Direct Repeats Research Articles

Unveiling the intricate structural variability induced by repeat-mediated recombination in the complete mitochondrial genome of Cuscuta gronovii Willd

Cuscuta gronovii Willd., a member of the Convolvulaceae family, is noted for its potential medicinal and nutritional benefits. In this study, we utilized a combination of Illumina and Oxford Nanopore sequencing technologies to successfully assemble the complete circular mitochondrial genome (mitogenome) of C. gronovii. The mitogenome, spanning 304,467 base pairs, includes 54 genes: 33 protein-coding genes, three ribosomal RNA (rRNA) genes, and 18 transfer RNA (tRNA) genes. Beyond its primary circular structure, we discovered and validated several alternative genomic conformations, driven by five specific repeat sequences. Three inverted repeats were found to initiate rearrangements, resulting in the creation of seven distinct chromosomal structures, while two direct repeats split a larger molecule into two subgenomic entities. We also mapped 421 RNA editing sites across the protein-coding sequences, influencing 33 protein-coding genes with varying distribution, particularly noting high frequencies in the nad4 and ccmB genes. Sixteen of these RNA editing sites were experimentally validated through PCR amplification and Sanger sequencing, confirming their presence with 100 % accuracy. This research not only introduces the first mitochondrial genome of C. gronovii but also highlights its complex conformational variability induced by repeat-mediated recombination, providing a valuable genomic resource for further molecular breeding efforts and phylogenetic evolution within the genus Cuscuta.

The complete mitochondrial DNA of the carnivorous sponge Lycopodina hypogea is putatively complemented by microDNAs.

Carnivorous sponges (Porifera, Demospongiae, Cladorhizidae), contrary to the usual filter-feeding mechanism of sponges, are specialized in catching larger prey through adhesive surfaces or hook-like spicules. The mitochondrial DNA of sponges overall present several divergences from other metazoans, and while presenting unique features among major transitions, such as in calcarean and glass sponges, poriferan mitogenomes are relatively stable within their groups. Here, we report and discuss the mitogenome of Lycopodina hypogea (Vacelet & Boury-Esnault, 1996), which greatly vary from its subordinal counterparts in both structure and gene order. This mitogenome is seemingly multipartite into three chromosomes, two of them as microDNAs. The main chromosome, chrM1, is unusually large, 31,099 bp in length, has a unique gene order within Poecilosclerida, and presents two rRNA, 13 protein and 19 tRNA coding genes. Intergenic regions comprise approximately 40% of chrM1, bearing several terminal direct and inverted repeats (TDRr and TIRs) but holding no vestiges of former mitochondrial sequences, pseudogenes, or transposable elements. The nd4l and trnI(gau) genes are likely located in microDNAs thus comprising putative mitochondrial chromosomes chrM2, 291 bp, and chrM3, 140 bp, respectively. It is unclear which processes are responsible for the remarkable features of the of L. hypogea mitogenome, including a generalized gene rearrangement, long IGRs, and putative extrachromosomal genes in microDNAs.

Repeat mediated excision of gene drive elements for restoring wild-type populations.

Here, we demonstrate that single strand annealing (SSA) can be co-opted for the precise autocatalytic excision of a drive element. We have termed this technology Repeat Mediated Excision of a Drive Element (ReMEDE). By engineering direct repeats flanking the drive allele and inducing a double-strand DNA break (DSB) at a second endonuclease target site within the allele, we increased the utilization of SSA repair. ReMEDE was incorporated into the mutagenic chain reaction (MCR) gene drive targeting the yellow gene of Drosophila melanogaster, successfully replacing drive alleles with wild-type alleles. Sequencing across the Cas9 target site confirmed transgene excision by SSA after pair-mated outcrosses with yReMEDE females, revealing ~4% inheritance of an engineered silent TcG marker sequence. However, phenotypically wild-type flies with alleles of indeterminate biogenesis also were observed, retaining the TGG sequence (~16%) or harboring a silent gGG mutation (~0.5%) at the PAM site. Additionally, ~14% of alleles in the F2 flies were intact or uncut paternally inherited alleles, indicating limited maternal deposition of Cas9 RNP. Although ReMEDE requires further research and development, the technology has some promising features as a gene drive mitigation strategy, notably its potential to restore wild-type populations without additional transgenic releases or large-scale environmental modifications.

