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Information regarding Klebsiella aerogenes haboring carbapenemase in Japan is limited. A comprehensive nationwide survey was conducted from September 2014 to December 2022, and 67 non-duplicate strains of carbapenem-resistant K. aerogenes were isolated from 57 healthcare facilities in Japan. Through genetic testing and whole-genome sequencing, six strains were found to possess carbapenemases, including imipenemase (IMP)-1, IMP-6, New Delhi metallo-β-lactamase (NDM)-1, and NDM-5. The strain harboring blaNDM-5 was the novel strain ST709, which belongs to the clonal complex of the predominant ST4 in China. The novel integron containing blaIMP-1 featured the oxacillinase-101 gene, which is a previously unreported structure, with an IncN4 plasmid type. However, integrons found in the strains possessing blaIMP-6, which were the most commonly identified, matched those reported domestically in Klebsiella pneumoniae, suggesting the prevalence of identical integrons. Transposons containing blaNDM are similar or identical to the transposon structure of K. aerogenes harboring blaNDM-5 previously reported in Japan, suggesting that the same type of transposon could have been transmitted to K. aerogenes in Japan. This investigation analyzed mobile genetic elements, such as integrons and transposons, to understand the spread of carbapenemases, highlighting the growing challenge of carbapenem-resistant Enterobacterales in Japan and underscoring the critical need for ongoing surveillance to control these pathogens. [ABSTRACT FROM AUTHOR]

Background The ascomycete fungus Anisogramma anomala causes Eastern Filbert Blight (EFB) on hazelnut (Corylus spp.) trees. It is a minor disease on its native host, the American hazelnut (C. americana), but is highly destructive on the commercially important European hazelnut (C. avellana). In North America, EFB has historically limited commercial production of hazelnut to west of the Rocky Mountains. A. anomala is an obligately biotrophic fungus that has not been grown in continuous culture, rendering its study challenging. There is a 15-month latency before symptoms appear on infected hazelnut trees, and only a sexual reproductive stage has been observed. Here we report the sequencing, annotation, and characterization of its genome. Results The genome of A. anomala was assembled into 108 scafolds totaling 342,498,352 nt with a GC content of 34.46%. Scafold N50 was 33.3 Mb and L50 was 5. Nineteen scafolds with lengths over 1 Mb constituted 99% of the assembly. Telomere sequences were identifed on both ends of two scafolds and on one end of another 10 scafolds. Flow cytometry estimated the genome size of A. anomala at 370 Mb. The genome exhibits two-speed evolution, with 93% of the assembly as AT-rich regions (32.9% GC) and the other 7% as GC-rich (57.1% GC). The AT-rich regions consist predominantly of repeats with low gene content, while 90% of predicted protein coding genes were identifed in GC-rich regions. Copia-like retrotransposons accounted for more than half of the genome. Evidence of repeat-induced point mutation (RIP) was identifed throughout the AT-rich regions, and two copies of the rid gene and one of dim-2, the key genes in the RIP mutation pathway, were identifed in the genome. Consistent with its homothallic sexual reproduction cycle, both MAT1-1 and MAT1-2 idiomorphs were found. We identifed a large suite of genes likely involved in pathogenicity, including 614 carbohydrate active enzymes, 762 secreted proteins and 165 efectors. Conclusions This study reveals the genomic structure, composition, and putative gene function of the important pathogen A. anomala. It provides insight into the molecular basis of the pathogen’s life cycle and a solid foundation for studying EFB. [ABSTRACT FROM AUTHOR]

This study aimed to identify genes related to acetic acid tolerance and elucidate the tolerance mechanism in yeast Saccharomyces cerevisiae as an appropriate eukaryote model. In this study, one tolerant strain exposed to acetic acid stress was isolated by screening a transposon-mediated mutant library and the disrupted gene was identified to be RGD1. In addition, this phenotype related to acetic acid tolerance was confirmed by deletion and overexpressing of this corresponding gene. This mutant strain showed activation of Hog1p and increased expression of the MSN2/4 and STRE-mediated genes such as CTT1 and HSP12 in response to acetic acid stress. [ABSTRACT FROM AUTHOR]

Transposable elements (TEs) are major components of eukaryotic genomes and are implicated in a range of evolutionary processes. Yet, TE annotation and characterization remain challenging, particularly for nonspecialists, since existing pipelines are typically complicated to install, run, and extract data from. Current methods of automated TE annotation are also subject to issues that reduce overall quality, particularly (i) fragmented and overlapping TE annotations, leading to erroneous estimates of TE count and coverage, and (ii) repeat models represented by short sections of total TE length, with poor capture of 5′ and 3′ ends. To address these issues, we present Earl Grey, a fully automated TE annotation pipeline designed for user-friendly curation and annotation of TEs in eukaryotic genome assemblies. Using nine simulated genomes and an annotation of Drosophila melanogaster , we show that Earl Grey outperforms current widely used TE annotation methodologies in ameliorating the issues mentioned above while scoring highly in benchmarking for TE annotation and classification and being robust across genomic contexts. Earl Grey provides a comprehensive and fully automated TE annotation toolkit that provides researchers with paper-ready summary figures and outputs in standard formats compatible with other bioinformatics tools. Earl Grey has a modular format, with great scope for the inclusion of additional modules focused on further quality control and tailored analyses in future releases. [ABSTRACT FROM AUTHOR]

The endoplasmic reticulum (ER) is the largest membrane system in eukaryotic cells and is the primary site for the biosynthesis of lipids and carbohydrates, as well as for the folding, assembly, modification, and transport of secreted and integrated membrane proteins. The ER membrane complex (EMC) on the ER membrane is an ER multiprotein complex that affects the quality control of membrane proteins, which is abundant and widely preserved. Its disruption has been found to affect a wide range of processes, including protein and lipid synthesis, organelle communication, endoplasmic reticulum stress, and viral maturation, and may lead to neurodevelopmental disorders and cancer. Therefore, EMC has attracted the attention of many scholars and become a hot field. In this paper, we summarized the main contributions of the research of EMC in the past nearly 15 years, and reviewed the structure and function of EMC as well as its related diseases. We hope this review will promote further progress of research on EMC. [ABSTRACT FROM AUTHOR]

C3‐C4 intermediate photosynthesis has evolved at least five times convergently in the Brassicaceae, despite this family lacking bona fide C4 species. The establishment of this carbon concentrating mechanism is known to require a complex suite of ultrastructural modifications, as well as changes in spatial expression patterns, which are both thought to be underpinned by a reconfiguration of existing gene‐regulatory networks. However, to date, the mechanisms which underpin the reconfiguration of these gene networks are largely unknown.In this study, we used a pan‐genomic association approach to identify genomic features that could confer differential gene expression towards the C3‐C4 intermediate state by analysing eight C3 species and seven C3‐C4 species from five independent origins in the Brassicaceae.We found a strong correlation between transposable element (TE) insertions in cis‐regulatory regions and C3‐C4 intermediacy. Specifically, our study revealed 113 gene models in which the presence of a TE within a gene correlates with C3‐C4 intermediate photosynthesis. In this set, genes involved in the photorespiratory glycine shuttle are enriched, including the glycine decarboxylase P‐protein whose expression domain undergoes a spatial shift during the transition to C3‐C4 photosynthesis. When further interrogating this gene, we discovered independent TE insertions in its upstream region which we conclude to be responsible for causing the spatial shift in GLDP1 gene expression.Our findings hint at a pivotal role of TEs in the evolution of C3‐C4 intermediacy, especially in mediating differential spatial gene expression. [ABSTRACT FROM AUTHOR]

