Adaptive immunity of type VI CRISPR-Cas systems associated with reverse transcriptase-Cas1 fusion proteins.

Cas13-containing type VI CRISPR-Cas systems specifically target RNA; however, the mechanism of spacer acquisition remains unclear. We have previously reported the association of reverse transcriptase-Cas1 (RT-Cas1) fusion proteins with certain types of VI-A systems. Here, we show that RT-Cas1 fusion proteins are also recruited by type VI-B systems in bacteria from gut microbiomes, constituting a VI-B1 variant system that includes a CorA-encoding locus in addition to the CRISPR array and the RT-Cas1/Cas2 adaptation module.

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.

DNA cleavage and reverse splicing of ribonucleoprotein particles reconstituted 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. , 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.

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.

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.

Bacterial Group II Introns: Identification and Mobility Assay.

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.

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

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.

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.

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

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.

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.

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.

Relevance of the branch point adenosine, coordination loop, and 3' exon binding site for in vivo excision of the Sinorhizobium meliloti group II intron RmInt1.

Excision of the bacterial group II intron RmInt1 has been demonstrated in vivo, resulting in the formation of both intron lariat and putative intron RNA circles. We show here that the bulged adenosine in domain VI of RmInt1 is required for splicing via the branching pathway, but branch site mutants produce small numbers of RNA molecules in which the first G residue of the intron is linked to the last C residue. Mutations in the coordination loop in domain I reduced splicing efficiency, but branched templates clearly predominated among splicing products.

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.

An alternative intron-exon pairing scheme implied by unexpected in vitro activities of group II intron RmInt1 from Sinorhizobium meliloti.

RmInt1 is a mobile group II intron which interrupts ISRm2011-2, another mobile element from the bacterium Sinorhizobium meliloti. Ribozyme constructs derived from intron RmInt1 self-splice in vitro when incubated under permissive conditions, but the excised intron and ligated exons are largely replaced by unconventional products. These include a slightly shorter, 5'-end truncated 3' exon, truncated variants of the linear and lariat forms of the intron-3' exon reaction intermediate, as well as presumably circular molecules derived from the latter.

Identification and characterization of bacterial class E group II introns.

Group II introns are catalytic RNAs and mobile genetic elements. Phylogenetic characterization of group II intron-encoded reverse transcriptases (RTs) established seven classes: the mitochondrial class, chloroplast-like classes 1 and 2, and bacterial classes A, B, C, and D. In this study, we identified and characterized a new bacterial class of group II introns, bacterial class E, on the basis of phylogenetic analysis of the intron-encoded protein (IEP) RT and determination of a consensus intron RNA structure.