Endonuclease Gene Research Articles

A putative endonuclease reduces the efficiency of oral RNA interference in Nilaparvata lugens.

The RNA interference (RNAi) efficiency of double-stranded RNA (dsRNA) delivery to insects by various methods is different and the reduced efficacy of feeding dsRNA is partly due to the presence of DNA/RNA non-specific endonuclease in the insect gut. However, the mechanism leading to the low RNAi efficiency of Nilaparvata lugens by feeding remains elusive. In this study, we identified a putatively DNA/RNA non-specific endonuclease gene in the N. lugens genome database that was highly expressed in the first nymphal instar and the midgut. Different expression levels of NldsRNase after feeding and injection suggested that NldsRNase might interfere with oral RNAi in N. lugens. A co-delivery RNAi strategy further revealed that the presence of NldsRNase reduces RNAi efficiency. In vitro dsRNA degradation experiments also showed that the stability of dsRNA was higher in a gut mixture from nymphs injected with dsNldsRNase. These results support the idea that the low oral RNAi response observed in N. lugens is likely due to the presence of NldsRNase. Our study provides insight into the differences in RNAi response between the injection and feeding of dsRNA in N. lugens and sheds light on the mechanisms underlying the reduced efficacy of RNAi via feeding. These findings may help to inform the development of more-effective RNAi-based strategies controlling N. lugens and other insect pests. © 2024 Society of Chemical Industry.

Insights into phage-bacteria interaction in cold seep Gigantidas platifrons through metagenomics and transcriptome analyses

Viruses are crucial for regulating deep-sea microbial communities and biogeochemical cycles. However, their roles are still less characterized in deep-sea holobionts. Bathymodioline mussels are endemic species inhabiting cold seeps and harboring endosymbionts in gill epithelial cells for nutrition. This study unveiled a diverse array of viruses in the gill tissues of Gigantidas platifrons mussels and analyzed the viral metagenome and transcriptome from the gill tissues of Gigantidas platifrons mussels collected from a cold seep in the South Sea. The mussel gills contained various viruses including Baculoviridae, Rountreeviridae, Myoviridae and Siphovirdae, but the active viromes were Myoviridae, Siphoviridae, and Podoviridae belonging to the order Caudovirales. The overall viral community structure showed significant variation among environments with different methane concentrations. Transcriptome analysis indicated high expression of viral structural genes, integrase, and restriction endonuclease genes in a high methane concentration environment, suggesting frequent virus infection and replication. Furthermore, two viruses (GP-phage-contig14 and GP-phage-contig72) interacted with Gigantidas platifrons methanotrophic gill symbionts (bathymodiolin mussels host intracellular methanotrophic Gammaproteobacteria in their gills), showing high expression levels, and have huge different expression in different methane concentrations. Additionally, single-stranded DNA viruses may play a potential auxiliary role in the virus–host interaction using indirect bioinformatics methods. Moreover, the Cro and DNA methylase genes had phylogenetic similarity between the virus and Gigantidas platifrons methanotrophic gill symbionts. This study also explored a variety of viruses in the gill tissues of Gigantidas platifrons and revealed that bacteria interacted with the viruses during the symbiosis with Gigantidas platifrons. This study provides fundamental insights into the interplay of microorganisms within Gigantidas platifrons mussels in deep sea.

The complete mitochondrial genome of a species of Cirrhipathes de Blainville, 1830 from Kauaʻi, Hawaiʻi (Hexacorallia: Antipatharia)

This study reports the first mitogenome from the antipatharian (black coral) genus Cirrhipathes (GenBank accession number ON653414). The 20,452 bp mitochondrial genome of Cirrhipathes cf. anguina LS-2022 consists of 13 protein-coding genes, two rRNA genes, and two tRNA genes (trnM and trnW). The mitogenome is typical of other antipatharian families, including an A + T biased (64.1%) base composition and cytochrome c oxidase subunit I (COX1) intron with embedded homing endonuclease gene (HEG). A phylogenetic tree based on complete mitogenome sequences of currently available antipatharians indicates Cirrhipathes cf. anguina LS-2022 is sister and closely related to Stichopathes sp. SCBUCN-8849. However, it seems unlikely that intergeneric taxa share 99.97% similarity across their complete mitogenomes, raising questions about the current taxonomy of this group. This study highlights the need for additional vouchered antipatharian species to be sequenced so phylogenetic relationships can be compared with accepted taxonomy.