Suppression of Huntington's Disease Somatic Instability by Transcriptional Repression and Direct CAG Repeat Binding.

Huntington's disease (HD) arises from a CAG expansion in the huntingtin ( HTT ) gene beyond a critical threshold. A major thrust of current HD therapeutic development is lowering levels of mutant HTT mRNA (m HTT ) and protein (mHTT) with the aim of reducing the toxicity of these product(s). Human genetic data also support a key role for somatic instability (SI) in HTT 's CAG repeat - whereby it lengthens with age in specific somatic cell types - as a key driver of age of motor dysfunction onset. Thus, an attractive HD therapy would address both mHTT toxicity and SI, but to date the relationship between SI and HTT lowering remains unexplored. Here, we investigated multiple therapeutically-relevant HTT-lowering modalities to establish the relationship between HTT lowering and SI in HD knock-in mice. We find that repressing transcription of mutant Htt (m Htt ) provides robust protection from SI, using diverse genetic and pharmacological approaches (antisense oligonucleotides, CRISPR-Cas9 genome editing, the Lac repressor, and virally delivered zinc finger transcriptional repressor proteins, ZFPs). However, we find that small interfering RNA (siRNA), a potent HTT-lowering treatment, lowers HTT levels without influencing SI and that SI is also normal in mice lacking 50% of total HTT levels, suggesting HTT levels, per se , do not modulate SI in trans . Remarkably, modified ZFPs that bind the m Htt locus, but lack a repressive domain, robustly protect from SI, despite not reducing HTT mRNA or protein levels. These results have important therapeutic implications in HD, as they suggest that DNA-targeted HTT-lowering treatments may have significant advantages compared to other HTT-lowering approaches, and that interaction of a DNA-binding protein and HTT' s CAG repeats may provide protection from SI while sparing HTT expression.

Fine-tuning the evolutionary stability of recombinant herpesviral transmissible vaccines.

Spillover of infectious diseases from wild animal populations constitutes a long-standing threat to human health for which few globally viable solutions have been developed. The use of oral baits laden with conventional vaccines distributed en masse represents one success story but is costly and practicable primarily for rabies risk reduction in North American and European carnivores. Efforts to expand vaccination to control pathogens within less accessible wildlife populations have raised interest in a new kind of vaccine capable of spreading pathogen-specific immunity through autonomous spread. However, such 'transmissible' vaccines raise concerns about the irrevocable release of genetically modified viruses into the environment. Herein, we explore the feasibility of an intrinsic strategy for transgene control within these vaccines based on the genetic destabilizing effect of cis-acting sequences flanking the heterologous transgene of interest. While suitable for the control of transgene stability within all types of DNA-viral vectored vaccines, this strategy has particular applicability to transmissible vaccines. Using a combination of experiments, mathematical modelling and whole-genome sequencing, we show that the rate of transgene loss can be controlled by varying the lengths of the direct repeat sequences. This opens a way for fine-tuning the lifespan of a transmissible vaccine in the wild.

CRISPR-Cas systems in enterococci.

Enterococci are members of the microbiota of humans and other animals. They can also be found in the environment, associated with food, healthcare infections, and hospital settings. Due to their wide distribution, they are inserted in the One Health context. The selective pressure caused by the extensive use of antimicrobial agents in humans, animals, and agriculture has increased the frequency of resistance to various drugs among enterococcal species. CRISPR-Cas system, an important prokaryotic defense mechanism against the entry of mobile genetic elements, may prevent the acquisition of genes involved in antimicrobial resistance and virulence. This system has been increasingly used as a gene editing tool, which can be used as a way to recognize and inactivate genes of interest. Here, we conduct a review on CRISPR systems found in enterococci, considering their occurrence, structure and organization, mechanisms of action and use as a genetic engineering technology. Type II-A CRISPR-Cas systems were shown to be the most frequent among enterococcal species, and the orphan CRISPR2 was the most commonly found system (54.1%) among enterococcal species, especially in Enterococcus faecalis. Distribution of CRISPR systems varied among species. CRISPR systems had 1 to 20 spacers, with size between 23 and 37bp and direct repeat sequences from 25 to 37bp. Several applications of the CRISPR-Cas biotechnology have been described in enterococci, mostly in vitro, using this editing tool to target resistance- and virulence-related genes.