Pathogenic Aeromonas spp. are the etiological agents of Motile Aeromonas Septicemia (MAS). This study aimed to identify the pathogen of diseased tadpoles (Quasipaa spinosa) and the antibiotic-resistance characteristics of this bacterium. A Gram-negative bacterium, named strain QST31, was isolated from the ascites of diseased tadpoles and was identified as Aeromonas media based on physiological and biochemical tests, as well as molecular identification. Artificial infection experiments showed that strain QST31 was highly virulent to tadpoles, with an LC50 of 2.56 × 107 CFU/mL. The antimicrobial susceptibility of strain QST31 was evaluated using the disk diffusion method, and the results indicated that strain QST31 was resistant to 28 antibacterial agents. In addition, the whole genome of strain QST31 was sequenced, and the presence of antimicrobial resistance genes, integron, and transposon was investigated. Genes involved in adherence, hemolysis, type II secretion system (T2SS), T6SS, iron uptake system, and quorum sensing were identified in the genome of strain QST31. More than 12 antimicrobial resistance genes were predicted in the genome of strain QST31. Interestingly, a novel Tn7709 transposon harboring sul1, aadA16, catB3, blaOXA-21, aac(6′)-IIa, and tet(A) genes was identified. In conclusion, this is the first report on the isolation and identification of pathogenic A. media with multidrug resistance genes from diseased tadpoles. The results revealed that preventing and controlling aquatic animal diseases caused by multidrug resistance A. media will be a huge challenge in the future. [ABSTRACT FROM AUTHOR]

New transposon insertions are deleterious to genome stability. The RNAdirected DNA methylation (RdDM) pathway evolved to regulate transposon activity via DNA methylation. However, current studies have not yet clearly described the transposition regulation. ONSEN is a heat-activated retrotransposon that is activated at 37°C. The plant-specific SUPPRESSOR OF VARIEGATION 3-9 HOMOLOG (SUVH) family proteins function downstream of the RdDM pathway. The SUVH protein families are linked to TE silencing by two pathways, one through DNA methylation and the other through chromatin remodeling. In this study, we analyzed the regulation of ONSEN activity by SUVH2. We observed that ONSEN transcripts were increased; however, there was no transpositional activity in Arabidopsis suvh2 mutant. The suvh2 mutant produced siRNAs from the ONSEN locus under heat stress, suggesting that siRNAs are involved in suppressing transposition. These results provide new insights into the regulatory mechanisms of retrotransposons that involve siRNA in the RdDM pathway. [ABSTRACT FROM AUTHOR]

The type-I interferon (IFN) response constitutes the major innate immune pathway against viruses in mammals. Despite its critical importance for antiviral defence, this pathway is inactive during early embryonic development. There seems to be an incompatibility between the IFN response and pluripotency, the ability of embryonic cells to develop into any cell type of an adult organism. Instead, pluripotent cells employ alternative ways to defend against viruses that are typically associated with safeguard mechanisms against transposable elements. The absence of an inducible IFN response in pluripotent cells and the constitutive activation of the alternative antiviral pathways have led to the hypothesis that embryonic cells are highly resistant to viruses. However, some findings challenge this interpretation. We have performed a meta-analysis that suggests that the susceptibility of pluripotent cells to viruses is directly correlated with the presence of receptors or co-receptors for viral adhesion and entry. These results challenge the current view of pluripotent cells as intrinsically resistant to infections and raise the fundamental question of why these cells have sacrificed the major antiviral defence pathway if this renders them susceptible to viruses. [ABSTRACT FROM AUTHOR]

Pseudomonas aeruginosa is a leading cause of hospital-acquired infections worldwide. Biofilm production, antibiotic resistance, and a wide range of virulence factors contribute to their persistence in nosocomial environments. We describe an outbreak caused by a multidrug-resistant P. aeruginosa strain in an ICU. Antibiotic susceptibility was determined and blaPER-1 and qnrVC were amplified via PCR. Clonality was determined using PFGE and biofilm formation was studied with a static model. A combination of antibiotics was assessed on both planktonic cells and biofilms. WGS was performed on five isolates. All isolates were clonally related, resistant to ceftazidime, cefepime, amikacin, and ceftolozane-tazobactam, and harbored blaPER-1; 11/19 possessed qnrVC. Meropenem and ciprofloxacin reduced the biofilm biomass; however, the response to antibiotic combinations with rifampicin was different between planktonic cells and biofilms. WGS revealed that the isolates belonged to ST309 and serotype O11. blaPER-1 and qnrVC6 were associated with a tandem of ISCR1 as part of a complex class one integron, with aac(6′)-Il and ltrA as gene cassettes. The structure was associated upstream and downstream with Tn4662 and flanked by direct repeats, suggesting its horizontal mobilization capability as a composite transposon. ST309 is considered an emerging high-risk clone that should be monitored in the Americas. [ABSTRACT FROM AUTHOR]

Abstract The sixth Japanese meeting on host–transposon interactions, titled “Biological Function and Evolution through Interactions between Hosts and Transposable Elements,” was held on August 24th and 25th, 2023, at the National Institute of Genetics as well as online. This meeting was supported by the National Institute of Genetics and aimed to bring together researchers studying the diverse roles of TEs in genome function and evolution, as well as host defense systems against TE mobility, TE bursts during evolution, and intron mobility in mammals, insects, land plants, yeast, protozoa, and bacteria. Here, we have presented the highlights of the discussion. Organizers: Kenji Ichiyanagi, Yoko Ikeda, and Kuniaki Saito.

Abstract Background The genomes of many major crops including barley (Hordeum vulgare) consist of numerous transposons. Despite their important roles in crop genome evolution and morphological variations, most of these elements are silent or truncated and unable to be mobile in host genomes. Thus far, only a very limited number of active transposons were identified in plants. Results We analyzed the barley full-length cDNA (FLcDNA) sequences and detected 71 unique FLcDNAs exhibiting significant sequence similarity to the extant transposase proteins. These FLcDNAs were then used to search against the genome of a malting barley cultivar ‘Morex’, seven new intact transposons were identified. Sequence alignments indicated that six intact transposons contained the entire FLcDNAs whereas another one served as 3’ untranslated region (3’ UTR) of a barley gene. Our reverse transcription-PCR (RT-PCR) experiment further confirmed the expression of these six transposons and revealed their differential expression. We conducted genome-wide transposon comparisons and detected polymorphisms of three transposon families between the genomes of ‘Morex’ and other three genotypes including the wild barley (Hordeum spontaneum, B1K-04-12) and two cultivated barley varieties, ‘Golden Promise’ and ‘Lasa Goumang’. Lastly, we screened the transcripts of all annotated barley genes and found that some transposons may serve as the coding regions (CDSs) or UTRs of barley genes. Conclusion We identified six newly expressed transposons in the barley genome and revealed the recent mobility of three transposon families. Our efforts provide a valuable resource for understanding the effects of transposons on barley genome evolution and for developing novel molecular tools for barley genetic improvement and other research.