Parallel evolution and enhanced virulence upon in vivo passage of an RNA virus in Drosophila melanogaster.

Virus evolution is strongly affected by antagonistic co-evolution of virus and host. Host immunity positively selects for viruses that evade the immune response, which in turn may drive counter-adaptations in host immune genes. We investigated how host immune pressure shapes virus populations, using the fruit fly Drosophila melanogaster and its natural pathogen Drosophila C virus (DCV), as a model. We performed an experimental evolution study in which DCV was serially passaged for ten generations in three fly genotypes differing in their antiviral RNAi response: wild-type flies and flies in which the endonuclease gene Dicer-2 was either overexpressed or inactivated. All evolved virus populations replicated more efficiently in vivo and were more virulent than the parental stock. The number of polymorphisms increased in all three host genotypes with passage number, which was most pronounced in Dicer-2 knockout flies. Mutational analysis showed strong parallel evolution, as mutations accumulated in a specific region of the VP3 capsid protein in every lineage in a host genotype-independent manner. The parental tyrosine at position ninety-five of VP3 was substituted with either one of five different amino acids in fourteen out of fifteen lineages. However, no consistent amino acid changes were observed in the viral RNAi suppressor gene 1A, nor elsewhere in the genome in any of the host backgrounds. Our study indicates that the RNAi response restricts the sequence space that can be explored by viral populations. Moreover, our study illustrates how evolution towards higher virulence can be a highly reproducible, yet unpredictable process.

SITE-directed mutagenesis for producing grain sorgum mutants with improved kafirine digestibility

The use of genome editing technologies opens wide opportunities for the targeted mutagenesis in important agricultural crops. In the context of global warming, sorghum, an important drought- and heat-tolerant crop is of particular importance. However, compared to other cereals, sorghum grain has a lower nutritional value, due to the resistance of its storage proteins (kafirins) to proteolytic digestion. A decrease in the synthesis of kafirins as a result of mutations or the expression of the RNAi genetic constructs modifies the ultrastructure of protein bodies and improves their digestibility by proteases. To obtain mutants with improved protein digestibility, we created four binary vectors for site-directed mutagenesis of the k1C5 and gKAF1 genes encoding α- and γ-kafirin, respectively. Each of these vectors contained the cas9 endonuclease gene and a guide RNA targeted the nucleotide sequences encoding the kafirin signal polypeptides. By means of agrobacterial transformation, the created vectors were introduced into the genome of the grain sorghum cv. Avans. 14 transgenic plants were regenerated. Sequencing of 5 regenerants obtained using a vector for the k1C5 mutagenesis revealed 3 plants with mutations. The offspring of these mutants had a higher digestibility of grain proteins in vitro (86–92%) compared to the initial cv. Avans (63–67%). Notably, the T1 plants lacked the cas9 gene and the bar marker gene, which indicates the production of mutants with the edited k1C5 gene sequence, which lack the genetic construct that induced this mutation. Two mutants with mutations in the gKAF1 sequence were obtained. Thus, using the CRISPR/Cas technology, we have obtained mutants with improved digestibility of kafirins, which can be used in practical sorghum breeding.

Engineered Gut Symbiotic Bacterium-Mediated RNAi for Effective Control of Anopheles Mosquito Larvae.

