Calle Profesor Albareda 1, 18008 Granada, Spain.

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.
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http://dx.doi.org/10.1186/s40064-015-1062-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4468178PMC
June 2015

Metagenomic assessment of the potential microbial nitrogen pathways in the rhizosphere of a mediterranean forest after a wildfire.

Microb Ecol 2015 May 3;69(4):895-904. Epub 2015 Mar 3.

Grupo de Ecología Genética de la Rizosfera, Departamento de Microbiología del Suelo y Sistemas Simbióticos, Estación Experimental del Zaidín, Consejo Superior de Investigaciones Científicas, calle Profesor Albareda 1, E-18008, Granada, Spain.

Wildfires are frequent in the forests of the Mediterranean Basin and have greatly influenced this ecosystem. Changes to the physical and chemical properties of the soil, due to fire and post-fire conditions, result in alterations of both the bacterial communities and the nitrogen cycle. We explored the effects of a holm oak forest wildfire on the rhizospheric bacterial communities involved in the nitrogen cycle. Metagenomic data of the genes involved in the nitrogen cycle showed that both the undisturbed and burned rhizospheres had a conservative nitrogen cycle with a larger number of sequences related to the nitrogen incorporation pathways and a lower number for nitrogen output. However, the burned rhizosphere showed a statistically significant increase in the number of sequences for nitrogen incorporation (allantoin utilization and nitrogen fixation) and a significantly lower number of sequences for denitrification and dissimilatory nitrite reductase subsystems, possibly in order to compensate for nitrogen loss from the soil after burning. The genetic potential for nitrogen incorporation into the ecosystem was assessed through the diversity of the nitrogenase reductase enzyme, which is encoded by the nifH gene. We found that nifH gene diversity and richness were lower in burned than in undisturbed rhizospheric soils. The structure of the bacterial communities involved in the nitrogen cycle showed a statistically significant increase of Actinobacteria and Firmicutes phyla after the wildfire. Both approaches showed the important role of gram-positive bacteria in the ecosystem after a wildfire.
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http://dx.doi.org/10.1007/s00248-015-0586-7DOI Listing
May 2015

Insights into the strategies used by related group II introns to adapt successfully for the colonisation of a bacterial genome.

RNA Biol 2014 31;11(8):1061-71. Epub 2014 Oct 31.

a Grupo de Ecología Genética; Estación Experimental del Zaidín; Consejo Superior de Investigaciones Científicas ; Granada , Spain.

Group II introns are self-splicing RNAs and site-specific mobile retroelements found in bacterial and organellar genomes. The group II intron RmInt1 is present at high copy number in Sinorhizobium meliloti species, and has a multifunctional intron-encoded protein (IEP) with reverse transcriptase/maturase activities, but lacking the DNA-binding and endonuclease domains. We characterized two RmInt1-related group II introns RmInt2 from S. meliloti strain GR4 and Sr.md.I1 from S. medicae strain WSM419 in terms of splicing and mobility activities. We used both wild-type and engineered intron-donor constructs based on ribozyme ΔORF-coding sequence derivatives, and we determined the DNA target requirements for RmInt2, the element most distantly related to RmInt1. The excision and mobility patterns of intron-donor constructs expressing different combinations of IEP and intron RNA provided experimental evidence for the co-operation of IEPs and intron RNAs from related elements in intron splicing and, in some cases, in intron homing. We were also able to identify the DNA target regions recognized by these IEPs lacking the DNA endonuclease domain. Our results provide new insight into the versatility of related group II introns and the possible co-operation between these elements to facilitate the colonization of bacterial genomes.
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http://dx.doi.org/10.4161/rna.32092DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4615759PMC
August 2015

Comprehensive phylogenetic analysis of bacterial reverse transcriptases.

PLoS One 2014 25;9(11):e114083. Epub 2014 Nov 25.

Grupo de Ecología Genética de la Rizosfera, Estación Experimental del Zaidín, Consejo Superior de Investigaciones Científicas (CSIC), Granada, Spain.

