UG/Abi: a highly diverse family of prokaryotic reverse transcriptases associated with defense functions.

Reverse transcriptases (RTs) are enzymes capable of synthesizing DNA using RNA as a template. Within the last few years, a burst of research has led to the discovery of novel prokaryotic RTs with diverse antiviral properties, such as DRTs (Defense-associated RTs), which belong to the so-called group of unknown RTs (UG) and are closely related to the Abortive Infection system (Abi) RTs. In this work, we performed a systematic analysis of UG and Abi RTs, increasing the number of UG/Abi members up to 42 highly diverse groups, most of which are predicted to be functionally associated with other gene(s) or domain(s). Based on this information, we classified these systems into three major classes. In addition, we reveal that most of these groups are associated with defense functions and/or mobile genetic elements, and demonstrate the antiphage role of four novel groups. Besides, we highlight the presence of one of these systems in novel families of human gut viruses infecting members of the Bacteroidetes and Firmicutes phyla. This work lays the foundation for a comprehensive and unified understanding of these highly diverse RTs with enormous biotechnological potential.

PADLOC: a web server for the identification of antiviral defence systems in microbial genomes.

Most bacteria and archaea possess multiple antiviral defence systems that protect against infection by phages, archaeal viruses and mobile genetic elements. Our understanding of the diversity of defence systems has increased greatly in the last few years, and many more systems likely await discovery. To identify defence-related genes, we recently developed the Prokaryotic Antiviral Defence LOCator (PADLOC) bioinformatics tool. To increase the accessibility of PADLOC, we describe here the PADLOC web server (freely available at https://padloc.otago.ac.nz), allowing users to analyse whole genomes, metagenomic contigs, plasmids, phages and archaeal viruses. The web server includes a more than 5-fold increase in defence system types detected (since the first release) and expanded functionality enabling detection of CRISPR arrays and retron ncRNAs. Here, we provide user information such as input options, description of the multiple outputs, limitations and considerations for interpretation of the results, and guidance for subsequent analyses. The PADLOC web server also houses a precomputed database of the defence systems in > 230,000 RefSeq genomes. These data reveal two taxa, Campylobacterota and Spriochaetota, with unusual defence system diversity and abundance. Overall, the PADLOC web server provides a convenient and accessible resource for the detection of antiviral defence systems.

Identification of Group II Intron RmInt1 Binding Sites in a Bacterial Genome.

RmInt1 is a group II intron encoding a reverse transcriptase protein (IEP) lacking the C-terminal endonuclease domain. RmInt1 is an efficient mobile retroelement that predominantly reverse splices into the transient single-stranded DNA at the template for lagging strand DNA synthesis during host replication, a process facilitated by the interaction of the RmInt1 IEP with DnaN at the replication fork. It has been suggested that group II intron ribonucleoprotein particles bind DNA nonspecifically, and then scan for their correct target site. In this study, we investigated RmInt1 binding sites throughout the Sinorhizobium meliloti genome, by chromatin-immunoprecipitation coupled with next-generation sequencing. We found that RmInt1 binding sites cluster around the bidirectional replication origin of each of the three replicons comprising the S. meliloti genome. Our results provide new evidence linking group II intron mobility to host DNA replication.

Prokaryotic reverse transcriptases: from retroelements to specialized defense systems.

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.

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

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.

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

We report here the complete genome sequence of the salt-tolerant Sinorhizobium meliloti strain AK21, isolated from nodules of Medicago sativa L. subsp. ambigua 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.

Cirugía de Latarjet-Bankart artroscópico: evaluación tomográfica de la posición del bloque óseo. Primera experiencia chilena / Arthroscopic Latarjet-Bankart procedure

Introducción: Evaluar la precisión de la cirugía artroscópica de Latarjet­Bankart, mediante tomografía computada post operatoria. Describir resultados y complicaciones en la primera serie prospectiva de esta técnica en Chile.Material y Método: Quince pacientes fueron sometidos a cirugía de estabilización mediante la técnica artroscópica de Latarjet­Bankart. Se realizó una evaluación de la posición del injerto de coracoides mediante tomografía computada considerando los índices: ángulo tornillo - superficie articular, tornillo excesivamente largo, distancia línea articular - borde injerto en plano axial (método tangente), distancia borde injerto ­ superficie articular (método del círculo) en planos axial y coronal y relación posición injerto ­ diámetro glenoideo.Resultados: Se obtuvo un ángulo tornillo ­ superficie promedio de 32.9°. En un paciente se objetivó un tornillo de largo excesivo (+ 4 mm). De acuerdo al método tangente axial la distancia fue de 0 mm [0 mm ­ 2,5 mm], método circulo axial 0 mm [-0,8 mm ­ 1 mm], circulo coronal 0 mm [-1 mm ­ 2 mm]. En el 100% de los casos la posición injerto ­ diámetro glenoideo, fue bajo el 50% o subecuatorial. El injerto se encontró en posición "flush" en todos los pacientes. En un paciente fue necesario convertir a cirugía abierta. En un paciente ocurrió una factura parcial del injerto y un paciente presento una fractura de glenoides y una plexitis transitoria de 5 semanas. Un 13% de los pacientes presentó recurrencia al seguimiento a los 2 años.Conclusión: Es factible realizar esta técnica quirúrgica de manera artroscópica, con una baja necesidad de conversión y complicaciones, logrando una posición óptima del injerto.Tipo de estudio: Serie de Casos. Nivel de Evidencia: IV

Introduction: Evaluate the feasibility and precision of arthroscopic Latarjet-Bankart surgery, using computed tomography. Describe outcomes and complications in the first prospective series of this technique in Chile.Method: Fifteen patients underwent surgery using the arthroscopic Latarjet-Bankart technique. We evaluated the position of the coracoid graft by Computed Tomography, considering the following indices: screw angle - joint surface, excessively long screw incidence, joint line distance - graft edge in axial plane (tangent method), distance graft edge - joint surface (circle method) in axial and coronal planes, graft position relationship - glenoid diameter. Results: A screw angle - surface 32.9 ° was obtained. In one patient a screw of excessive length (+ 4mm). According to the axial tangent method the distance was 0mm [0mm - 2.5mm], axial circle method 0mm [-0.8mm - 1mm], coronal circle 0mm [-1mm - 2mm], in 100% of the cases the graft position - glenoid diameter was under 50% or subequatorial. The graft was found in the "flush" position in all patients. In one patient was necessary to convert to open surgery. One patient with partial graft fracture. One patient had a major complication, which was a glenoid fracture and a transient plexitis of 5 weeks. Instability recurrence was observed in 13% of patients at 2 years follow-up.Conclusion: It's feasible to perform this technique arthroscopically, with a low conversion and complications rate, obtaining an optimal position of the graft. Type study: Case Series. Level of Evidence: IV

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

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.

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.

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.

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

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-cas 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.

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

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 ß-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 ß-sliding clamp of the DNA replication machinery, favouring reverse splicing into the transient ssDNA at DNA replication forks.

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

The RmInt1 group II intron is an efficient self-splicing mobile retroelement that catalyzes its own excision as lariat, linear and circular molecules. In vivo, 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 in vitro 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.

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

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.

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

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.

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.

The Reverse Transcriptases Associated with CRISPR-Cas Systems.

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.

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

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.

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.

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

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.