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The molecular basis of orchid flower development involves a specific regulatory program in which MADS-box transcription factors play a central role. The recent 'perianth code' model hypothesizes that two types of higher-order heterotetrameric complexes, namely SP complex and L complex, play pivotal roles in the orchid perianth organ formation. Therefore, we explored their roles and searched for other components of the regulatory network. Through the combined analysis for transposase-accessible chromatin with high-throughput sequencing and RNA sequencing of the lip-like petal and lip from Phalaenopsis equestris var.trilip, transcription factor-(TF) genes involved in lip development were revealed. PeNAC67 encoding a NAC-type TF and PeSCL23 encoding a GRAS-type TF were differentially expressed between the lip-like petal and the lip. PeNAC67 interacted with and stabilized PeMADS3, which positively regulated the development of lip-like petal to lip. PeSCL23 and PeNAC67 competitively bound with PeKAN2 and positively regulated the development of lip-like petal to petal by affecting the level of PeMADS3. PeKAN2 as an important TF that interacts with PeMADS3 and PeMADS9 can promote lip development. These results extend the 'perianth code' model and shed light on the complex regulation of orchid flower development. [ABSTRACT FROM AUTHOR]

The co-cultivation of sake yeast (AK25, K901, K1401, or K1801 strain) and the kuratsuki Bacillus A-10 and/or Priestia B-12 strains in koji solution was performed to demonstrate the effects of these two kuratsuki bacteria on sake taste. The results showed that the Brix and acidity patterns of sake preparations produced with and without these kuratsuki bacteria were very similar. This indicated that the addition of these kuratsuki bacteria did not inhibit ethanol fermentation or organic acid production by sake yeast. A taste recognition device showed that the effects of these kuratsuki bacteria on the saltiness and sourness of sake were greater than those on other taste properties. Astringency stimulation and saltiness of sake produced using the sake yeast K901 were increased by Bacillus A-10 and decreased by Priestia B-12. Except for these two cases, the taste intensities of sake preparations produced with the Bacillus A-10 and Priestia B-12 strains were very similar, but differed from those of sake produced with kuratsuki Kocuria. These results support our hypothesis that the flavor and taste of sake can be controlled by utilizing the interactions between kuratsuki bacteria and sake yeast. For crating the desired sake taste, a combination of kuratsuki bacteria and sake yeast should be considered. [ABSTRACT FROM AUTHOR]

Simple Summary: Streptococcus suis can cause dangerous infections in swine and humans. Among numerous pathogenic serotypes, serotype 8 is one of the predominant serotypes isolated from different hosts. However, little is known about its pathogenicity and genomic characterization. In this study, we first provided complete genomes of two serotype 8 strains: virulent and non-virulent. Several virulence-associated genes are located in mobile genetic elements that can form circular DNA intermediates, indicating the possibility of horizontal transmission among S. suis strains. Mouse infection experiments confirmed the roles of two new virulence-associated genes in S. suis virulence. These findings contribute to understanding the genomic characterization of S. suis serotype 8 and the pathogenic properties of S. suis. Streptococcus suis is an important zoonotic pathogen that can cause meningitis and septicemia in swine and humans. Among numerous pathogenic serotypes, S. suis serotype 8 has distinctive characteristics such as a high detection rate and causing multi-host infection. There is no complete genome of serotype 8 strains so far. In this study, the complete genome of two S. suis serotype 8 strains, virulent strain 2018WUSS151 and non-virulent strain WUSS030, were sequenced. Comparative genomic analysis showed that the homology of the two genomes reaches 99.68%, and the main difference is the distinctive prophages. There are 83 genes unique to virulent strain 2018WUSS151, including three putative virulence-associated genes (PVGs). Two PVGs, padR and marR, are passenger genes in ISSsu2 family transposons that are able to form circular DNA intermediates during transposition, indicating the possibility of horizontal transmission among S. suis strains. The deletion mutant of PVGs marR or atpase attenuated the virulence of serotype 2 virulent SC070731 in a mouse infection model, confirming their role in S. suis virulence. These findings contribute to clarifying the genomic characterization of S. suis serotype 8 and S. suis pathogenesis. [ABSTRACT FROM AUTHOR]

Streptococcus suis is an important zoonotic pathogen that can cause meningitis and septicemia in swine and humans. Among numerous pathogenic serotypes, S. suis serotype 8 has distinctive characteristics such as a high detection rate and causing multi-host infection. There is no complete genome of serotype 8 strains so far. In this study, the complete genome of two S. suis serotype 8 strains, virulent strain 2018WUSS151 and non-virulent strain WUSS030, were sequenced. Comparative genomic analysis showed that the homology of the two genomes reaches 99.68%, and the main difference is the distinctive prophages. There are 83 genes unique to virulent strain 2018WUSS151, including three putative virulence-associated genes (PVGs). Two PVGs, padR and marR, are passenger genes in ISSsu2 family transposons that are able to form circular DNA intermediates during transposition, indicating the possibility of horizontal transmission among S. suis strains. The deletion mutant of PVGs marR or atpase attenuated the virulence of serotype 2 virulent SC070731 in a mouse infection model, confirming their role in S. suis virulence. These findings contribute to clarifying the genomic characterization of S. suis serotype 8 and S. suis pathogenesis.