Pseudomonas putida KT2440 has long been studied for its diverse and robust metabolisms, yet many genes and proteins imparting these growth capacities remain uncharacterized. Using pooled mutant fitness assays, we identified genes and proteins involved in the assimilation of 52 different nitrogen containing compounds. To assay amino acid biosynthesis, 19 amino acid drop-out conditions were also tested. From these 71 conditions, significant fitness phenotypes were elicited in 672 different genes including 100 transcriptional regulators and 112 transport-related proteins. We divide these conditions into 6 classes, and propose assimilatory pathways for the compounds based on this wealth of genetic data. To complement these data, we characterize the substrate range of three promiscuous aminotransferases relevant to metabolic engineering efforts in vitro. Furthermore, we examine the specificity of five transcriptional regulators, explaining some fitness data results and exploring their potential to be developed into useful synthetic biology tools. In addition, we use manifold learning to create an interactive visualization tool for interpreting our BarSeq data, which will improve the accessibility and utility of this work to other researchers. IMPORTANCE Understanding the genetic basis of P. putida's diverse metabolism is imperative for us to reach its full potential as a host for metabolic engineering. Many target molecules of the bioeconomy and their precursors contain nitrogen. This study provides functional evidence linking hundreds of genes to their roles in the metabolism of nitrogenous compounds, and provides an interactive tool for visualizing these data. We further characterize several aminotransferases, lactamases, and regulators, which are of particular interest for metabolic engineering.

The cryptic β-glucoside GFB (bglGFB) operon in Escherichia coli (E. coli) can be activated by mutations arising under starvation conditions in the presence of an aromatic β-glucoside. This may involve the insertion of an insertion sequence (IS) element into a "stress-induced DNA duplex destabilization" (SIDD) region upstream of the operon promoter, although other types of mutations can also activate the bgl operon. Here, we show that increased expression of the bglG gene, encoding a well-characterized transcriptional antiterminator, dramatically increases the frequency of both IS-mediated and IS-independent Bgl+ mutations occurring on salicin- and arbutin-containing agar plates. Both mutation rates increased with increasing levels of bglG expression but IS-mediated mutations were more prevalent at lower BglG levels. Mutations depended on the presence of both BglG and an aromatic β-glucoside, and bglG expression did not influence IS insertion in other IS-activated operons tested. The N-terminal mRNA-binding domain of BglG was essential for mutational activation, and alteration of BglG's binding site in the mRNA nearly abolished Bgl+ mutant appearances. Increased bglG expression promoted residual bgl operon expression in parallel with the increases in mutation rates. Possible mechanisms are proposed explaining how BglG enhances the frequencies of bgl operon activating mutations.

The evolution of CRISPR-Cas systems and their possible origin from mobile genetic elements of transposons are currently being actively studied. Recently, unique systems have been discovered in a new group of Asgardarchaea that presumably function as transposons and contain Cas1-like proteins. In this study, the genetic and biochemical technologies were used, along with electrophoresis, affinity chromatography, and high-resolution mass spectrometry, to obtain and partially characterize a recombinant version of one of these proteins, Cas1_3. In particular, it was shown to have the ATPase activity, the quantitative value of the latter being determined by the spectrophotometric method. The results obtained may be useful in understanding the mechanisms of functioning the potential ancestor of CRISPR-Cas systems.

Summary: Diatoms are eukaryotic microalgae responsible for nearly half of the marine productivity. RNA interference (RNAi) is a mechanism of regulation of gene expression mediated by small RNAs (sRNAs) processed by the endoribonuclease Dicer (DCR). To date, the mechanism and physiological role of RNAi in diatoms are unknown.We mined diatom genomes and transcriptomes for key RNAi effectors and retraced their phylogenetic history. We generated DCR knockout lines in the model diatom species Phaeodactylum tricornutum and analyzed their mRNA and sRNA populations, repression‐associated histone marks, and acclimatory response to nitrogen starvation.Diatoms presented a diversification of key RNAi effectors whose distribution across species suggests the presence of distinct RNAi pathways. P. tricornutum DCR was found to process 26–31‐nt‐long double‐stranded sRNAs originating mostly from transposons covered by repression‐associated epigenetic marks. In parallel, P. tricornutum DCR was necessary for the maintenance of the repression‐associated histone marks H3K9me2/3 and H3K27me3. Finally, PtDCR‐KO lines presented a compromised recovery post nitrogen starvation suggesting a role for P. tricornutum DCR in the acclimation to nutrient stress.Our study characterized the molecular function of the single DCR homolog of P. tricornutum suggesting an association between RNAi and heterochromatin maintenance in this model diatom species. [ABSTRACT FROM AUTHOR]

Simple Summary: Transposable elements (TEs) are mobile genetic elements that play vital role in defining and contributing to the size, shape and structure of both prokaryotic and eukaryotic genomes in nature. The piggyBac (PB), a superfamily of DNA transposons, has been isolated over the years from various organisms including insects, fungi and plants. These piggyBac transposon systems have high efficiency with a wide usage in the study of gene therapies, mutagenesis and transgenesis. Currently, there is limited information available on DNA transposons in small (compact) genomes of animals. Therefore, this study aims to annotate the PB transposons in small genomes of animals, revealing their evolution profiles in both vertebrate and invertebrate genomes. TEs, including DNA transposons, are major contributors of genome expansions, and have played a very significant role in shaping the evolution of animal genomes, due to their capacity to jump from one genomic position to the other. In this study, we investigated the evolution landscapes of PB transposons, including their distribution, diversity, activity and structure organization in 79 species of small (compact) genomes of animals comprising both vertebrate and invertebrates. Overall, 212 PB transposon types were detected from almost half (37) of the total number of the small genome species (79) investigated. The detected PB transposon types, which were unevenly distributed in various genera and phyla, have been classified into seven distinct clades or families with good bootstrap support (>80%). The PB transposon types that were identified have a length ranging from 1.23 kb to 9.51 kb. They encode transposases of approximately ≥500 amino acids in length, and possess terminal inverted repeats (TIRs) ranging from 4 bp to 24 bp. Though some of the transposon types have long TIRs (528 bp), they still maintain the consistent and reliable 4 bp target site duplication (TSD) of TTAA. However, PiggyBac-2_Cvir transposon originating from the Crassostrea virginica species exhibits a unique TSD of TATG. The TIRs of the transposons in all the seven families display high divergence, with a highly conserved 5′ end motif. The core transposase domains (DDD) were better conserved among the seven different families compared to the other protein domains, which were less prevalent in the vertebrate genome. The divergent evolution dynamics analysis also indicated that the majority of the PB transposon types identified in this study are either relatively young or old, with some being active. Additionally, numerous invasions of PB transposons were found in the genomes of both vertebrate and invertebrate animals. The data reveals that the PB superfamily is widely distributed in these species. PB transposons exhibit high diversity and activity in the small genomes of animals, and might play a crucial role in shaping the evolution of these small genomes of animals. [ABSTRACT FROM AUTHOR]

The sixth Japanese meeting on host–transposon interactions, titled "Biological Function and Evolution through Interactions between Hosts and Transposable Elements," was held on August 24th and 25th, 2023, at the National Institute of Genetics as well as online. This meeting was supported by the National Institute of Genetics and aimed to bring together researchers studying the diverse roles of TEs in genome function and evolution, as well as host defense systems against TE mobility, TE bursts during evolution, and intron mobility in mammals, insects, land plants, yeast, protozoa, and bacteria. Here, we have presented the highlights of the discussion. Organizers: Kenji Ichiyanagi, Yoko Ikeda, and Kuniaki Saito. [ABSTRACT FROM AUTHOR]