Anopheles mosquitoes are the primary vectors for the transmission of malaria parasites, which poses a devastating burden on global public health and welfare. The recent invasion of Anopheles stephensi in Africa has made malaria eradication more challenging due to its outdoor biting behavior and widespread resistance to insecticides. To address this issue, we developed a new approach for mosquito larvae control using gut microbiota-mediated RNA interference (RNAi). We engineered a mosquito symbiotic gut bacterium, Serratia fonticola, by deleting its RNase III gene to produce double-stranded RNAs (dsRNAs) in the mosquito larval gut. We found that the engineered S. fonticola strains can stably colonize mosquito larval guts and produce dsRNAs dsMet or dsEcR to activate RNAi and effectively suppress the expression of methoprene-tolerant gene Met and ecdysone receptor gene EcR, which encode receptors for juvenile hormone and ecdysone pathways in mosquitoes, respectively. Importantly, the engineered S. fonticola strains markedly inhibit the development of A. stephensi larvae and leads to a high mortality, providing an effective dsRNA delivery system for silencing genes in insects and a novel RNAi-mediated pest control strategy. Collectively, our symbiont-mediated RNAi (smRNAi) approach offers an innovative and sustainable method for controlling mosquito larvae and provides a promising strategy for combating malaria. IMPORTANCE Mosquitoes are vectors for various diseases, imposing a significant threat to public health globally. The recent invasion of A. stephensi in Africa has made malaria eradication more challenging due to its outdoor biting behavior and widespread resistance to insecticides. RNA interference (RNAi) is a promising approach that uses dsRNA to silence specific genes in pests. This study presents the use of a gut symbiotic bacterium, Serratia fonticola, as an efficient delivery system of dsRNA for RNAi-mediated pest control. The knockout of RNase III, a dsRNA-specific endonuclease gene, in S. fonticola using CRISPR-Cas9 led to efficient dsRNA production. Engineered strains of S. fonticola can colonize the mosquito larval gut and effectively suppress the expression of two critical genes, Met and EcR, which inhibit mosquito development and cause high mortality in mosquito larvae. This study highlights the potential of exploring the mosquito microbiota as a source of dsRNA for RNAi-based pest control.

Nuclease genes occupy boundaries of genetic exchange between bacteriophages.

Homing endonuclease genes (HEGs) are ubiquitous selfish elements that generate targeted double-stranded DNA breaks, facilitating the recombination of the HEG DNA sequence into the break site and contributing to the evolutionary dynamics of HEG-encoding genomes. Bacteriophages (phages) are well-documented to carry HEGs, with the paramount characterization of HEGs being focused on those encoded by coliphage T4. Recently, it has been observed that the highly sampled vibriophage, ICP1, is similarly enriched with HEGs distinct from T4's. Here, we examined the HEGs encoded by ICP1 and diverse phages, proposing HEG-driven mechanisms that contribute to phage evolution. Relative to ICP1 and T4, we found a variable distribution of HEGs across phages, with HEGs frequently encoded proximal to or within essential genes. We identified large regions (> 10kb) of high nucleotide identity flanked by HEGs, deemed HEG islands, which we hypothesize to be mobilized by the activity of flanking HEGs. Finally, we found examples of domain swapping between phage-encoded HEGs and genes encoded by other phages and phage satellites. We anticipate that HEGs have a larger impact on the evolutionary trajectory of phages than previously appreciated and that future work investigating the role of HEGs in phage evolution will continue to highlight these observations.

Multiple rearrangements and low inter- and intra-species mitogenome sequence variation in the Heterobasidion annosum s.l. species complex.