Much less is known about reverse transcriptases (RTs) in prokaryotes than in eukaryotes, with most prokaryotic enzymes still uncharacterized. Two surveys involving BLAST searches for RT genes in prokaryotic genomes revealed the presence of large numbers of diverse, uncharacterized RTs and RT-like sequences. Here, using consistent annotation across all sequenced bacterial species from GenBank and other sources via RAST, available from the PATRIC (Pathogenic Resource Integration Center) platform, we have compiled the data for currently annotated reverse transcriptases from completely sequenced bacterial genomes. RT sequences are broadly distributed across bacterial phyla, but green sulfur bacteria and cyanobacteria have the highest levels of RT sequence diversity (≤85% identity) per genome. By contrast, phylum Actinobacteria, for which a large number of genomes have been sequenced, was found to have a low RT sequence diversity. Phylogenetic analyses revealed that bacterial RTs could be classified into 17 main groups: group II introns, retrons/retron-like RTs, diversity-generating retroelements (DGRs), Abi-like RTs, CRISPR-Cas-associated RTs, group II-like RTs (G2L), and 11 other groups of RTs of unknown function. Proteobacteria had the highest potential functional diversity, as they possessed most of the RT groups. Group II introns and DGRs were the most widely distributed RTs in bacterial phyla. Our results provide insights into bacterial RT phylogeny and the basis for an update of annotation systems based on sequence/domain homology.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0114083PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4244168PMC
January 2016

In vitro characterization of the splicing efficiency and fidelity of the RmInt1 group II intron as a means of controlling the dispersion of its host mobile element.

RNA 2014 Dec 21;20(12):2000-10. Epub 2014 Oct 21.

Grupo de Ecología Genética, Estación Experimental del Zaidín, Consejo Superior de Investigaciones Científicas, 18008 Granada, Spain

Group II introns are catalytic RNAs that are excised from their precursors in a protein-dependent manner in vivo. Certain group II introns can also react in a protein-independent manner under nonphysiological conditions in vitro. The efficiency and fidelity of the splicing reaction is crucial, to guarantee the correct formation and expression of the protein-coding mRNA. RmInt1 is an efficient mobile intron found within the ISRm2011-2 insertion sequence in the symbiotic bacterium Sinorhizobium meliloti. The RmInt1 intron self-splices in vitro, but this reaction generates side products due to a predicted cryptic IBS1* sequence within the 3' exon. We engineered an RmInt1 intron lacking the cryptic IBS1* sequence, which improved the fidelity of the splicing reaction. However, atypical circular forms of similar electrophoretic mobility to the lariat intron were nevertheless observed. We analyzed a run of four cytidine residues at the 3' splice site potentially responsible for a lack of fidelity at this site leading to the formation of circular intron forms. We showed that mutations of residues base-pairing in the tertiary EBS3-IBS3 interaction increased the efficiency and fidelity of the splicing reaction. Our results indicate that RmInt1 has developed strategies for decreasing its splicing efficiency and fidelity. RmInt1 makes use of unproductive splicing reactions to limit the transposition of the insertion sequence into which it inserts itself in its natural context, thereby preventing potentially harmful dispersion of ISRm2011-2 throughout the genome of its host.
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http://dx.doi.org/10.1261/rna.047407.114DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4238363PMC
December 2014

Complete Genome Sequence of the RmInt1 Group II Intronless Sinorhizobium meliloti Strain RMO17.

Genome Announc 2014 Oct 9;2(5). Epub 2014 Oct 9.

Grupo de Ecología Genética de la Rizosfera, Estación Experimental del Zaidín, Consejo Superior de Investigaciones Científicas (CSIC), Granada, Spain.