Butterfly wing patterns derive from a deeply conserved developmental ground plan yet are diverse and evolve rapidly. It is poorly understood how gene regulatory architectures can accommodate both deep homology and adaptive change. To address this, we characterized the cis-regulatory evolution of the color pattern gene WntA in nymphalid butterflies. Comparative assay for transposase-accessible chromatin using sequencing (ATAC-seq) and in vivo deletions spanning 46 cis-regulatory elements across five species revealed deep homology of ground plan-determining sequences, except in monarch butterflies. Furthermore, noncoding deletions displayed both positive and negative regulatory effects that were often broad in nature. Our results provide little support for models predicting rapid enhancer turnover and suggest that deeply ancestral, multifunctional noncoding elements can underlie rapidly evolving trait systems. [ABSTRACT FROM AUTHOR]

Single-cell sequencing technologies have rapidly progressed in recent years, and been applied to characterize stem cells in a number of organs. Somatic (postnatal) stem cells are generally identified using combinations of cell surface markers and transcription factors. However, it has been challenging to define micro-heterogeneity within “stem cell” populations, each of which stands at a different level of differentiation. As stem cells become defined at a single-cell level, their differentiation path becomes clearly defined. Here, this viewpoint discusses the potential synergy of single-cell sequencing analyses with in vivo lineage-tracing approaches, with an emphasis on practical considerations in stem cell biology. [ABSTRACT FROM AUTHOR]

ABSTRACT Transposition is a major mechanism of horizontal gene mobility in prokaryotes. However, exploration of the genes mobilized by transposons (cargo) is hampered by the difficulty in delineating integrated transposons from their surrounding genetic context. Here, we present a computational approach that allowed us to identify the boundaries of 6,549 Tn7-like transposons. We found that 96% of these transposons carry at least one cargo gene. Delineation of distinct communities in a gene-sharing network demonstrates how transposons function as a conduit of genes between phylogenetically distant hosts. Comparative analysis of the cargo genes reveals significant enrichment of mobile genetic elements (MGEs) nested within Tn7-like transposons, such as insertion sequences and toxin-antitoxin modules, and of genes involved in recombination, anti-MGE defense, and antibiotic resistance. More unexpectedly, cargo also includes genes encoding central carbon metabolism enzymes. Twenty-two Tn7-like transposons carry both an anti-MGE defense system and antibiotic resistance genes, illustrating how bacteria can overcome these combined pressures upon acquisition of a single transposon. This work substantially expands the distribution of Tn7-like transposons, defines their evolutionary relationships, and provides a large-scale functional classification of prokaryotic genes mobilized by transposition. IMPORTANCE Transposons are major vehicles of horizontal gene transfer that, in addition to genes directly involved in transposition, carry cargo genes. However, characterization of these genes is hampered by the difficulty of identification of transposon boundaries. We developed a computational approach for detecting transposon ends and applied it to perform a comprehensive census of the cargo genes of Tn7-like transposons, a large class of bacterial mobile genetic elements (MGE), many of which employ a unique, CRISPR-mediated mechanism of site-specific transposition. The cargo genes encompass a striking diversity of MGE, defense, and antibiotic resistance systems. Unexpectedly, we also identified cargo genes encoding metabolic enzymes. Thus, Tn7-like transposons mobilize a vast repertoire of genes that can have multiple effects on the host bacteria.

Mealybugs are insects that maintain intracellular bacterial symbionts to supplement their nutrient-poor plant sap diets. Some mealybugs have a single betaproteobacterial endosymbiont, a Candidatus Tremblaya species (hereafter Tremblaya) that alone provides the insect with its required nutrients. Other mealybugs have two nutritional endosymbionts that together provision these same nutrients, where Tremblaya has gained a gammaproteobacterial partner that resides in its cytoplasm. Previous work had established that Pseudococcus longispinus mealybugs maintain not one but two species of gammaproteobacterial endosymbionts along with Tremblaya. Preliminary genomic analyses suggested that these two gammaproteobacterial endosymbionts have large genomes with features consistent with a relatively recent origin as insect endosymbionts, but the patterns of genomic complementarity between members of the symbiosis and their relative cellular locations were unknown. Here, using long-read sequencing and various types of microscopy, we show that the two gammaproteobacterial symbionts of P. longispinus are mixed together within Tremblaya cells, and that their genomes are somewhat reduced in size compared with their closest nonendosymbiotic relatives. Both gammaproteobacterial genomes contain thousands of pseudogenes, consistent with a relatively recent shift from a free-living to an endosymbiotic lifestyle. Biosynthetic pathways of key metabolites are partitioned in complex interdependent patterns among the two gammaproteobacterial genomes, the Tremblaya genome, and horizontally acquired bacterial genes that are encoded on the mealybug nuclear genome. Although these two gammaproteobacterial endosymbionts have been acquired recently in evolutionary time, they have already evolved codependencies with each other, Tremblaya , and their insect host. [ABSTRACT FROM AUTHOR]

The ability and efficiency of targeted nucleases to perform sequence replacements or insertions into the genome are limited. This limited efficiency for sequence replacements or insertions can be explained by the dependency on DNA repair pathways, the possibility of cellular toxicity, and unwanted activation of proto‐oncogenes. The piggyBac (PB) transposase uses a very efficient enzymatic mechanism to integrate DNA fragments into the genome in a random manner. In this study, we fused an RNA‐guided catalytically inactive Cas9 (dCas9) to the PB transposase and used dual sgRNAs to localize this molecule to specific genomic targets. We designed and used a promoter/reporter complementation assay to register and recover cells harboring‐specific integrations, where only by complementation upon correct genomic integration, the reporter can be activated. Using an RNA‐guided piggyBac transposase and dual sgRNAs, we were able to achieve site‐directed integrations in the human ROSA26 safe harbor region in 0.32% of cells. These findings show that the methodology used in this study can be used for targeting genomic regions. An application for this finding could be in cancer cells to insert sequences into specific target regions that are intended to be destroyed, or to place promoter cargos behind the tumor suppressor genes to activate them. [ABSTRACT FROM AUTHOR]