High-energy heavy ion beams are a new type of physical mutagen that can produce a wide range of phenotypic variations. In order to understand the mechanism of high-energy heavy ion beams, we resequenced the whole genome of individual plants with obvious phenotypic variations in rice. The sequence alignment results revealed a large number of SNPs and InDels, as well as genetic variations related to grain type and heading date. The distribution of SNP and InDel on chromosomes is random, but they often occur in the up/downstream regions and the intergenic region. Mutagenesis can cause changes in transposons such as Dasheng, mPing, Osr13 and RIRE2, affecting the stability of the genome. This study obtained the major gene mutation types, discovered differentially active transposons, screened out gene variants related to phenotype, and explored the mechanism of high-energy heavy ion beam radiation on rice genes. [ABSTRACT FROM AUTHOR]

Transposable elements (TEs) are mobile DNA sequences that can insert themselves into various locations within the genome, causing mutations that may provide advantages or disadvantages to individuals and species. The insertion of TEs can result in genetic variation that may affect a wide range of human traits including genetic disorders. Understanding the role of TEs in human biology is crucial for both evolutionary and medical research. This review discusses the involvement of TEs in human traits and disease susceptibility, as well as methods for functional analysis of TEs. [ABSTRACT FROM AUTHOR]

Polintons are double-stranded DNA, virus-like self-synthesizing transposons widely found in eukaryotic genomes. Recent metagenomic discoveries of Polinton-like viruses are consistent with the hypothesis that Polintons invade eukaryotic host genomes through infectious viral particles. Nematode genomes contain multiple copies of Polintons and provide an opportunity to explore the natural distribution and evolution of Polintons during this process. We performed an extensive search of Polintons across nematode genomes, identifying multiple full-length Polinton copies in several species. We provide evidence of both ancient Polinton integrations and recent mobility in strains of the same nematode species. In addition to the major nematode Polinton family, we identified a group of Polintons that are overall closely related to the major family but encode a distinct protein-primed DNA polymerase B (pPolB) that is related to homologs from a different group of Polintons present outside of the Nematoda. Phylogenetic analyses on the pPolBs support the evolutionary scenarios in which these extrinsic pPolBs that seem to derive from Polinton families present in oomycetes and molluscs replaced the canonical pPolB in subsets of Polintons found in terrestrial and marine nematodes, respectively, suggesting interphylum horizontal gene transfers. The pPolBs of the terrestrial nematode and oomycete Polintons share a unique feature, an insertion of an HNH nuclease domain, whereas the pPolBs in the marine nematode Polintons share an insertion of a VSR nuclease domain with marine mollusc pPolBs. We hypothesize that horizontal gene transfer occurs among Polintons from widely different but cohabiting hosts. [ABSTRACT FROM AUTHOR]

Background: The genomes of many major crops including barley (Hordeum vulgare) consist of numerous transposons. Despite their important roles in crop genome evolution and morphological variations, most of these elements are silent or truncated and unable to be mobile in host genomes. Thus far, only a very limited number of active transposons were identified in plants. Results: We analyzed the barley full-length cDNA (FLcDNA) sequences and detected 71 unique FLcDNAs exhibiting significant sequence similarity to the extant transposase proteins. These FLcDNAs were then used to search against the genome of a malting barley cultivar 'Morex', seven new intact transposons were identified. Sequence alignments indicated that six intact transposons contained the entire FLcDNAs whereas another one served as 3' untranslated region (3' UTR) of a barley gene. Our reverse transcription-PCR (RT-PCR) experiment further confirmed the expression of these six transposons and revealed their differential expression. We conducted genome-wide transposon comparisons and detected polymorphisms of three transposon families between the genomes of 'Morex' and other three genotypes including the wild barley (Hordeum spontaneum, B1K-04-12) and two cultivated barley varieties, 'Golden Promise' and 'Lasa Goumang'. Lastly, we screened the transcripts of all annotated barley genes and found that some transposons may serve as the coding regions (CDSs) or UTRs of barley genes. Conclusion: We identified six newly expressed transposons in the barley genome and revealed the recent mobility of three transposon families. Our efforts provide a valuable resource for understanding the effects of transposons on barley genome evolution and for developing novel molecular tools for barley genetic improvement and other research. [ABSTRACT FROM AUTHOR]

Summary: Petal senescence is the final stage of flower development. Transcriptional regulation plays key roles in this process. However, whether and how post‐transcriptional regulation involved is still largely unknown. Here, we identified an ethylene‐induced NAC family transcription factor DcNAP in carnation (Dianthus caryophyllus L.). One allele, DcNAP‐dTdic1, has an insertion of a dTdic1 transposon in its second exon. The dTdic1 transposon disrupts the structure of DcNAP and causes alternative splicing, which transcribes multiple domain‐deleted variants (DcNAP2 and others). Conversely, the wild type allele DcNAP transcribes DcNAP1 encoding an intact NAC domain. Silencing DcNAP1 delays and overexpressing DcNAP1 accelerates petal senescence in carnation, while silencing and overexpressing DcNAP2 have the opposite effects, respectively. Further, DcNAP2 could interact with DcNAP1 and interfere the binding and activation activity of DcNAP1 to the promoters of its downstream target ethylene biosynthesis genes DcACS1 and DcACO1. Lastly, ethylene signalling core transcriptional factor DcEIL3‐1 can activate the expression of DcNAP1 and DcNAP2 in the same way by binding their promoters. In summary, we discovered a novel mechanism by which DcNAP regulates carnation petal senescence at the post‐transcriptional level. It may also provide a useful strategy to manipulate the NAC domains of NAC transcription factors for crop genetic improvement. [ABSTRACT FROM AUTHOR]

As the importance of the gastrointestinal microbiome in human health and disease has been revealed via association studies, genetic manipulation tools for gut bacteria are much needed to define mechanisms and show causality. Exogenous DNA delivery and DNA manipulation using traditional strategies and novel clustered regularly interspaced short palindromic repeats (CRISPR)-Cas-based approaches have shown tractability in both model bacteria and non-model ('wild') bacteria. The development of genetic manipulation tools in gut bacteria is crucial to overcome barriers to 'taming' the microbiome, which allows us not only to understand host–microbiome interactions but also to accelerate microbiome engineering for novel therapeutics. Studies of the gut microbiota have revealed associations between specific bacterial species or community compositions with health and disease, yet the causal mechanisms underlying microbiota gene–host interactions remain poorly understood. This is partly due to limited genetic manipulation (GM) tools for gut bacteria. Here, we review current advances and challenges in the development of GM approaches, including clustered regularly interspaced short palindromic repeats (CRISPR)-Cas and transposase-based systems in either model or non-model gut bacteria. By overcoming barriers to 'taming' the gut microbiome, GM tools allow molecular understanding of host–microbiome associations and accelerate microbiome engineering for clinical treatment of cancer and metabolic disorders. Finally, we provide perspectives on the future development of GM for gut microbiome species, where more effort should be placed on assembling a generalized GM pipeline to accelerate the application of groundbreaking GM tools in non-model gut bacteria towards both basic understanding and clinical translation. [ABSTRACT FROM AUTHOR]