We report the complete genome sequence of the RmInt1 group II intronless Sinorhizobium meliloti strain RMO17 isolated from Medicago orbicularis nodules from Spanish soil. The genome consists of 6.73 Mb distributed between a single chromosome and two megaplasmids (the chromid pSymB and pSymA).
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http://dx.doi.org/10.1128/genomeA.01001-14DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4192382PMC
October 2014

Genome-wide profiling of Hfq-binding RNAs uncovers extensive post-transcriptional rewiring of major stress response and symbiotic regulons in Sinorhizobium meliloti.

RNA Biol 2014 26;11(5):563-79. Epub 2014 Feb 26.

Grupo de Ecología Genética de la Rizosfera; Estación Experimental del Zaidín; Consejo Superior de Investigaciones Científicas; CSIC, Granada, Spain.

The RNA chaperone Hfq is a global post-transcriptional regulator in bacteria. Here, we used RNAseq to analyze RNA populations from the legume symbiont Sinorhizobium meliloti that were co-immunoprecipitated (CoIP-RNA) with a FLAG-tagged Hfq in five growth/stress conditions. Hfq-bound transcripts (1315) were largely identified in stressed bacteria and derived from small RNAs (sRNAs), both trans-encoded (6.4%) and antisense (asRNAs; 6.3%), and mRNAs (86%). Pull-down with Hfq recovered a small proportion of annotated S. meliloti sRNAs (14% of trans-sRNAs and 2% of asRNAs) suggesting a discrete impact of this protein in sRNA pathways. Nonetheless, Hfq selectively stabilized CoIP-enriched sRNAs, anticipating that these interactions are functionally significant. Transcription of 26 Hfq-bound sRNAs was predicted to occur from promoters recognized by the major stress σ factors σ(E2) or σ(H1/2). Recovery rates of sRNAs in each of the CoIP-RNA libraries suggest a large impact of Hfq-assisted riboregulation in S. meliloti osmoadaptation. Hfq directly targeted 18% of the predicted S. meliloti mRNAs, which encode functionally diverse proteins involved in transport and metabolism, σ(E2)-dependent stress responses, quorum sensing, flagella biosynthesis, ribosome, and membrane assembly or symbiotic nitrogen fixation. Canonical targeting of the 5' regions of two of the ABC transporter mRNAs by the homologous Hfq-binding AbcR1 and AbcR2 sRNAs leading to inhibition of protein synthesis was confirmed in vivo. We therefore provide a comprehensive resource for the systems-level deciphering of hitherto unexplored S. meliloti stress and symbiotic post-transcriptional regulons and the identification of Hfq-dependent sRNA-mRNA regulatory pairs.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4152363PMC
http://dx.doi.org/10.4161/rna.28239DOI Listing
March 2015

Use of the computer-retargeted group II intron RmInt1 of Sinorhizobium meliloti for gene targeting.

RNA Biol 2014 7;11(4):391-401. Epub 2014 Mar 7.

Grupo de Ecología Genética, Estación Experimental del Zaidín; Consejo Superior de Investigaciones Científicas; Granada, Spain.

Gene-targeting vectors derived from mobile group II introns capable of forming a ribonucleoprotein (RNP) complex containing excised intron lariat RNA and an intron-encoded protein (IEP) with reverse transcriptase (RT), maturase, and endonuclease (En) activities have been described. RmInt1 is an efficient mobile group II intron with an IEP lacking the En domain. We performed a comprehensive study of the rules governing RmInt1 target site recognition based on selection experiments with donor and recipient plasmid libraries, with randomization of the elements of the intron RNA involved in target recognition and the wild-type target site. The data obtained were used to develop a computer algorithm for identifying potential RmInt1 targets in any DNA sequence. Using this algorithm, we modified RmInt1 for the efficient recognition of DNA target sites at different locations in the Sinorhizobium meliloti chromosome. The retargeted RmInt1 integrated efficiently into the chromosome, regardless of the location of the target gene. Our results suggest that RmInt1 could be efficiently adapted for gene targeting.
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http://dx.doi.org/10.4161/rna.28373DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4075523PMC
January 2015
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