PromA plasmids are broad host range (BHR) plasmids, which are often cryptic and hence have an uncertain ecological role. We present three novel PromA γ plasmids which carry genes associated with degradation of the phenylurea herbicide linuron, two of which originated from unrelated Hydrogenophaga hosts isolated from different environments (pPBL-H3-2 and pBPS33-2), and one (pEN1) which was exogenously captured from an on-farm biopurification system (BPS). Hydrogenophaga sp. plasmid pBPS33-2 carries all three necessary gene clusters (hylA, dca, ccd) determining the three main steps for conversion of linuron to Krebs cycle intermediates, while pEN1 only determines the initial linuron hydrolysis step. Hydrogenophaga sp. plasmid pPBL-H3-2 exists as two variants, both containing ccd but with the hylA and dca gene modules interchanged between each other at exactly the same location. Linuron catabolic gene clusters that determine the same step were identical on all plasmids, encompassed in differently arranged constellations and characterized by the presence of multiple IS1071 elements. In all plasmids except pEN1, the insertion spot of the catabolic genes in the PromA γ plasmids was the same. Highly similar PromA plasmids carrying the linuron degrading gene cargo at the same insertion spot were previously identified in linuron degrading Variovorax sp. Interestingly, in both Hydrogenophaga populations not every PromA plasmid copy carries catabolic genes. The results indicate that PromA plasmids are important vehicles of linuron catabolic gene dissemination, rather than being cryptic and only important for the mobilization of other plasmids.

Abstract Background Next-generation sequencing (NGS) has revolutionized almost all fields of biology, agriculture and medicine, and is widely utilized to analyse genetic variation. Over the past decade, the NGS pipeline has been steadily improved, and the entire process is currently relatively straightforward. However, NGS instrumentation still requires upfront library preparation, which can be a laborious process, requiring significant hands-on time. Herein, we present a simple but robust approach to streamline library preparation by utilizing surface bound transposases to construct DNA libraries directly on a flowcell surface. Results The surface bound transposases directly fragment genomic DNA while simultaneously attaching the library molecules to the flowcell. We sequenced and analysed a Drosophila genome library generated by this surface tagmentation approach, and we showed that our surface bound library quality was comparable to the quality of the library from a commercial kit. In addition to the time and cost savings, our approach does not require PCR amplification of the library, which eliminates potential problems associated with PCR duplicates. Conclusions We described the first study to construct libraries directly on a flowcell. We believe our technique could be incorporated into the existing Illumina sequencing pipeline to simplify the workflow, reduce costs, and improve data quality.

The use of Assay for Transposase-Accessible Chromatin (ATAC-seq) to profile chromatin accessibility has surged over the past years, but its applicability to tissues has been very limited. With the intent of preserving nuclear architecture during long-term storage, cryopreserved nuclei preparations from chicken lung were used to optimize ATAC-seq. Sequencing data were compared with existing DNase-seq, ChIP-seq, and RNA-seq data to evaluate library quality, ultimately resulting in a modified ATAC-seq method capable of generating high quality chromatin accessibility data from cryopreserved nuclei preparations. Using this method, nucleosome-free regions (NFR) identified in chicken lung overlapped half of DNase-I hypersensitive sites, coincided with active histone modifications, and specifically marked actively expressed genes. Notably, sequencing only the subnucleosomal fraction dramatically improved signal, while separation of subnucleosomal reads post-sequencing did not improve signal or peak calling. The broader applicability of this modified ATAC-seq technique was tested using cryopreserved nuclei preparations from pig tissues, resulting in NFR that were highly consistent among biological replicates. Furthermore, tissue-specific NFR were enriched for binding motifs of transcription factors related to tissue-specific functions, and marked genes functionally enriched for tissue-specific processes. Overall, these results provide insights into the optimization of ATAC-seq and a platform for profiling open chromatin in animal tissues. [ABSTRACT FROM AUTHOR]

PromA plasmids are broad host range (BHR) plasmids, which are often cryptic and hence have an uncertain ecological role. We present three novel PromA γ plasmids which carry genes associated with degradation of the phenylurea herbicide linuron, two of which originated from unrelated Hydrogenophaga hosts isolated from different environments (pPBL-H3-2 and pBPS33-2), and one (pEN1) which was exogenously captured from an on-farm biopurification system (BPS). Hydrogenophaga sp. plasmid pBPS33-2 carries all three necessary gene clusters (hylA, dca, ccd) determining the three main steps for conversion of linuron to Krebs cycle intermediates, while pEN1 only determines the initial linuron hydrolysis step. Hydrogenophaga sp. plasmid pPBL-H3-2 exists as two variants, both containing ccd but with the hylA and dca gene modules interchanged between each other at exactly the same location. Linuron catabolic gene clusters that determine the same step were identical on all plasmids, encompassed in differently arranged constellations and characterized by the presence of multiple IS 1071 elements. In all plasmids except pEN1, the insertion spot of the catabolic genes in the PromA γ plasmids was the same. Highly similar PromA plasmids carrying the linuron degrading gene cargo at the same insertion spot were previously identified in linuron degrading Variovorax sp. Interestingly, in both Hydrogenophaga populations not every PromA plasmid copy carries catabolic genes. The results indicate that PromA plasmids are important vehicles of linuron catabolic gene dissemination, rather than being cryptic and only important for the mobilization of other plasmids. [ABSTRACT FROM AUTHOR]