Disruption of mgrB gene by ISkpn14 sourced from a blaKPC−2 carrying plasmid mediating polymyxin resistance against carbapenem-resistant Klebsiella pneumoniae during treatment: study on the underlying mechanisms

BackgroundCarbapenem-resistant Klebsiella pneumoniae (CRKP) infections poses global challenges, with limited options available for targeted therapy. Polymyxin was been regarded as one of the most important last-resort antimicrobial agents. Many factors could accelerate the resistance evolution of polymyxin. Insertion sequence (IS) inserted into mgrB is the main polymyxin resistance mechanism in K. pneumoniae. In this study, two CRKPs (KP31157 and KP31311) were isolated from the urine of a patient, shifting from susceptible to resistant as the mgrB inserted by ISkpn14. We intended to explore the origin of the IS and underlying mechanisms resulting in polymyxin resistance.MethodsThe within-host evolution relationship and molecular features of both CRKPs were determined by pulsed-field gel electrophoresis (PFGE) and whole-genome sequencing (WGS). pKP31311_KPC-2 plasmid genome structures contained in the above two CRKPs were aligned with the homologic plasmids, retrieved from the NCBI genome database via comparative genomic analysis. The plasmids encoding ISkpn14 elements flanked by direct repeat (DR) or not were analyzed. The mRNA expression, plasmid curing and in vitro antibiotics inducing experiment were employed to understand the potential mechanism of polymyxin resistance.ResultsBoth strains, sharing homology, exhibited polymyxin resistance due to the insertion of ISkpn14 into the mgrB gene, influenced by minocycline exposure. Minocycline and tigecycline could accelerate polymyxin resistance (P < 0.05), validated by an in vitro induction experiment. The ISkpn14 without DR flanked expressed about 4 times higher than that with DR. The frequency of the mgrB insertion induced by polymyxin was significantly reduced (0 strain detected) after the blaKPC−2-carrying plasmid was eliminated.ConclusionsThis study provides direct experimental evidence that the ISkpn14 element causing mgrB inactivation and polymyxin resistance in K. pneumoniae originates from blaKPC−2-carrying plasmids. Minocycline exposure will accelerate the evolution of polymyxin resistance. Understanding the dynamics of IS transposition and its association with antibiotic exposure is crucial for developing effective strategies to reduce the emergence of polymyxin resistance in CRKP.

Pipolins are bimodular platforms that maintain a reservoir of defense systems exchangeable with various bacterial genetic mobile elements.

Defense genes gather in diverse types of genomic islands in bacteria and provide immunity against viruses and other genetic mobile elements. Here, we disclose pipolins, previously found in diverse bacterial phyla and encoding a primer-independent PolB, as a new category of widespread defense islands. The analysis of the occurrence and structure of pipolins revealed that they are commonly integrative elements flanked by direct repeats in Gammaproteobacteria genomes, mainly Escherichia, Vibrio or Aeromonas, often taking up known mobile elements integration hotspots. Remarkably, integrase dynamics correlates with alternative integration spots and enables diverse lifestyles, from integrative to mobilizable and plasmid pipolins, such as in members of the genera Limosilactobacillus, Pseudosulfitobacter or Staphylococcus. Pipolins harbor a minimal core and a large cargo module enriched for defense factors. In addition, analysis of the weighted gene repertoire relatedness revealed that many of these defense factors are actively exchanged with other mobile elements. These findings indicate pipolins and, potentially other defense islands, act as orthogonal reservoirs of defense genes, potentially transferable to immune autonomous MGEs, suggesting complementary exchange mechanisms for defense genes in bacterial populations.

The epistatic relationship of Drosophila melanogaster CtIP and Rif1 in homology-directed repair of DNA double-strand breaks.