Short interspersed nuclear elements (SINEs) and long interspersed nuclear elements (LINE-1s) are the abundant and well-characterized repetitive elements in the human genome. For this review, all relevant original research studies were assessed by searching electronic databases, including PubMed, Google Scholar, and Web of Science, by using relevant keywords. Accumulating evidence indicates that the disorder of gene expression regulated by these repetitive sequences is one of the causes of the diseases of visual system dysfunction, including retinal degenerations, glaucoma, retinitis punctata albescens, retinitis pigmentosa, geographic atrophy, and age-related macular degeneration, suggesting that SINEs and LINE-1s may have great potential implications in ophthalmology. Alu elements belonging to the SINEs are present in more than one million copies, comprising 10% of the human genome. This study offers recent advances in Alu and LINE-1 mechanisms in the development of eye diseases. The current study could advance our knowledge of the roles of SINEs and LINE-1s in the developing process of eye diseases, suggesting new diagnostic biomarkers, therapeutic strategies, and significant points for future studies. This study reveals the Alu and LINE-1 interspersed repetitive sequences involved in the diseases of visual system dysfunction. This study shows the disorder of gene expression regulated by SINEs and LINE-1s sequences is one of the causes of the diseases of visual system dysfunction. This study suggests recent advances in Alu and LINE-1 mechanisms are involved in eye diseases. [ABSTRACT FROM AUTHOR]

The plasmid known as pAsa5 is present in Aeromonas salmonicida subsp. salmonicida, a fish pathogen. The pAsa5 plasmid carries genes that are essential for the bacterium's virulence. Recombination events are known to occur in pAsa5, resulting in the loss of certain segments or the acquisition of additional genetic elements. For example, the transposon carried by the large pAsa8 plasmid was found to be inserted into the pAsa5 plasmid in the SHY16-3432 strain, enabling the addition of antibiotic resistance genes to this plasmid, which does not normally possess any. In this study, we present the isolation of additional strains carrying pAsa8. Further analyses of these strains revealed that a fusion between pAsa5 and the complete version of pAsa8 is possible. The pAsa8 transposon insertion in pAsa5 seen in the SHY16-3432 strain appears to be an aberrant event compared to the fusion of the two full-length plasmids. A 22-nucleotide sequence, present in both plasmids, serves as the site for the fusion of the two plasmids. Moreover, it is possible to introduce pAsa8 through conjugation into naive strains of A. salmonicida subsp. salmonicida and once the plasmid is within a new strain, the fusion with pAsa5 is detectable. This study reveals a previously unexplored aspect of pAsa5 plasmid biology, highlighting an additional risk for the spread of antibiotic resistance genes in A. salmonicida subsp. salmonicida. [ABSTRACT FROM AUTHOR]

Natural killer cells are a promising platform for cancer immunotherapy. Natural killer cells have high intrinsic killing capability, and the insertion of a chimeric antigen receptor can further enhance their antitumor potential. In first-in-human trials, chimeric antigen receptor-natural killer cells demonstrated strong clinical activity without therapy-induced side effects. The applicability of natural killer cells as an "off-the-shelf product makes them highly attractive for gene-engineered cell therapies. Traditionally, viral transduction has been used for gene editing; however, the use of viral vectors remains a safety concern and is associated with high costs and regulatory requirements. Here, we review the current landscape of nonviral approaches for chimeric antigen receptor- natural killer cell generation. This includes transfection of vector particles and electroporation of mRNA and DNA vectors, resulting in transient modification and chimeric antigen receptor expression. In addition, using nonviral transposon technologies, natural killer cells can be stably modified ensuring long-lasting chimeric antigen receptor expression. Finally, we discuss CRISPR/Cas9 tools to edit key genes for natural killer cell functionality. [ABSTRACT FROM AUTHOR]

Transposable elements (TEs) are mobile sequences that engender widespread mutations and thus are a major hazard that must be silenced. The most abundant active class of TEs in mammalian genomes is long interspersed element class 1 (LINE1). Here, we report that LINE1 transposition is suppressed in the male germline by transcription factors encoded by a rapidly evolving X-linked homeobox gene cluster. LINE1 transposition is repressed by many members of this RHOX transcription factor family, including those with different patterns of expression during spermatogenesis. One family member-RHOX10-suppresses LINE1 transposition during fetal development in vivo when the germline would otherwise be susceptible to LINE1 activation because of epigenetic reprogramming. We provide evidence that RHOX10 suppresses LINE transposition by inducing Piwil2, which encodes a key component in the Piwi-interacting RNA pathway that protects against TEs. The ability of RHOX transcription factors to suppress LINE1 is conserved in humans but is lost in RHOXF2 mutants from several infertile human patients, raising the possibility that loss of RHOXF2 causes human infertility by allowing uncontrolled LINE1 expression in the germline. Together, our results support a model in which the Rhox gene cluster is in an evolutionary arms race with TEs, resulting in expansion of the Rhox gene cluster to suppress TEs in different biological contexts.

ABSTRACT: Objectives: Emergence of multidrug-resistant (MDR) Salmonella enterica serovar Indiana has raised global concern. Mobile genetic elements (MGEs) play vital roles in accelerating the dissemination of resistance genes in bacteria communities. The study aims to improve our understanding of the underlying resistance mechanisms and characterize the MGEs in a MDR S. Indiana isolate. Methods: Here, we report the characteristics of a MDR pathogenic S. Indiana isolate. The antimicrobial susceptibility pattern of S. Indiana QT6365 was determined. The genomic structure of the chromosome and the plasmid, serotype, and multi-locus sequence type were analysed by whole genome sequencing. The circular form derived from IS26-flanked transposon was confirmed by reverse polymerase chain reaction and sequencing. Results: S. Indiana QT6365 exhibited resistance to all tested antimicrobials except for aztreonam, amikacin, polymyxin, and tigecycline, was defined as MDR, and belonged to ST17. S. Indiana QT6365 was closely related with food resource S. Indiana C629 with similar resistance gene profiles. Multiple resistance genes are mainly carried by a novel transposon Tn7540 located on the chromosome and an IncHI2/HI2A/N plasmid. Sequence analysis and the formed circular intermediate suggested Tn7540 might be generated through homologous recombination by IS26-bounded translocatable units (IS26-fosA-IS26-intI1-dfrA12-aadA2-sul1-ISCR1-blaNDM-9-IS26). Conclusions: To the best of our knowledge, this is the first report of the novel chromosomal transposon possessing blaNDM-9 and fosA3 in S. Indiana isolated from human specimen, which might facilitate the dissemination of resistance genes and should arouse serious awareness.