Relatively little is known about the role of sponge microbiomes in the Antarctic marine environment, where sponges may dominate the benthic landscape. Specifically, we understand little about how taxonomic and functional diversity contributes to the symbiotic lifestyle and aids in nutrient cycling. Here we use functional metagenomics to investigate the community composition and metabolic potential of microbiomes from two abundant Antarctic sponges, Leucetta antarctica and Myxilla sp. Genomic and taxonomic analyses show that both sponges harbor a distinct microbial community with high fungal abundance, which differs from the surrounding seawater. Functional analyses reveal both sponge-associated microbial communities are enriched in functions related to the symbiotic lifestyle (e.g., CRISPR system, Eukaryotic-like proteins, and transposases), and in functions important for nutrient cycling. Both sponge microbiomes possessed genes necessary to perform processes important to nitrogen cycling (i.e., ammonia oxidation, nitrite oxidation, and denitrification), and carbon fixation. The latter indicates that Antarctic sponge microorganisms prefer light-independent pathways for CO2 fixation mediated by chemoautotrophic microorganisms. Together, these results show how the unique metabolic potential of two Antarctic sponge microbiomes help these sponge holobionts survive in these inhospitable environments, and contribute to major nutrient cycles of these ecosystems. [ABSTRACT FROM AUTHOR]

Transposases are ubiquitous mobile genetic elements responsible for genome development, driving rearrangements, such as insertions, deletions and translocations. Across species evolution, some transposases are tamed by their host and are made part of complex cellular systems. The proliferation of retroviruses is also dependent on transposase related enzymes termed integrases. Recombination-activating gene protein (RAG)1 and metnase are just two examples of transposase domestication and together with retroviral integrases (INs), they belong to the DDE polynucleotidyl transferases superfamily. They share mechanistic and structural features linked to the RNase H-like fold, harboring a DDE(D) metal dependent catalytic motif. Recent antiretroviral compounds target the catalytic domain of integrase, but they also have the potential of inhibiting other related enzymes. In this review, we report the activity of different classes of integrase inhibitors on various DDE transposases. Computational simulations are useful to predict the extent of off-target activity and have been employed to study the interactions between RAG1 recombinase and compounds from three different pharmacologic classes. We demonstrate that strand-transfer inhibitors display a higher affinity towards the RAG1 RNase H domain, as suggested by experimental data compared to allosteric inhibitors. While interference with RAG1 and 2 recombination is associated with a negative impact on immune function, the inhibition of metnase or HTLV-1 integrase opens the way for the development of novel therapies for refractory cancers. [ABSTRACT FROM AUTHOR]

Microbial community function in freshwater sediments is influenced by the presence and persistence of anthropogenic pollutants, yet simultaneously imposes significant control on their transformation. Thus, microbes provide valuable ecosystem services in terms of biodegradation and bioindicators of compromised habitats. From a remediation perspective it is valuable to leverage the suite of microbial genes at the transcriptional level that are active in either natural versus stressed environments to provide insight into the cycling and fate of contaminants. Metatranscriptomic analysis of total bacterial and archaeal messenger RNA (mRNA) is a useful tool in this facet and was applied to sediments sampled from the Detroit River; a binational Area of Concern (AOC) in the Great Lakes. Previously established sediment surveys and modelling delineated the river into contaminant gradients based on concentrations of polycyclic aromatic hydrocarbons (PAHs), polychlorinated biphenyls (PCBs), and metals. Differential expression analysis through DESeq2 revealed that microbial transcripts associated with nitrate reduction, methanogenesis, and beta-oxidation were significant in legacy polluted sediments and linked with energetic pathways key in the generation of cellular currencies (acetyl-CoA, succinyl-CoA). Gluconeogenesis and polyester synthesis also showed high abundance in contaminated regions, along with increased expression of stress response genes and transposons, despite decreases in community α-diversity. Aromatic cleavage genes were detected, but in low abundance across the contaminant gradient. These results suggest that microbial communities within the Detroit River exploit unique anabolic and catabolic pathways to derive and store energy from legacy organic contaminants while simultaneously recruiting stress-response and gene transfer mechanisms to cope with xenobiotic pressures. By coupling well-resolved chemical datasets with metatranscriptomics, this study adds to the spatial understanding of in-situ microbial activities in pristine and perturbed freshwater sediments. Unlabelled Image • Geospatial model confirmed contaminant gradient in selected Detroit River sediments. • Microbial diversity decreased with increasing metals, PAHs, and PCBs. • Metatranscriptomics revealed linked microbial contaminant degradation pathways. • Nitrate reduction and methanogenesis are key processes in community energetics. • Microbial transposons showed significant increased abundance in polluted sediments. [ABSTRACT FROM AUTHOR]

Frankia strainsinducetheformationofnitrogen-fixingnodulesonrootsofactinorhizalplants.Phylogenetically, Frankia strainscanbe grouped in four clusters. The earliest divergent cluster, cluster-2, has a particularly wide host range. The analysis of cluster-2 strains has been hampered by the fact that with two exceptions, they could never be cultured. In this study, 12 Frankia-enriched meta genomes of Frankia cluster-2 strains or strain assemblages were sequenced based on seven inoculum sources. Sequences obtained via DNA isolated from whole nodules were compared with those of DNA isolated from fractionated preparations enhanced in the Frankia symbiotic structures. The results show that cluster-2 inocula represent groups of strains, and that strains not represented in symbiotic structures, that is, unable to perform symbiotic nitrogen fixation, may still be able to colonize nodules. Transposase gene abundance was compared in the different Frankia-enriched metagenomes with the result that North American strains contain more transposase genes than Eurasian strains. An analysis of the evolution and distribution of the host plants indicated that bursts of transposition may have coincided with niche competition with other cluster-2 Frankia strains. The first genome of an inoculum from the Southern Hemisphere, obtained from nodules of Coriaria papuana in Papua New Guinea, represents a novel species, postulated as Candidatus Frankia meridionalis. All Frankia-enriched metagenomes obtained in this study contained homologs of the canonical nod genes nodABC; the North American genomes also contained the sulfotransferase gene nodH, while the genome from the Southern Hemisphere only contained nodC and a truncated copy of nodB. [ABSTRACT FROM AUTHOR]