Mitochondria are essential organelles in the eukaryotic cells and responsible for the energy production but are also involved in many other functions including virulence of some fungal species. Although the evolution of fungal mitogenomes have been studied at some taxonomic levels there are still many things to be learned from studies of closely related species. In this study, we have analyzed 60 mitogenomes in the five species of the Heterobasidion annosum sensu lato complex that all are necrotrophic pathogens on conifers. Compared to other fungal genera the genomic and genetic variation between and within species in the complex was low except for multiple rearrangements. Several translocations of large blocks with core genes have occurred between the five species and rearrangements were frequent in intergenic areas. Mitogenome lengths ranged between 108 878 to 116 176 bp, mostly as a result of intron variation. There was a high degree of homology of introns, homing endonuclease genes, and intergenic ORFs among the five Heterobasidion species. Three intergenic ORFs with unknown function (uORF6, uORF8 and uORF9) were found in all five species and was located in conserved synteny blocks. A 13 bp long GC-containing self-complementary palindrome was discovered in many places in the five species that were optional in presence/absence. The within species variation is very low, among 48 H. parviporum mitogenomes, there was only one single intron exchange, and SNP frequency was 0.28% and indel frequency 0.043%. The overall low variation in the Heterobasidion annosum sensu lato complex suggests a slow evolution of the mitogenome.

Mitochondrial genomes in the iconic reindeer lichens: Architecture, variation, and synteny across multiple evolutionary scales

ABSTRACT Variation in mitochondrial genome composition across intraspecific, interspecific, and higher taxonomic scales has been little studied in lichen obligate symbioses. Cladonia is one of the most diverse and ecologically important lichen genera, with over 500 species representing an array of unique morphologies and chemical profiles. Here, we assess mitochondrial genome diversity and variation in this flagship genus, with focused sampling of two clades of the “true” reindeer lichens, Cladonia subgenus Cladina, and additional genomes from nine outgroup taxa. We describe composition and architecture at the gene and the genome scale, examining patterns in organellar genome size in larger taxonomic groups in Ascomycota. Mitochondrial genomes of Cladonia, Pilophorus, and Stereocaulon were consistently larger than those of Lepraria and contained more introns, suggesting a selective pressure in asexual morphology in Lepraria driving it toward genomic simplification. Collectively, lichen mitochondrial genomes were larger than most other fungal life strategies, reaffirming the notion that coevolutionary streamlining does not correlate to genome size reductions. Genomes from Cladonia ravenelii and Stereocaulon pileatum exhibited ATP9 duplication, bearing paralogs that may still be functional. Homing endonuclease genes (HEGs), though scarce in Lepraria, were diverse and abundant in Cladonia, exhibiting variable evolutionary histories that were sometimes independent of the mitochondrial evolutionary history. Intraspecific HEG diversity was also high, with C. rangiferina especially bearing a range of HEGs with one unique to the species. This study reveals a rich history of events that have transformed mitochondrial genomes of Cladonia and related genera, allowing future study alongside a wealth of assembled genomes.

Functional annotation and comparative analysis of four Botrytis cinerea mitogenomes reported from Punjab, Pakistan

Botrytis cinerea is one of the top phytopathogenic fungus which ubiquitously cause grey mold on a variety of horticultural plants. The mechanism of respiration in the fungus occurs within the mitochondria. Mitogenomes serve as a key molecular marker for the investigation of fungal evolutionary patterns. This study aimed at the complete assembly, characterization, and comparative relationship of four mitogenomes of Botrytis cinerea strains including Kst5C, Kst14A, Kst32B, Kst33A, respectively. High throughput sequencing of four mitogenomes allowed the full assembly and annotation of these sequences. The total genome length of these 4 isolates Kst5C Kst14A, Kst32B, Kst33A was 69,986 bp, 77,303 bp, 76,204 bp and 55, 226 bp respectively. The distribution of features represented 2 ribosomal RNA genes,14 respiration encoding proteins, 1 mitochondrial ribosomal protein-encoding gene, along with varying numbers of transfer RNA genes, protein-coding genes, mobile intronic regions and homing endonuclease genes including LAGLIDADG and GIY-YIG domains were found in all four mitogenomes. The comparative analyses performed also decipher significant results for four mitogenomes among fungal isolates included in the study. This is the first report on the detailed annotation of mitogenomes as a proof for investigation of variation patterns present with in the B. cinerea causing grey mold on strawberries in Pakistan. This study will also contribute to the rapid evolutionary analysis and population patterns present among Botrytis cinerea.

DNA Methylation and RNA-DNA Hybrids Regulate the Single-Molecule Localization of a DNA Methyltransferase on the Bacterial Nucleoid.