Double-strand breaks (DSBs) are genotoxic DNA lesions that pose significant threats to genomic stability, necessitating precise and efficient repair mechanisms to prevent cell death or mutations. DSBs are repaired through nonhomologous end-joining (NHEJ) or homology-directed repair (HDR), which includes homologous recombination (HR) and single-strand annealing (SSA). CtIP and Rif1 are conserved proteins implicated in DSB repair pathway choice, possibly through redundant roles in promoting DNA end-resection required for HDR. Although the roles of these proteins have been well-established in other organisms, the role of Rif1 and its potential redundancies with CtIP in Drosophila melanogaster remain elusive. To examine the roles of DmCtIP and DmRif1 in DSB repair, this study employed the direct repeat of white (DR-white) assay, tracking across indels by decomposition (TIDE) analysis, and P{wIw_2 kb 3'} assay to track repair outcomes in HR, NHEJ, and SSA, respectively. These experiments were performed in DmCtIPΔ/Δ single mutants, DmRif1Δ/Δ single mutants, and DmRif1Δ/Δ; DmCtIPΔ/Δ double mutants. This work demonstrates significant defects in both HR and SSA repair in DmCtIPΔ/Δ and DmRif1Δ/Δ single mutants. However, experiments in DmRif1Δ/Δ; DmCtIPΔ/Δ double mutants reveal that DmCtIP is epistatic to DmRif1 in promoting HDR. Overall, this study concludes that DmRif1 and DmCtIP do not perform their activities in a redundant pathway, but rather DmCtIP is the main driver in promoting HR and SSA, most likely through its role in end resection.

PAM-relaxed and temperature-tolerant CRISPR-Mb3Cas12a single transcript unit systems for efficient singular and multiplexed genome editing in rice, maize, and tomato.

Class 2 Type V-A CRISPR-Cas (Cas12a) nucleases are powerful genome editing tools, particularly effective in A/T-rich genomic regions, complementing the widely used CRISPR-Cas9 in plants. To enhance the utility of Cas12a, we investigate three Cas12a orthologs-Mb3Cas12a, PrCas12a, and HkCas12a-in plants. Protospacer adjacent motif (PAM) requirements, editing efficiencies, and editing profiles are compared in rice. Among these orthologs, Mb3Cas12a exhibits high editing efficiency at target sites with a simpler, relaxed TTV PAM which is less restrictive than the canonical TTTV PAM of LbCas12a and AsCas12a. To optimize Mb3Cas12a, we develop an efficient single transcription unit (STU) system by refining the linker between Mb3Cas12a and CRISPR RNA (crRNA), nuclear localization signal (NLS), and direct repeat (DR). This optimized system enables precise genome editing in rice, particularly for fine-tuning target gene expression by editing promoter regions. Further, we introduced Arginine (R) substitutions at Aspartic acid (D) 172, Asparagine (N) 573, and Lysine (K) 579 of Mb3Cas12a, creating two temperature-tolerant variants: Mb3Cas12a-R (D172R) and Mb3Cas12a-RRR (D172R/N573R/K579R). These variants demonstrate significantly improved editing efficiency at lower temperatures (22 °C and 28 °C) in rice cells, with Mb3Cas12a-RRR showing the best performance. We extend this approach by developing efficient Mb3Cas12a-RRR STU systems in maize and tomato, achieving biallelic mutants targeting single or multiple genes in T0 lines cultivated at 28 °C and 25 °C, respectively. This study significantly expands Cas12a's targeting capabilities in plant genome editing, providing valuable tools for future research and practical applications.

Context effects on repair of 5'-overhang DNA double-strand breaks induced by Cas12a in Arabidopsis.

Sequence-specific endonucleases have been key to the study of the mechanisms and control of DNA double-strand break (DSB) repair and recombination, and the availability of CRISPR-Cas nucleases over the last decade has driven rapid progress in the understanding and application of targeted recombination in many organisms, including plants. We present here an analysis of recombination at targeted chromosomal 5' overhang DSB generated by the FnCas12a endonuclease in the plant, Arabidopsis thaliana. The much-studied Cas9 nuclease cleaves DNA to generate blunt-ended DSBs, but relatively less is known about the repair of other types of breaks, such as those with 5'-overhanging ends. Sequencing the repaired breaks clearly shows that the majority of repaired DSB carry small deletions and are thus repaired locally by end-joining recombination, confirmed by Nanopore sequencing of larger amplicons. Paired DSBs generate deletions at one or both cut-sites, as well as deletions and reinsertions of the deleted segment between the two cuts, visible as inversions. While differences are seen in the details, overall the deletion patterns are similar between repair at single-cut and double-cut events, notwithstanding the fact that only the former involve cohesive DNA overhangs. A strikingly different repair pattern is however observed at breaks flanked by direct repeats. This change in sequence context results in the presence of a major alternative class of repair events, corresponding to highly efficient repair by single-strand annealing recombination.