CRISPR-Cas nucleases are powerful tools for manipulating nucleic acids; however, targeted insertion of DNA remains a challenge, as it requires host cell repair machinery. Here we characterize a CRISPR-associated transposase from cyanobacteria Scytonema hofmanni (ShCAST) that consists of Tn7-like transposase subunits and the type V-K CRISPR effector (Cas12k). ShCAST catalyzes RNA-guided DNA transposition by unidirectionally inserting segments of DNA 60 to 66 base pairs downstream of the protospacer. ShCAST integrates DNA into targeted sites in the Escherichia coli genome with frequencies of up to 80% without positive selection. This work expands our understanding of the functional diversity of CRISPR-Cas systems and establishes a paradigm for precision DNA insertion. [ABSTRACT FROM AUTHOR]

Capsules allow bacteria to colonize novel environments, to withstand numerous stresses, and to resist antibiotics. Yet, even though genetic exchanges with other cells should be adaptive under such circumstances, it has been suggested that capsules lower the rates of homologous recombination and horizontal gene transfer. We analysed over one hundred pan-genomes and thousands of bacterial genomes for the evidence of an association between genetic exchanges (or lack thereof) and the presence of a capsule system. We found that bacteria encoding capsules have larger pan-genomes, higher rates of horizontal gene transfer, and higher rates of homologous recombination in their core genomes. Accordingly, genomes encoding capsules have more plasmids, conjugative elements, transposases, prophages, and integrons. Furthermore, capsular loci are frequent in plasmids, and can be found in prophages. These results are valid for Bacteria, independently of their ability to be naturally transformable. Since we have shown previously that capsules are commonly present in nosocomial pathogens, we analysed their co-occurrence with antibiotic resistance genes. Genomes encoding capsules have more antibiotic resistance genes, especially those encoding efflux pumps, and they constitute the majority of the most worrisome nosocomial bacteria. We conclude that bacteria with capsule systems are more genetically diverse and have fast-evolving gene repertoires, which may further contribute to their success in colonizing novel niches such as humans under antibiotic therapy. [ABSTRACT FROM AUTHOR]

Acute hepatopancreatic necrosis disease (AHPND) is a lethal shrimp disease. The pathogenic agent of this disease is a special Vibrio parahaemolyticus strain that contains a pVA1 plasmid. The protein products of two toxin genes in pVA1, pirAvp and pirBvp, targeted the shrimp’s hepatopancreatic cells and were identified as the major virulence factors. However, in addition to pirAvp and pirBvp, pVA1 also contains about ~90 other open-reading frames (ORFs), which may encode functional proteins. NCBI BLASTp annotations of the functional roles of 40 pVA1 genes reveal transposases, conjugation factors, and antirestriction proteins that are involved in horizontal gene transfer, plasmid transmission, and maintenance, as well as components of type II and III secretion systems that may facilitate the toxic effects of pVA1-containing Vibrio spp. There is also evidence of a post-segregational killing (PSK) system that would ensure that only pVA1 plasmid-containing bacteria could survive after segregation. Here, in this review, we assess the functional importance of these pVA1 genes and consider those which might be worthy of further study.

In Enterobacteriaceae, the blaOXA-48-like genes have been identified on plasmids in different regions of the world. The OXA-370 is a plasmid-encoded OXA-48-like enzyme reported in two distinct regions of Brazil. Recently, we demonstrate that the blaOXA-370 gene is disseminated among several Enterobacteriaceae species and clones, indicating a high potential for dissemination. In this work, we described for the first time the complete nucleotide sequence of six plasmids harboring the blaOXA-370 gene. Complete DNA sequencing using the Illumina platform and annotation of the plasmids showed that they belonged to incompatibility groups IncX and had in average 70 kbp. The blaOXA-370 gene is located in a composite transposon containing four genes encoding transposases, named Tn6435. In this study, highly similar plasmids were detected in different Enterobacteriaceae genera. [ABSTRACT FROM AUTHOR]

Abstract Mu-like transposable phages and prophages have been isolated from, or predicted, in a wide range of bacterial phyla. However, related B3-like transposable phages, which differ in their genome organisation and the DDE transposase and transposition activator they code for have thus far been restricted to a very limited set of hosts. Through sequence similarity searches, we have now expanded the number of predicted B3-like prophages and uncovered a third genomic organisation. These new genomes, although only prophages, further illustrate the previously reported mosaicism existing in the proposed "Saltoviridae" family of Caudovirales , and further support the proposal to move morphology criteria (contractile vs. flexible or short tail, i.e. Myo -vs. Sipho - or Podoviridae) from the family to the subfamily level in the taxonomic classification of the Caudovirales. [ABSTRACT FROM AUTHOR]