Bacterial DNA methyltransferases (MTases) function in restriction modification systems, cell cycle control, and the regulation of gene expression. DnmA is a recently described DNA MTase that forms N6-methyladenosine at nonpalindromic 5'-GACGAG-3' sites in Bacillus subtilis, yet how DnmA activity is regulated is unknown. To address DnmA regulation, we tested substrate binding in vitro and found that DnmA binds poorly to methylated DNA and to an RNA-DNA hybrid with the DNA recognition sequence. Further, DnmA variants with amino acid substitutions that disrupt cognate sequence recognition or catalysis also bind poorly to DNA. Using superresolution fluorescence microscopy and single-molecule tracking of DnmA-PAmCherry, we characterized the subcellular DnmA diffusion and detected its preferential localization to the replisome region and the nucleoid. Under conditions where the chromosome is highly methylated, upon RNA-DNA hybrid accumulation, or with a DnmA variant with severely limited DNA binding activity, DnmA is excluded from the nucleoid, demonstrating that prior methylation or accumulation of RNA-DNA hybrids regulates the association of DnmA with the chromosome in vivo. Furthermore, despite the high percentage of methylated recognition sites and the proximity to putative endonuclease genes conserved across bacterial species, we find that DnmA fails to protect B. subtilis against phage predation, suggesting that DnmA is functionally an orphan MTase involved in regulating gene expression. Our work explores the regulation of a bacterial DNA MTase and identifies prior methylation and RNA-DNA hybrids as regulators of MTase localization. These MTase regulatory features could be common across biology. IMPORTANCE DNA methyltransferases (MTases) influence gene expression, cell cycle control, and host defense through DNA modification. Predicted MTases are pervasive across bacterial genomes, but the vast majority remain uncharacterized. Here, we show that in the soil microorganism Bacillus subtilis, the DNA MTase dnmA and neighboring genes are remnants of a phage defense system that no longer protects against phage predation. This result suggests that portions of the bacterial methylome may originate from inactive restriction modification systems that have maintained methylation activity. Analysis of DnmA movement in vivo shows that active DnmA localizes in the nucleoid, suggesting that DnmA can search for recognition sequences throughout the nucleoid region with some preference for the replisome. Our results further show that prior DNA methylation and RNA-DNA hybrids regulate DnmA dynamics and nucleoid localization, providing new insight into how DNA methylation is coordinated within the cellular environment.

Correlation of ERCC5 polymorphisms and linkage disequilibrium associated with overall survival and clinical outcome to chemotherapy in breast cancer.

ERCC5 is a DNA endonuclease and nucleotide excision repair gene; its mutations lead to a lack of activity by this enzyme, causing oxidative DNA damage. This study aimed to assess the role of four selected single nucleotide polymorphisms (SNPs) in ERCC5 and their linkage disequilibrium associated with survival analysis and clinical outcomes in breast cancer. Four SNPs (rs751402, rs17655, rs2094258, and rs873601) of the ERCC5 gene were analyzed using the PCR-RFLP technique, followed by sequencing in 430 breast cancer (BC) cases and 430 cancer-free individuals. Statistical analysis was performed using MedCalc 17 and SPSS version 24, while bioinformatic analysis of linkage disequilibrium was performed using Haploview software 4.2. Multivariate analysis showed that the rs751402 and rs2094258 polymorphisms were significantly associated with an elevated risk of BC (P < 0.001), while the other two SNPs, rs17655 and rs873601, did not show any association (P > 0.001). Survival analysis revealed that rs751402 and rs2094258 had longer overall survival periods (P <0.001) than rs17655 and rs873601. Moreover, rs751402 and rs2094258 also had significantly longer overall survival (log-rank test, P < 0.005) for all three survival functions (positive family history, ER+PR status, and use of contraceptives), while rs17655 and rs873601 did not show any significant association. Only rs873601 showed a strong negative correlation with all the chemotherapeutic groups. The current results suggest that variations in ERCC5 may contribute to BC development and that their genetic anomalies may be associated with cancer risk and may be used as a biomarker of clinical outcome.