Abstract Background Murraya paniculata (L.) Jack, commonly called orange jessamine in the family Rutaceae, is an important ornamental plant in tropical and subtropical regions which is famous for its strong fragrance. Although genome assemblies have been reported for many Rutaceae species, mainly in the genus Citrus, full genomic information has not been reported for M. paniculata, which is a prerequisite for in-depth genetic studies on Murraya and manipulation using genetic engineering techniques. Here, we report a high-quality chromosome-level genome assembly of M. paniculata and aim to provide insights on the molecular mechanisms of flower volatile biosynthesis. Results The genome assembly with a contig N50 of 18.25 Mb consists of 9 pseudomolecules and has a total length of 216.86 Mb. Phylogenetic analysis revealed that M. paniculata diverged from the common ancestor approximately 25 million years ago and has not undergone any species-specific whole genome duplication events. Genome structural annotation and comparative genomics analysis revealed that there are obvious differences in transposon contents among the genomes of M. paniculata and Citrus species, especially in the upstream regions of genes. Research on the flower volatiles of M. paniculata and C. maxima at three flowering stages revealed significant differences in volatile composition with the flowers of C. maxima lacking benzaldehyde and phenylacetaldehyde. Notably, there are transposons inserted in the upstream region of the phenylacetaldehyde synthase (PAAS) genes Cg1g029630 and Cg1g029640 in C. maxima, but not in the upstream region of three PAAS genes Me2G_2379, Me2G_2381, and Me2G_2382 in M. paniculata. Our results indicated that compared to the low expression levels of PAAS genes in C. maxima, the higher expression levels of the three PAAS genes in M. paniculata are the main factor affecting the phenylacetaldehyde biosynthesis and causing the content difference of phenylacetaldehyde. The phenylacetaldehyde synthetic activities of the enzymes encoded by M. paniculata PAAS genes were validated by in vitro analyses. Conclusions Our study provides useful genomic resources of M. paniculata for further research on Rutaceae plants, identifies new PAAS genes, and provides insights into how transposons contribute to variations in flower volatiles among Murraya and Citrus plants.

Arbuscular mycorrhizal fungi (AMF) (subphylum Glomeromycotina)1 are among the most prominent symbionts and form the Arbuscular Mycorrhizal symbiosis (AMS) with over 70% of known land plants.2,3 AMS allows plants to efficiently acquire poorly soluble soil nutrients4 and AMF to receive photosynthetically fixed carbohydrates. This plant-fungus symbiosis dates back more than 400 million years5 and is thought to be one of the key innovations that allowed the colonization of lands by plants.6 Genomic and genetic analyses of diverse plant species started to reveal the molecular mechanisms that allowed the evolution of this symbiosis on the host side, but how and when AMS abilities emerged in AMF remain elusive. Comparative phylogenomics could be used to understand the evolution of AMS.7,8 However, the availability of genome data covering basal AMF phylogenetic nodes (Archaeosporales, Paraglomerales) is presently based on fragmentary protein coding datasets.9Geosiphon pyriformis (Archaeosporales) is the only fungus known to produce endosymbiosis with nitrogen-fixing cyanobacteria (Nostoc punctiforme) presumably representing the ancestral AMF state.10-12 Unlike other AMF, it forms long fungal cells ("bladders") that enclose cyanobacteria. Once in the bladder, the cyanobacteria are photosynthetically active and fix nitrogen, receiving inorganic nutrients and water from the fungus. Arguably, G. pyriformis represents an ideal candidate to investigate the origin of AMS and the emergence of a unique endosymbiosis. Here, we aimed to advance knowledge in these questions by sequencing the genome of G. pyriformis, using a re-discovered isolate.

ABSTRACT Group B Streptococcus (GBS) is a major contributor to sepsis, meningitis, and pneumonia in newborns. Indexed bacterial mutant libraries accelerate pathogenesis research by allowing rapid screening of genes that contribute to disease. In this study, we created and characterized a large-scale GBS indexed library of Himar1 mini-transposon mutant strains grown as monocultures from a mixed transposon insertion library that we had previously used for transposon-genome junction sequencing. We used a high-throughput workflow to identify transposon insertion sites in chromosomal DNA purified from individual mutants. Following quality control steps and the removal of isogenic duplicate strains, we isolated 1,919 monocultures of unique transposon insertion mutants with even dispersion across the GBS genome. Our final library, stored in barcoded, traceable glycerol stocks, contains interruptions of 878 genes and 253 intergenic regions. We also validated select library mutants with confirmatory PCR and, when possible, specific phenotypic testing. While the library contains only sparse interruptions of essential or near-essential genes, the genes represented in the set span a wide range of predicted functional categories, including roles in metabolism, structure, and virulence. In conclusion, we developed and applied a high-throughput molecular analysis and bioinformatic pipeline to generate a GBS indexed library of unprecedented scale that we believe will be a useful genetic tool for fellow GBS researchers. IMPORTANCE Group B Streptococcus (GBS) is a significant global cause of serious infections, most of which affect pregnant women, newborns, and infants. Studying GBS genetic mutant strains is a valuable approach for learning more about how these infections are caused and is a key step toward developing more effective preventative and treatment strategies. In this resource report, we describe a newly created library of defined GBS genetic mutants, containing over 1,900 genetic variants, each with a unique disruption to its chromosome. An indexed library of this scale is unprecedented in the GBS field; it includes strains with mutations in hundreds of genes whose potential functions in human disease remain unknown. We have made this resource freely available to the broader research community through deposition in a publicly funded bacterial maintenance and distribution repository.

Polyploidization is important to the evolution of plants. Subgenome dominance is a distinct phenomenon associated with most allopolyploids. A gene on the dominant subgenome tends to express to higher RNA levels in all organs as compared to the expression of its syntenic paralogue (homoeolog). The mechanism that underlies the formation of subgenome dominance remains unknown, but there is evidence for the involvement of transposon/DNA methylation density differences nearby the genes of parents as being causal. The subgenome with lower density of transposon and methylation near genes is positively associated with subgenome dominance. Here, we generated eight generations of allotetraploid progenies from the merging of parental genomes Brassica rapa and Brassica oleracea. We found that transposon/methylation density differ near genes between the parental (rapa:oleracea) existed in the wide hybrid, persisted in the neotetraploids (the synthetic Brassica napus), but these neotetraploids expressed no expected subgenome dominance. This absence of B. rapa vs. B. oleracea subgenome dominance is particularly significant because, while there is no negative relationship between transposon/methylation level and subgenome dominance in the neotetraploids, the more ancient parental subgenomes for all Brassica did show differences in transposon/methylation densities near genes and did express, in the same samples of cells, biased gene expression diagnostic of subgenome dominance. We conclude that subgenome differences in methylated transposon near genes are not sufficient to initiate the biased gene expressions defining subgenome dominance. Our result was unexpected, and we suggest a "nuclear chimera" model to explain our data. [ABSTRACT FROM AUTHOR]