The development of Acidithiobacillus ferrooxidans as a non-model host organism for synthetic biology is hampered by a lack of genetic tools and techniques. New plating and liquid-based selection methods were developed to improve the identification of transformed cell lines. Enabled by these methods, a hyperactive transposase was used to generate mutants with integrated genes for the expression of the superfolder green fluorescent protein (sfGFP) gene or a 2-keto decarboxylase (KDC) gene, which enabled the production and secretion of isobutyric acid (IBA). An inverse PCR method was used to identify the insertion sites of the KDC gene in several mutants, leading to the identification of a region on the chromosome that may be suitable for future genetic insertions. These results demonstrate that functional exogenous metabolic genes have been chromosomally integrated into A. ferrooxidans, and this advance will facilitate the future development of these cells for new biotechnology applications. [ABSTRACT FROM AUTHOR]

The pancreas and liver are closely related developmentally and trans-differentiation of cells from one tissue into the cells of the other has been documented to occur after injury or exposure to selected growth factors or glucocorticoid hormones. To generate a readily-expandable source of human hepatocyte-like (H-13) cells, the human pancreatic adenocarcinoma cell (HPAC) line was stably transfected with a construct encoding the variant 2 hepatocyte nuclear factor 4 α (HNF4α) using a piggyBac vector and transient expression of a transposase. Through induction of transgene HNF4α regulated via an upstream glucocorticoid response element in combination with existing modulating effects of glucocorticoid, H-13 cells were converted into quantitatively similar hepatocyte-like (H-13/H) cells based on expression of a variety of hepatocyte proteins. H-13/H cells also demonstrated the ability to store glycogen and lipids. These data provide proof of concept that regulated expression of genes associated with hepatocyte phenotype could be used to generate quantitatively functional human hepatocyte-like cells using a readily expandable cell source and simple culture protocol. This approach would have utility in Toxicology and Hepatology research. [ABSTRACT FROM AUTHOR]

Abstract The role of the nuclease, HARBI1-like protein (mjHARBI1-like) in the innate immunity of Marsupenaeus japonicus was explored in this study. The 1361 bp cDNA sequence of mjHARBI1-like was cloned from M. japonicus using RACE. RT-qPCR analysis results showed that the gills and hepatopancreas of M. japonicus were the main tissues where mjHARBI1-like is expressed. In addition, it was also found that white spot syndrome virus (WSSV) or Vibrio alginolyticus challenge could stimulate mjHARBI1-like expression. After mjHARBI1-likewas inhibited, expression of immune genes such as toll, p53, myosin, and proPO were significantly downregulated ( P < 0.01). However, in shrimp hemocytes, hemocyanin and tumor necrosis factor-α (TNF-α) were up-regulated significantly ( P < 0.01). This study demonstrated that mjHARBI1-like plays a key role in the progression of WSSV and V. alginolyticus infection. Specifically, the cumulative mortality of WSSV-infected and V. alginolyticus -infected shrimp was significantly advanced by double-strand RNA interference (dsRNAi) of mjHARBI1-like. Apoptosis studies indicated that mjHARBI1-dsRNA treatment caused a reduction in hemocyte apoptosis in bacterial and viral groups. In addition, phagocytosis experiments illustrated that mjHARBI1-dsRNA treatment led to a lower phagocytosis rate in hemocytes of V. alginolyticus -challenged shrimp. It was also found that knockdown of mjHARBI1-like inhibited shrimp phenoloxidase (PO) activity, superoxide dismutase (SOD) activity, and total hemocyte count (THC) after WSSV or V. alginolyticus infection. These data indicate a regulative role of mjHARBI1-likein the immunity of shrimp in response to pathogen infection. Resultantly, it was concluded that mjHARBI1-like might have a positive effect on the anti -WSSV immune response of shrimp by regulating apoptosis, THC, PO activity, and SOD activity. Additionally, mjHARBI1-like might promote anti - V. alginolyticus infection by participating in regulating phagocytosis, apoptosis, SOD activity, PO activity, and THC. Highlights • The 1361 bp cDNA sequence of HARBI1-like was cloned from M. japonicus. • The mortality of WSSV-infected and V. alginolyticus -infected shrimp was significantly advanced by RNAi of mjHARBI1-like. • The mjHARBI1-dsRNA treatment caused a reduction in hemocyte apoptosis in bacterial and viral groups. • The mjHARBI1-dsRNA treatment led to a lower phagocytosis rate in hemocytes of V. alginolyticus -challenged shrimp. • The mjHARBI1-dsRNA treatment inhibited PO activity, SOD activity, and THC after WSSV or V. alginolyticus infection. [ABSTRACT FROM AUTHOR]

Even in times of advanced site-specific genome editing tools, the improvement of DNA transposases is still on high demand in the field of transgenesis: especially in emerging model systems where evaluated integrase landing sites have not yet been created and more importantly in non-model organisms such as agricultural pests and disease vectors, in which reliable sequence information and genome annotations are still pending. In fact, random insertional mutagenesis is essential to identify new genomic locations that are not influenced by position effects and thus can serve as future stable transgene integration sites. In this respect, a hyperactive version of the most widely used piggyBac transposase (PBase) has been engineered. The hyperactive version (hyPBase) is currently available with the original insect codon-based coding sequence ( i hyPBase ) as well as in a mammalian codon-optimized ( m hyPBase ) version. Both facilitate significantly higher rates of transposition when expressed in mammalian in vitro and in vivo systems compared to the classical PBase at similar protein levels. Here we demonstrate that the usage of helper plasmids encoding the hyPBase - irrespective of the codon-usage - also strikingly increases the rate of successful germline transformation in the Mediterranean fruit fly (Medfly) Ceratitis capitata , the red flour beetle Tribolium castaneum, and the vinegar fly Drosophila melanogaster . hyPBase-encoding helpers are therefore highly suitable for the generation of transgenic strains of diverse insect orders. Depending on the species, we achieved up to 15-fold higher germline transformation rates compared to PBase and generated hard to obtain transgenic T. castaneum strains that express constructs affecting fitness and viability. Moreover, previously reported high sterility rates supposedly caused by hyPBase (iPB7), encoded by i hyPBase, could not be confirmed by our study. Therefore, we value hyPBase as an effective genetic engineering tool that we highly recommend for insect transgenesis. [ABSTRACT FROM AUTHOR]