“Molecular Characterization And Its Association To Ap Endonuclease-1 (Ape1) And X-Ray Cross-Complementing Group 1 (Xrcc1), As A Dna Repair Protein From Patients With The Risk Of Age-Related Cataract At A Tertiary Care Centre, Uttar Pradesh”

Introduction: Age-related cataract is the leading cause of blindness in the world. (AP)-endonuclease APE1 and the X-ray cross-complementing group 1 (XRCC1) are the key enzymes taking part in the repair of DNA damage. The association of X-ray cross-complementing group 1 (XRCC1) and AP endonuclease-1 (APE1) plays a critical role as polymorphisms of DNA repair genes decreasing the ability to repair DNA damage, leaving human body a greatly increased susceptibility to cancer or age-related diseases.&#x0D; Aim and objective: To study the Molecular characterization and its Association to AP endonuclease-1 (APE) and X-ray cross-complementing group 1 (XRCC1), as a DNA Repair protein from patients with the risk of age-related cataract at a Tertiary Care Centre, Uttar Pradesh.&#x0D; Material and methods: This was a case control study carried out in the Department of Anatomy &amp; Department of Ophthalmology of RMCH&amp;RC for a period of 2 year i.e., June 2020 to June 2022. A total of 500 clinical suspected cases were included in our study in which 250 patients were confirmed as cataract positive patients whereas 250 were the Controls. The Demographic profiles of the cases were recorded after duly obtaining consent. The 5ml of venous blood was collected in Ethylene diamine tetraacetic acid tubes.&#x0D; DNA was extracted by using Qiagen DNA Extraction kit as per manufactures guidelines. The AP endonuclease-1 (APE) and X-ray cross-complementing group 1 (XRCC1) gene was detected by the conventional PCR.&#x0D; Results: In the present study the maximum number of cases were reported in the Females as compared to the Males with the maximum age group of 51-60 years being affected the most. The Expression of (AP) –endonuclease (APE1) gene was more 68.4 % as compared to X-ray cross-complementing group 1 (XRCC1) with 62.8 %. The increase expression indicates the presence of these genes in the DNA repair damage.&#x0D; Conclusion: The Expression of (AP)-endonuclease (APE1) gene was more 68.4% as compared to X-ray cross-complementing group 1 (XRCC1) with 62.8%. The increase expression indicates the presence of these genes in the DNA repair damage. So, there should be early screening and its molecular detection of the DNA repair gene, which will help the clinician in the early diagnosis as well as early treatment of the cataract.

A CRISPR endonuclease gene drive reveals distinct mechanisms of inheritance bias

CRISPR/Cas gene drives can bias transgene inheritance through different mechanisms. Homing drives are designed to replace a wild-type allele with a copy of a drive element on the homologous chromosome. In Aedes aegypti, the sex-determining locus is closely linked to the white gene, which was previously used as a target for a homing drive element (wGDe). Here, through an analysis using this linkage we show that in males inheritance bias of wGDe did not occur by homing, rather through increased propagation of the donor drive element. We test the same wGDe drive element with transgenes expressing Cas9 with germline regulatory elements sds3, bgcn, and nup50. We only find inheritance bias through homing, even with the identical nup50-Cas9 transgene. We propose that DNA repair outcomes may be more context dependent than anticipated and that other previously reported homing drives may, in fact, bias their inheritance through other mechanisms.

Driving down malaria transmission with engineered gene drives.