Complete analysis of G-quadruplex forming sequences in the gapless assembly of human chromosome Y

Recent advancements have finally delivered a complete human genome assembly, including the elusive Y chromosome. This accomplishment closes a significant knowledge gap. Prior efforts were hampered by challenges in sequencing repetitive DNA structures such as direct and inverted repeats. We used the G4Hunter algorithm to analyze the presence of G-quadruplex forming sequences (G4s) within the current human reference genome (GRCh38) and the new telomere-to-telomere (T2T) Y chromosome assemblies. This analysis served a dual purpose: identifying the location of potential G4s within the genomes and exploring their association with functionally annotated sequences. Compared to GRCh38, the T2T assembly exhibited a significantly higher prevalence of G-quadruplex forming sequences. Notably, these repeats were abundantly located around precursor RNA, exons, genes, and within protein binding sites. This remarkable co-occurrence of G4-forming sequences with these critical regulatory regions suggests their role in fundamental DNA regulation processes. Our findings indicate that the current human reference genome significantly underestimated the number of G4s, potentially overlooking their functional importance.

Transposon-mediated genic rearrangements underlie variation in small RNA pathways

Transposable elements (TEs) can alter host gene structure and expression, whereas host organisms develop mechanisms to repress TE activities. In the nematode Caenorhabditis elegans , a small interfering RNA pathway dependent on the helicase ERI-6/7 primarily silences retrotransposons and recent genes of likely viral origin. By studying gene expression variation among wild C. elegans strains, we found that structural variants and transposon remnants likely underlie expression variation in eri-6/7 and the pathway targets. We further found that multiple insertions of the DNA transposons, Polintons, reshuffled the eri-6/7 locus and induced inversion of eri-6 in some wild strains. In the inverted configuration, gene function was previously shown to be repaired by unusual trans-splicing mediated by direct repeats. We identified that these direct repeats originated from terminal inverted repeats of Polintons . Our findings highlight the role of host-transposon interactions in driving rapid host genome diversification among natural populations and shed light on evolutionary novelty in genes and splicing mechanisms.

CRISETR: an efficient technology for multiplexed refactoring of biosynthetic gene clusters.

The efficient refactoring of natural product biosynthetic gene clusters (BGCs) for activating silent BGCs is a central challenge for the discovery of new bioactive natural products. Herein, we have developed a simple and robust CRISETR (CRISPR/Cas9 and RecET-mediated Refactoring) technique, combining clustered regulatory interspaced short palindromic repeats (CRISPR)/Cas9 and RecET, for the multiplexed refactoring of natural product BGCs. By this approach, natural product BGCs can be refactored through the synergistic interaction between RecET-mediated efficient homologous recombination and the CRISPR/Cas9 system. We firstperformed a proof-of-concept validation of the ability of CRISETR, and CRISETR can achieve simultaneous replacement of four promoter sites and marker-free replacement of single promoter site in natural product BGCs. Subsequently, we applied CRISETR to the promoter engineering of the 74-kb daptomycin BGC containing a large number of direct repeat sequences forenhancingthe heterologous production of daptomycin. We used combinatorial design to build multiple refactored daptomycin BGCs with diverse combinations of promoters different in transcriptional strengths, and the yield of daptomycin was improved 20.4-fold in heterologous host Streptomyces coelicolor A3(2). In general, CRISETR exhibits enhanced tolerance to repetitive sequences within gene clusters, enabling efficient refactoring of diverse and complex BGCs, which would greatly accelerate discovery of novel bioactive metabolites present in microorganism.

In Vitro and In Vivo Assessments of Newly Isolated N4-like Bacteriophage against ST45 K62 Capsular-Type Carbapenem-Resistant Klebsiella pneumoniae: vB_kpnP_KPYAP-1.