Class II DNA Transposable Elements (TEs) are moved from one location to another in the genome by the action of transposase proteins that bind to repeat sequences at the ends of the elements. Although the location TE insertion is mostly random, the addition of DNA binding domains to the transposase proteins has allowed for targeted insertion of some elements. In this study, the Gal4 binding domain was added to the transposase proteins, ORF1 and TPase, which mobilize the mPing element from rice. The Gal4:TPase construct was capable of increasing the number of mPing insertions into the Gal2 and Gal4 promoter sequences in yeast. While this confirms that mPing insertion preference can be manipulated, the target specificity is relatively low. Thus, the CRISPR/Cas9 system was tested for its ability to generate targeted insertion of mPing. A dCas9:TPase fusion protein had a low transposition rate suggesting that the addition of this large protein disrupts TPase function. Unfortunately, the use of a MS2 binding domain to localize the TPase to the MS2 hairpin containing gRNA failed to produce targeted insertion. Thus, our results suggest that the addition of small DNA binding domain to the N-terminal of TPase is the best strategy for targeted insertion of mPing. [ABSTRACT FROM AUTHOR]

Mycobacterium avium subsp. paratuberculosis (MAP) is a causative agent of Johne’s disease or paratuberculosis (PTB), which is a chronic debilitating disease in ruminants, that is characterized by incurable enteritis and persistent diarrhea. ISMap 02 is one of the major targets of PCR because it is present in multicopies (six copies) and known to be specific to MAP. However, in the present study, non-MAP mycobacteria were shown to be positive by ISMap 02 targeting PCR. Two bacterial isolates (Sample ID: BO-038 and BO-042) were cultured from bovine fecal samples that produced positive results in three of two ISMap 02 targeting PCR analyses with negative results in IS 900 real-time PCR. Species identification using 16S rRNA gene sequencing and hsp65 gene partial sequencing revealed that strains BO-038 and BO-042 were M. virginiense and M. nonchromogenicum , respectively, which both belong to the M. terrae complex (MTC). Moreover, the two isolates shared a novel insertion sequence (IS) with high similarity to some parts of nucleotide sequences of ISMap 02 , and IS was presumed to be identical to that present in M. heraklionense . Both the novel IS and ISMap 02 were characterized as IS 1182 family members, and several sequences similar to ISMap 02 were identified by BLAST analysis. In addition, the DDE transposase of the novel IS showed great similarity in the N-terminal portion with the IS 5 / 1182 DDE transposase of other mycobacteria. These results suggest that ISMap 02 has a conserved region with similarity to other ISs, and that the diagnostic value of the primer sets targeting that region should be re-addressed. [ABSTRACT FROM AUTHOR]

Summary: Rapid spread of resistance to vancomycin has generated difficult to treat bacterial pathogens worldwide. Though vancomycin resistance is often conferred by the conjugative transposon Tn1549, it is yet unclear whether Tn1549 moves actively between bacteria. Here we demonstrate, through development of an in vivo assay system, that a mini‐Tn1549 can transpose in E. coli away from its natural Gram‐positive host. We find the transposon‐encoded INT enzyme and its catalytic tyrosine Y380 to be essential for transposition. A second Tn1549 protein, XIS is important for efficient and accurate transposition. We further show that DNA flanking the left transposon end is critical for excision, with changes to nucleotides 7 and 9 impairing movement. These mutations could be partially compensated for by changing the final nucleotide of the right transposon end, implying concerted excision of the two ends. With changes in these essential DNA sequences, or without XIS, a large amount of flanking DNA transposes with Tn1549. This rescues mobility and allows the transposon to capture and transfer flanking genomic DNA. We further identify the transposon integration target sites as TTTT‐N6‐AAAA. Overall, our results provide molecular insights into conjugative transposition and the adaptability of Tn1549 for efficient antibiotic resistance transfer. [ABSTRACT FROM AUTHOR]

Mobile genetic elements (MGEs) are key agents in the spread of antibiotic resistance genes (ARGs) across environments. Here we used metagenomics to compare the river resistome (collection of all ARGs) and mobilome ( e.g. , integrases, transposases, integron integrases and insertion sequence common region “IS CR ” elements) between samples collected upstream ( n = 6) and downstream ( n = 6) of an urban wastewater treatment plant (UWWTP). In comparison to upstream metagenomes, downstream metagenomes showed a drastic increase in the abundance of ARGs, as well as markers of MGEs, particularly integron integrases and IS CR elements. These changes were accompanied by a concomitant prevalence of 16S rRNA gene signatures of bacteria affiliated to families encompassing well-known human and animal pathogens. Our results confirm that chronic discharges of treated wastewater severely impact the river resistome affecting not only the abundance and diversity of ARGs but also their potential spread by enriching the river mobilome in a wide variety of MGEs. [ABSTRACT FROM AUTHOR]