The last century has witnessed the introduction, establishment and expansion of mosquito-borne diseases into diverse new geographic ranges. Malaria is transmitted by female Anopheles mosquitoes. Despite making great strides over the past few decades in reducing the burden of malaria, transmission is now on the rise again, in part owing to the emergence of mosquito resistance to insecticides, antimalarial drug resistance and, more recently, the challenges of the COVID-19 pandemic, which resulted in the reduced implementation efficiency of various control programs. The utility of genetically engineered gene drive mosquitoes as tools to decrease the burden of malaria by controlling the disease-transmitting mosquitoes is being evaluated. To date, there has been remarkable progress in the development of CRISPR/Cas9-based homing endonuclease designs in malaria mosquitoes due to successful proof-of-principle and multigenerational experiments. In this review, we examine the lessons learnt from the development of current CRISPR/Cas9-based homing endonuclease gene drives, providing a framework for the development of gene drive systems for the targeted control of wild malaria-transmitting mosquito populations that overcome challenges such as with evolving drive-resistance. We also discuss the additional substantial works required to progress the development of gene drive systems from scientific discovery to further study and subsequent field application in endemic settings.

Mobile group I introns at nuclear rDNA position L2066 harbor sense and antisense homing endonuclease genes intervened by spliceosomal introns

BackgroundMobile group I introns encode homing endonucleases that confer intron mobility initiated by a double-strand break in the intron-lacking allele at the site of insertion. Nuclear ribosomal DNA of some fungi and protists contain mobile group I introns harboring His-Cys homing endonuclease genes (HEGs). An intriguing question is how protein-coding genes embedded in nuclear ribosomal DNA become expressed. To address this gap of knowledge we analyzed nuclear L2066 group I introns from myxomycetes and ascomycetes.ResultsA total of 34 introns were investigated, including two identified mobile-type introns in myxomycetes with HEGs oriented in sense or antisense directions. Intriguingly, both HEGs are interrupted by spliceosomal introns. The intron in Didymium squamulosum, which harbors an antisense oriented HEG, was investigated in more detail. The group I intron RNA self-splices in vitro, thus generating ligated exons and full-length intron circles. The intron HEG is expressed in vivo in Didymium cells, which involves removal of a 47-nt spliceosomal intron (I-47) and 3′ polyadenylation of the mRNA. The D. squamulosum HEG (lacking the I-47 intron) was over-expressed in E. coli, and the corresponding protein was purified and shown to confer endonuclease activity. The homing endonuclease was shown to cleave an intron-lacking DNA and to produce a pentanucleotide 3′ overhang at the intron insertion site.ConclusionsThe L2066 family of nuclear group I introns all belong to the group IE subclass. The D. squamulosum L2066 intron contains major hallmarks of a true mobile group I intron by encoding a His-Cys homing endonuclease that generates a double-strand break at the DNA insertion site. We propose a potential model to explain how an antisense HEG becomes expressed from a nuclear ribosomal DNA locus.

Extended genomic analyses of the broad-host-range phages vB_KmiM-2Di and vB_KmiM-4Dii reveal slopekviruses have highly conserved genomes.

High levels of antimicrobial resistance among members of the Klebsiella oxytoca complex (KoC) have led to renewed interest in the use of bacteriophage (phage) therapy to tackle infections caused by these bacteria. In this study we characterized two lytic phages, vB_KmiM-2Di and vB_KmiM-4Dii, that were isolated from sewage water against two GES-5-positive Klebsiella michiganensis strains (PS_Koxy2 and PS_Koxy4, respectively). ViPTree analysis showed both phages belonged to the genus Slopekvirus. rpoB gene-based sequence analysis of 108 presumptive K. oxytoca isolates (n=59 clinical, n=49 veterinary) found K. michiganensis to be more prevalent (46 % clinical and 43 % veterinary, respectively) than K. oxytoca (40 % clinical and 6 % veterinary, respectively). Host range analysis against these 108 isolates found both vB_KmiM-2Di and vB_KmiM-4Dii showed broad lytic activity against KoC species. Several hypothetical homing endonuclease genes were encoded within the genomes of both phages, which may contribute to their broad host range. Differences in the tail fibre protein may explain the non-identical host range of the two phages. Pangenome analysis of 24 slopekviruses found that genomes within this genus are highly conserved, with more than 50 % of all predicted coding sequences representing core genes at ≥95 % identity and ≥70 % coverage. Given their broad host ranges, our results suggest vB_KmiM-2Di and vB_KmiM-4Dii represent attractive potential therapeutics. In addition, current recommendations for phage-based pangenome analyses may require revision.