Despite the potential of CAR-T therapies for hematological malignancies, their efficacy in patients with relapse and refractory Acute Myeloid Leukemia has been limited. The aim of our study has been to develop and manufacture a CAR-T cell product that addresses some of the current limitations. We initially compared the phenotype of T cells from AML patients and healthy young and elderly controls. This analysis showed that T cells from AML patients displayed a predominantly effector phenotype, with increased expression of activation (CD69 and HLA-DR) and exhaustion markers (PD1 and LAG3), in contrast to the enriched memory phenotype observed in healthy donors. This differentiated and more exhausted phenotype was also observed, and corroborated by transcriptomic analyses, in CAR-T cells from AML patients engineered with an optimized CAR construct targeting CD33, resulting in a decreased in vivo antitumoral efficacy evaluated in xenograft AML models. To overcome some of these limitations we have combined CRISPR-based genome editing technologies with virus-free genetransfer strategies using Sleeping Beauty transposons, to generate CAR-T cells depleted of HLA-I and TCR complexes (HLA-IKO/TCRKO CAR-T cells) for allogeneic approaches. Our optimized protocol allows one-step generation of edited CAR-T cells that show a similar phenotypic profile to non-edited CAR-T cells, with equivalent in vitro and in vivo antitumoral efficacy. Moreover, genomic analysis of edited CAR-T cells revealed a safe integration profile of the vector, with no preferences for specific genomic regions, with highly specific editing of the HLA-I and TCR, without significant off-target sites. Finally, the production of edited CAR-T cells at a larger scale allowed the generation and selection of enough HLA-IKO/TCRKO CAR-T cells that would be compatible with clinical applications. In summary, our results demonstrate that CAR-T cells from AML patients, although functional, present phenotypic and functional features that could compromise their antitumoral efficacy, compared to CAR-T cells from healthy donors. The combination of CRISPR technologies with transposonbased delivery strategies allows the generation of HLA-IKO/TCRKO CAR-T cells, compatible with allogeneic approaches, that would represent a promising option for AML treatment. [ABSTRACT FROM AUTHOR]

Summary: Polyploidization and transposon elements contribute to shape plant genome diversity and secondary metabolic variation in some edible crops. However, the specific contribution of these variations to the chemo‐diversity of Lamiaceae, particularly in economic shrubs, is still poorly documented. The rich essential oils (EOs) of Lavandula plants are distinguished by monoterpenoids among the main EO‐producing species, L. angustifolia (LA), L. × intermedia (LX) and L. latifolia (LL). Herein, the first allele‐aware chromosome‐level genome was assembled using a lavandin cultivar 'Super' and its hybrid origin was verified by two complete subgenomes (LX‐LA and LX‐LL). Genome‐wide phylogenetics confirmed that LL, like LA, underwent two lineage‐specific WGDs after the γ triplication event, and their speciation occurred after the last WGD. Chloroplast phylogenetic analysis indicated LA was the maternal source of 'Super', which produced premium EO (higher linalyl/lavandulyl acetate and lower 1,8‐cineole and camphor) close to LA. Gene expression, especially the monoterpenoid biosynthetic genes, showed bias to LX‐LA alleles. Asymmetric transposon insertions in two decoupling 'Super' subgenomes were responsible for speciation and monoterpenoid divergence of the progenitors. Both hybrid and parental evolutionary analysis revealed that LTR (long terminal repeat) retrotransposon associated with AAT gene loss cause no linalyl/lavandulyl acetate production in LL, and multi‐BDH copies retained by tandem duplication and DNA transposon resulted in higher camphor accumulation of LL. Advances in allelic variations of monoterpenoids have the potential to revolutionize future lavandin breeding and EO production. [ABSTRACT FROM AUTHOR]

To date, most reports of horizontal gene transfer (HGT) in fungi rely on genome sequence data and are therefore an indirect measure of HGT after the event has occurred. However, a novel group of class II‐like transposons known as Starships may soon alter this status quo. Starships are giant transposable elements that carry dozens of genes, some of which are host‐beneficial, and are linked to many recent HGT events in the fungal kingdom. These transposons remain active and mobile in many fungal genomes and their transposition has recently been shown to be driven by a conserved tyrosine‐recombinase called 'Captain'. This perspective explores some of the remaining unanswered questions about how these Starship transposons move, both within a genome and between different species. We seek to outline several experimental approaches that can be used to identify the genes essential for Starship‐mediated HGT and draw links to other recently discovered giant transposons outside of the fungal kingdom. [ABSTRACT FROM AUTHOR]

Recent advances in gene therapy have brought novel treatment options for cancer. However, the full potential of this approach has yet to be unlocked due to the limited payload capacity of commonly utilized viral vectors. Virus‐free DNA transposons, including piggyBac, have the potential to obviate these shortcomings. In this study, we improved a previously modified piggyBac system with superior transposition efficiency. We demonstrated that the internal domain sequences (IDS) within the 3′ terminal repeat domain of hyperactive piggyBac (hyPB) donor vector contain dominant enhancer elements. Plasmid‐free donor vector devoid of IDS was used in conjunction with a helper plasmid expressing Quantum PBase™ v2 to generate an optimal piggyBac system, Quantum pBac™ (qPB), for use in T cells. qPB outperformed hyPB in CD20/CD19 CAR‐T production in terms of performance as well as yield of the CAR‐T cells produced. Furthermore, qPB also produced CAR‐T cells with lower donor‐associated variabilities compared to lentiviral vector. Importantly, qPB yielded mainly CD8+ CAR‐TSCM cells, and the qPB‐produced CAR‐T cells effectively eliminated CD20/CD19‐expressing tumor cells both in vitro and in vivo. Our findings confirm qPB as a promising virus‐free vector system with an enhanced payload capacity to incorporate multiple genes. This highly efficient and potentially safe system will be expected to further advance gene therapy applications. [ABSTRACT FROM AUTHOR]

Transposable elements (TEs) are parasite DNA sequences that are able to move and multiply along the chromosomes of all genomes. They can be controlled by the host through the targeting of silencing epigenetic marks, which may affect the chromatin structure of neighboring sequences, including genes. In this study, we used transcriptomic and epigenomic high-throughput data produced from ovarian samples of several Drosophila melanogaster and Drosophila simulans wild-type strains, in order to finely quantify the influence of TE insertions on gene RNA levels and histone marks (H3K9me3 and H3K4me3). Our results reveal a stronger epigenetic effect of TEs on ortholog genes in D. simulans compared with D. melanogaster. At the same time, we uncover a larger contribution of TEs to gene H3K9me3 variance within genomes in D. melanogaster , which is evidenced by a stronger correlation of TE numbers around genes with the levels of this chromatin mark in D. melanogaster. Overall, this work contributes to the understanding of species-specific influence of TEs within genomes. It provides a new light on the considerable natural variability provided by TEs, which may be associated with contrasted adaptive and evolutionary potentials. [ABSTRACT FROM AUTHOR]

Research on various species has revealed a connection between genome size variation and the physiological and ecological characteristics of the species, suggesting that it could be a crucial factor influencing a species’ adaptability to different environments. Wheat, being one of the world’s three primary grains, holds significance in this regard. Investigating the genome size of wheat and analyzing the genetic factors contributing to its variation could offer valuable insights for enhancing wheat agronomic traits. This project has developed a conservative site ratio calculation approach to determine the size of the wheat genome. Additionally, it employs flow cytometry and k-mer distribution analysis to validate this method. Furthermore, the researchers use re-sequencing data to investigate the impact of environmental selection pressure and transposon dynamics on the variation in the size of the wheat genome. The findings from this study demonstrate a strong relationship between the size of the wheat genome and several environmental factors. These results serve as a valuable reference for understanding the development of variation in the size of the hetero-hexaploid wheat genome. Moreover, they contribute to advancing fundamental research on the genetic mechanisms underlying wheat characteristics. Additionally, the study paves the way for exploring new research directions in wheat breeding, which holds promise for future advancements in this field. [ABSTRACT FROM AUTHOR]