The rise of carbapenem-resistant Klebsiella pneumoniae (CRKP) presents a significant global challenge in clinical and healthcare settings, severely limiting treatment options. This study aimed to utilize a bacteriophage as an alternative therapy against carbapenem-resistant K. pneumoniae. A novel lytic N4-like Klebsiella phage, vB_kpnP_KPYAP-1 (KPYAP-1), was isolated from sewage. It demonstrated efficacy against the K62 serotype polysaccharide capsule of blaOXA-48-producing K. pneumoniae. KPYAP-1 forms small, clear plaques, has a latent period of 20 min, and reaches a growth plateau at 35 min, with a burst size of 473 plaque-forming units (PFUs) per infected cell. Phylogenetic analysis places KPYAP-1 in the Schitoviridae family, Enquatrovirinae subfamily, and Kaypoctavirus genus. KPYAP-1 employs an N4-like direct terminal repeat mechanism for genome packaging and encodes a large virion-encapsulated RNA polymerase. It lacks integrase or repressor genes, antibiotic resistance genes, bacterial virulence factors, and toxins, ensuring its safety for therapeutic use. Comparative genome analysis revealed that the KPYAP-1 genome is most similar to the KP8 genome, yet differs in tail fiber protein, indicating variations in host recognition. In a zebrafish infection model, KPYAP-1 significantly improved the survival rate of infected fish by 92% at a multiplicity of infection (MOI) of 10, demonstrating its potential for in vivo treatment. These results highlight KPYAP-1 as a promising candidate for developing phage-based therapies targeting carbapenemase-producing K. pneumoniae.

Characterization and functional insights of the novel RC-type plasmid pAnox1 from Anoxybacillus gonensis 05S15

The plasmid pAnox1, isolated from Anoxybacillus gonensis 05S15, was sequenced and characterized as a circular, double-stranded DNA molecule of 1592 base pairs with a GC content of 40.01 %. Despite its cryptic nature and small genome, bioinformatic analyses identified conserved motifs associated with replication-related proteins, though BLAST searches revealed no significant homology with other plasmids. The plasmid genome contains five putative Open Reading Frames (ORFs), four palindromic sequences, and two direct repeats on both strands, suggesting regulatory roles. Electron microscopy and Southern hybridization studies confirmed that pAnox1 follows a Rolling Circle (RC) replication mode. The study further demonstrated that the plasmid encodes three distinct transcripts: ORF-1 and ORF-3 are oriented in the same direction, while ORF-5 is on the opposite strand. RACE and LACE analyses revealed transcript lengths of 903 bp for ORF1, 499 bp for ORF3, and 211 bp for ORF5. Quantitative real-time PCR estimated the relative copy number of pAnox1 at 127 ± 2 copies per chromosomal equivalent. This novel RC-type plasmid in the Anoxybacillus genome holds promise as a cloning and expression vector for biotechnological applications and in vivo protein engineering.

Intra-Host Citrus Tristeza Virus Populations during Prolonged Infection Initiated by a Well-Defined Sequence Variant in Nicotiana benthamiana.

Due to the error-prone nature of viral RNA-dependent RNA polymerases, the replication of RNA viruses results in a diversity of viral genomes harboring point mutations, deletions, insertions, and genome rearrangements. Citrus tristeza virus (CTV), a causal agent of diseases of economically important citrus species, shows intrinsic genetic stability. While the virus appears to have some mechanism that limits the accumulation of single-nucleotide variants, the production of defective viral genomes (DVGs) during virus infection has been reported for certain variants of CTV. The intra-host diversity generated during plant infection with variant T36 (CTV-T36) remains unclear. To address this, we analyzed the RNA species accumulated in the initially infected and systemic leaves of Nicotiana benthamiana plants inoculated with an infectious cDNA clone of CTV-T36, which warranted that infection was initiated by a known, well-defined sequence variant of the virus. CTV-T36 limited the accumulation of single-nucleotide mutants during infection. With that, four types of DVGs-deletions, insertions, and copy- and snap-backs-were found in all the samples, with deletions and insertions being the most common types. Hot-spots across the genome for DVG recombination and short direct sequence repeats suggest that sequence complementarity could mediate DVG formation. In conclusion, our study illustrates the formation of diverse DVGs during CTV-T36 infection. To the best of our knowledge, this is the first study that has analyzed the genetic variability and recombination of a well-defined sequence variant of CTV in an herbaceous host.