Reactive oxygen signaling molecule inducible regulation of CRISPR-Cas9 gene editing.

We report development of a controllable gene editing tool that boronated gRNA, simply generated in situ, could regulate binding of gRNA molecules with either Cas9 endonuclease or target genes, thus serving as a modulator that can control CRISPR-Cas9 gene editing. Subsequent treatment with H2O2 facilitates the restoration of gene editing ability of the boronated gRNA to the level of using untreated gRNA. This is one of the few cases using small molecule to regulate CRISPR-Cas9 gene editing, which is a complement to the light approach, displaying great application potential. We develop a controllable gene editing tools based on the CRISPR-Cas9 gene editing system. This tool can be regulated by oxidative small molecule, i.e., H2O2. Compared with the light method, the application scope of our CRISPR-Cas9 systems have been widened with the small-molecule-triggered approaches, preventing the potential damage of cells or organism caused by UV light. In addition, the gain-of-function tools are expanding the gene code expansion for mechanistic studies of target enzymes since it provides a positive route to evaluate the activity of a given enzyme in dynamic and inversible regulation of targeting cellular processes.

Structural Organization of S516 Group I Introns in Myxomycetes.

Group I introns are mobile genetic elements encoding self-splicing ribozymes. Group I introns in nuclear genes are restricted to ribosomal DNA of eukaryotic microorganisms. For example, the myxomycetes, which represent a distinct protist phylum with a unique life strategy, are rich in nucleolar group I introns. We analyzed and compared 75 group I introns at position 516 in the small subunit ribosomal DNA from diverse and distantly related myxomycete taxa. A consensus secondary structure revealed a conserved group IC1 ribozyme core, but with a surprising RNA sequence complexity in the peripheral regions. Five S516 group I introns possess a twintron organization, where a His-Cys homing endonuclease gene insertion was interrupted by a small spliceosomal intron. Eleven S516 introns contained direct repeat arrays with varying lengths of the repeated motif, a varying copy number, and different structural organizations. Phylogenetic analyses of S516 introns and the corresponding host genes revealed a complex inheritance pattern, with both vertical and horizontal transfers. Finally, we reconstructed the evolutionary history of S516 nucleolar group I introns from insertion of mobile-type introns at unoccupied cognate sites, through homing endonuclease gene degradation and loss, and finally to the complete loss of introns. We conclude that myxomycete S516 introns represent a family of genetic elements with surprisingly dynamic structures despite a common function in RNA self-splicing.

An Inducible Microbacterium Prophage vB_MoxS-R1 Represents a Novel Lineage of Siphovirus.

Lytic and lysogenic infections are the main strategies used by viruses to interact with microbial hosts. The genetic information of prophages provides insights into the nature of phages and their potential influences on hosts. Here, the siphovirus vB_MoxS-R1 was induced from a Microbacterium strain isolated from an estuarine Synechococcus culture. vB_MoxS-R1 has a high replication capability, with an estimated burst size of 2000 virions per cell. vB_MoxS-R1 represents a novel phage genus-based genomic analysis. Six transcriptional regulator (TR) genes were predicted in the vB_MoxS-R1 genome. Four of these TR genes are involved in stress responses, virulence and amino acid transportation in bacteria, suggesting that they may play roles in regulating the host cell metabolism in response to external environmental changes. A glycerophosphodiester phosphodiesterase gene related to phosphorus acquisition was also identified in the vB_MoxS-R1 genome. The presence of six TR genes and the phosphorus-acquisition gene suggests that prophage vB_MoxS-R1 has the potential to influence survival and adaptation of its host during lysogeny. Possession of four endonuclease genes in the prophage genome suggests that vB_MoxS-R1 is likely involved in DNA recombination or gene conversion and further influences host evolution.