Multidrug-resistant Enterococcus faecalis (E. faecalis) often cause intestinal infections in cats. The aim of this study was to investigate a multidrug-resistant E. faecalis isolate for plasmidic and chromosomal antimicrobial resistance and their genetic environment. E. faecalis strain ESC1 was obtained from the feces of a cat. Antimicrobial susceptibility testing was carried out using the broth microdilution method. Conjugation experiments were performed using Escherichia coli and Staphylococcus aureus as receptors. Complete sequences of chromosomal DNA and plasmids were generated by whole genome sequencing (WGS) and bioinformatics analysis for the presence of drug resistance genes and mobile elements. Multidrug-resistant E. faecalis ESC1 contained a chromosome and three plasmids. The amino acid at position 80 of the parC gene on the chromosome was mutated from serine to isoleucine, and hence the amino acid mutation at this site led to the resistance of ESC1 strain to fluoroquinolones. Eleven antibiotic resistance genes were located on two plasmids. We identified a novel composite transposon carrying two aminoglycoside resistance genes aac(6′)-aph(2′′). This study reported the coexistence of a novel 5.4 kb composite transposon and a resistance plasmid with multiple homologous recombination in an isolate of E. faecalis ESC1. This data provides a basis for understanding the genomic signature and antimicrobial resistance mechanisms of this pathogen. [ABSTRACT FROM AUTHOR]

Increasing studies report an influence of transposable elements (TEs) on animal colouration. Current cases are dominated by mammals and insects, lab models, and domestic species. Recent studies reveal an influence of TEs on colouration in wild species, associated with fitness benefits including increased reproductive success, decreased predation risk, and optimised resource allocation. Most TE types influence animal colouration, but long terminal repeat/endogenous retrovirus elements are involved more often, potentially due to variation in insertion preferences or inclusion of regulatory sequences. Common genetic mechanisms involve TE-mediated regulatory change in domestic and wild species and disrupted splicing in lab strains. Accumulating genomic data will enable analyses across animal diversity, allowing testing of whether TEs are general contributors to the evolution of animal colouration and their importance relative to other forms of genetic variation. Transposable elements (TEs) are mobile genetic sequences present within host genomes. TEs can contribute to the evolution of host traits, since transposition is mutagenic and TEs often contain host regulatory and protein coding sequences. We review cases where TEs influence animal colouration, reporting major patterns and outstanding questions. TE-induced colouration phenotypes typically arise via introduction of novel regulatory sequences and splice sites, affecting pigment cell development or pigment synthesis. We discuss if particular TE types may be more frequently involved in the evolution of colour variation in animals, given that examples involving long terminal repeat (LTR) elements appear to dominate. Currently, examples of TE-induced colouration phenotypes in animals mainly concern model and domesticated insect and mammal species. However, several influential recent examples, coupled with increases in genome sequencing, suggest cases reported from wild species will increase considerably. [ABSTRACT FROM AUTHOR]

Leafy head is a unique type of plant architecture found in some vegetable crops, with leaves bending inward to form a compact head. The genetic and molecular mechanisms underlying leafy head in vegetables remain poorly understood. We genetically fine-mapped and cloned a major quantitative trait locus controlling heading in lettuce. The candidate gene (LsKN1) is a homolog of knotted 1 (KN1) from Zea mays Complementation and CRISPR/Cas9 knockout experiments confirmed the role of LsKN1 in heading. In heading lettuce, there is a CACTA-like transposon inserted into the first exon of LsKN1 (LsKN1▽). The transposon sequences act as a promoter rather than an enhancer and drive high expression of LsKN1▽. The enhanced expression of LsKN1▽ is necessary but not sufficient for heading in lettuce. Data from ChIP-sequencing, electrophoretic mobility shift assays, and dual luciferase assays indicate that the LsKN1▽ protein binds the promoter of LsAS1 and down-regulates its expression to alter leaf dorsoventrality. This study provides insight into plant leaf development and will be useful for studies on heading in other vegetable crops.

Many germ cells are eliminated during development, long before oogenesis or spermatogenesis. In mouse fetal testes, the majority of germ cell apoptosis coincides with the onset of male differentiation, suggesting coordination of these processes. We studied fetal germ-cell fates and discovered that both apoptosis and differentiation initiate in clonally related clusters. Lineage tracing confirmed that germ cells die as clones independent of intercellular bridges, suggesting that shared intrinsic properties are apoptotic determinants. We identified transcriptional heterogeneity among fetal germ cells that included an apoptosis-susceptible population characterized by failure to differentiate, whereas successful differentiation to prospermatogonia occurred through the expression of epigenetically regulated genes, including LINE1. Our results indicate that the fetal germ-cell fate is based on discrete cell-heritable identities. Elevated DNA methylation in the apoptosis-susceptible subpopulation supports our hypothesis that earlier errors in germ-cell epigenetic reprogramming derail differentiation in cellular progeny, leading to fetal apoptotic selection that ultimately improves the gamete quality.

With its ability to catabolize a wide variety of carbon sources and a growing engineering toolkit, Pseudomonas putida KT2440 is emerging as an important chassis organism for metabolic engineering. Despite advances in our understanding of the organism, many gaps remain in our knowledge of the genetic basis of its metabolic capabilities. The gaps are particularly noticeable in our understanding of both fatty acid and alcohol catabolism, where many paralogs putatively coding for similar enzymes coexist, making biochemical assignment via sequence homology difficult. To rapidly assign function to the enzymes responsible for these metabolisms, we leveraged random barcode transposon sequencing (RB-Tn-Seq). Global fitness analyses of transposon libraries grown on 13 fatty acids and 10 alcohols produced strong phenotypes for hundreds of genes. Fitness data from mutant pools grown on fatty acids of varying chain lengths indicated specific enzyme substrate preferences and enabled us to hypothesize that DUF1302/DUF1329 family proteins potentially function as esterases. From the data, we also postulate catabolic routes for the two biogasoline molecules isoprenol and isopentanol, which are catabolized via leucine metabolism after initial oxidation and activation with coenzyme A (CoA). Because fatty acids and alcohols may serve as both feedstocks and final products of metabolic-engineering efforts, the fitness data presented here will help guide future genomic modifications toward higher titers, rates, and yields.IMPORTANCE To engineer novel metabolic pathways into P. putida, a comprehensive understanding of the genetic basis of its versatile metabolism is essential. Here, we provide functional evidence for the putative roles of hundreds of genes involved in the fatty acid and alcohol metabolism of the bacterium. These data provide a framework facilitating precise genetic changes to prevent product degradation and to channel the flux of specific pathway intermediates as desired.

Regulation of transcription is the main mechanism responsible for precise control of gene expression. Whereas the majority of transcriptional regulation is mediated by DNA-binding transcription factors that bind to regulatory gene regions, an elegant alternative strategy employs small RNA guides, Piwi-interacting RNAs (piRNAs) to identify targets of transcriptional repression. Here, we show that in Drosophila the small ubiquitin-like protein SUMO and the SUMO E3 ligase Su(var)2-10 are required for piRNA-guided deposition of repressive chromatin marks and transcriptional silencing of piRNA targets. Su(var)2-10 links the piRNA-guided target recognition complex to the silencing effector by binding the piRNA/Piwi complex and inducing SUMO-dependent recruitment of the SetDB1/Wde histone methyltransferase effector. We propose that in Drosophila, the nuclear piRNA pathway has co-opted a conserved mechanism of SUMO-dependent recruitment of the SetDB1/Wde chromatin modifier to confer repression of genomic parasites.