Global Distribution and Diversity of Haloarchaeal pL6-Family Plasmids.

Australian isolates of Haloquadratum walsbyi, a square-shaped haloarchaeon, often harbor small cryptic plasmids of the pL6-family, approximately 6 kb in size, and five examples have been previously described. These plasmids exhibit a highly conserved gene arrangement and encode replicases similar to those of betapleolipoviruses. To assess their global distribution and recover more examples for analysis, fifteen additional plasmids were reconstructed from the metagenomes of seven hypersaline sites across four countries: Argentina, Australia, Puerto Rico, and Spain. Including the five previously described plasmids, the average plasmid size is 6002 bp, with an average G+C content of 52.5%. The tetramers GGCC and CTAG are either absent or significantly under-represented, except in the two plasmids with the highest %G+C. All plasmids share a similar arrangement of genes organized as outwardly facing replication and ATPase modules, but variations were observed in some core genes, such as F2, and some plasmids had acquired accessory genes. Two plasmids, pCOLO-c1 and pISLA-c6, shared 92.7% nt identity despite originating from Argentina and Spain, respectively. Numerous metagenomic CRISPR spacers matched sequences in the fifteen reconstructed plasmids, indicating frequent invasion of haloarchaea. Spacers could be assigned to haloarchaeal genera by mapping their associated direct repeats (DR), with half of these matching Haloquadratum. Finally, strand-specific metatranscriptome (RNA-seq) data could be used to demonstrate the active transcription of two pL6-family plasmids, including antisense transcripts.

Distinct horizontal transfer mechanisms for type I and type V CRISPR-associated transposons

CASTs use both CRISPR-associated proteins and Tn7-family transposons for RNA-guided vertical and horizontal transmission. CASTs encode minimal CRISPR arrays but can’t acquire new spacers. Here, we report that CASTs can co-opt defense-associated CRISPR arrays for horizontal transmission. A bioinformatic analysis shows that CASTs co-occur with defense-associated CRISPR systems, with the highest prevalence for type I-B and type V CAST sub-types. Using an E. coli quantitative transposition assay and in vitro reconstitution, we show that CASTs can use CRISPR RNAs from these defense systems. A high-resolution structure of the type I-F CAST-Cascade in complex with a type III-B CRISPR RNA reveals that Cas6 recognizes direct repeats via sequence-independent π − π interactions. In addition to using heterologous CRISPR arrays, type V CASTs can also transpose via an unguided mechanism, even when the S15 co-factor is over-expressed. Over-expressing S15 and the trans-activating CRISPR RNA or a single guide RNA reduces, but does not abrogate, off-target integration for type V CASTs. Our findings suggest that some CASTs may exploit defense-associated CRISPR arrays and that this fact must be considered when porting CASTs to heterologous bacterial hosts. More broadly, this work will guide further efforts to engineer the activity and specificity of CASTs for gene editing applications.

Horizontally transferred mitochondrial DNA tracts become circular by microhomology-mediated repair pathways.

The holoparasitic plant Lophophytum mirabile exhibits remarkable levels of mitochondrial horizontal gene transfer (HGT). Gathering comparative data from other individuals and host plants can provide insights into the HGT process. We sequenced the mitochondrial genome (mtDNA) from individuals of two species of Lophophytum and from mimosoid hosts. We applied a stringent phylogenomic approach to elucidate the origin of the whole mtDNAs, estimate the timing of the transfers, and understand the molecular mechanisms involved. Ancestral and recent HGT events replaced and enlarged the multichromosomal mtDNA of Lophophytum spp., with the foreign DNA ascending to 74%. A total of 14 foreign mitochondrial chromosomes originated from continuous regions in the host mtDNA flanked by short direct repeats. These foreign tracts are circularized by microhomology-mediated repair pathways and replicate independently until they are lost or they eventually recombine with other chromosomes. The foreign noncoding chromosomes are variably present in the population and likely evolve by genetic drift. We present the 'circle-mediated HGT' model in which foreign mitochondrial DNA tracts become circular and are maintained as plasmid-like molecules. This model challenges the conventional belief that foreign DNA must be integrated into the recipient genome for successful HGT.