The ecological competences in microbiomes are driven by the adaptive capabilities present within microbiome members. Horizontal gene transfer (HGT) promoted by plasmids provides a rapid adaptive strategy to microbiomes, an interesting feature considering the constantly changing conditions in most environments. This study examined the parA locus, found in the highly promiscuous PromA class of plasmids, as the insertion site for incoming genes. A novel PCR system was designed that enabled examining insertions into this locus. Microbiomes of mangrove sediments, salt marsh, mycosphere, and bulk soil revealed habitat-specific sets of insertions in this plasmid region. Furthermore, such habitats could be differentiated based on patterns of parA-inserted genes, and the genes carried by these plasmids. Thus, a suite of dioxygenase-related genes and transposase elements were found in oil-affected mangroves, whereas genes involved in nitrogen and carbon cycling were detected in salt marsh and soils. All genes detected could be associated with capabilities of members of the microbiome to adapt to and survive in each habitat. The methodology developed in this work was effective, sensitive, and practical, allowing detection of mobilized genes between microorganisms. [ABSTRACT FROM AUTHOR]

Efficient preparation of high-quality sequencing libraries that well represent the biological sample is a key step for using next-generation sequencing in research. Tn5 enables fast, robust, and highly efficient processing of limited input material while scaling to the parallel processing of hundreds of samples. Here, we present a robust Tn5 transposase purification strategy based on an N-terminal His6-Sumo3 tag. We demonstrate that libraries prepared with our in-house Tn5 are of the same quality as those processed with a commercially available kit (Nextera XT), while they dramatically reduce the cost of large-scale experiments. We introduce improved purification strategies for two versions of the Tn5 enzyme. The first version carries the previously reported point mutations E54K and L372P, and stably produces libraries of constant fragment size distribution, even if the Tn5-to-input molecule ratio varies. The second Tn5 construct carries an additional point mutation (R27S) in the DNA-binding domain. This construct allows for adjustment of the fragment size distribution based on enzyme concentration during tagmentation, a feature that opens new opportunities for use of Tn5 in customized experimental designs. We demonstrate the versatility of our Tn5 enzymes in different experimental settings, including a novel single-cell polyadenylation site mapping protocol as well as ultralow input DNA sequencing. [ABSTRACT FROM AUTHOR]

One hundred thirty-five single-spore isolates were collected from grape vineyards from 15 provinces or autonomous regions belonging to five viticulture climatic zones in China. All the isolates were identified as Botrytis cinerea based on their morphological and molecular characters. The 135 isolates were all heterothallic isolates. Seventy-one isolates belonged to the MAT1-1 type and 64 were characterized to MAT1-2 type. All the isolates studied belonged to Group II based on PCR-RFLP of the Bc-hch locus. The four TE genotypes, transposa, fioty-only, Flipper-only, and vacuma, comprised 51.9%, 33.3%, 10.4%, and 4.5%, respectively, of the total population. The frequency of transposa was highest in the total population and the most of any subpopulation (each viticulture climatic zone), and Borv-only was highest in warm areas (46.2%). Vacuma was mainly distributed in northern China, although in small amounts, and Flipper-only was mainly was distributed in humid tropical areas (42.9%). A microsatellite analysis of B. cinerea populations was performed to assess the genetic population structure. A total of 127 different MLGs were identified among 135 B. cinerea isolates, with a genotypic diversity of 0.9991. The transposa population isolates showed higher genetic diversity than other populations. Pairwise tests of genetic differentiation among four TE-type populations yielded generally low to high Gst values. All isolates belonged to two genetic clusters. The population microsatellite diversity and genetic structure had a certain correlation with the TE type and geographic origin. This is the first report of the genotypic diversity of B. cinerea isolates from grape vineyards across China. [ABSTRACT FROM AUTHOR]

The Tc1/mariner superfamily is one of the most widely distributed among the DNA transposons in both terrestrial and aquatic organisms. We studied the abundance of the Tc1/mariner elements in the genome of the gastropod Littorina saxatilis Olivi, 1792 (Gastropoda: Littorinimorpha). For this purpose, nucleotide sequences with a total length of 358877 bp were analyzed. Six sequences were found to be similar to the Tc1/mariner DNA transposons. These sequences were studied for structure, the presence of functional transposase, and the systematic position within the superfamily. In addition, the loci with high homology to the DNA transposons of the hAT, Sola, Ginger, EnSpm/CACTA, ISL2EU, Kolobok, Novosib, Zisupton, and Helitron superfamilies were identified. [ABSTRACT FROM AUTHOR]

A high level of transposon-mediated genome rearrangement is a common trait among microorganisms isolated from thermal environments, probably contributing to the extraordinary genomic plasticity and horizontal gene transfer (HGT) observed in these habitats. In this work, active and inactive insertion sequences (ISs) spanning the sequenced members of the genus Thermus were characterized, with special emphasis on three T. thermophilus strains: HB27, HB8, and NAR1. A large number of full ISs and fragments derived from different IS families were found, concentrating within megaplasmids present in most isolates. Potentially active ISs were identified through analysis of transposase integrity, and domestication-related transposition events of ISTth7 were identified in laboratory-adapted HB27 derivatives. Many partial copies of ISs appeared throughout the genome, which may serve as specific targets for homologous recombination contributing to genome rearrangement. Moreover, recruitment of IS1000 32 bp segments as spacers for CRISPR sequence was identified, pointing to the adaptability of these elements in the biology of these thermophiles. Further knowledge about the activity and functional diversity of ISs in this genus may contribute to the generation of engineered transposons as new genetic tools, and enrich our understanding of the outstanding plasticity shown by these thermophiles.

Background: HIV-1 Integrase (IN) interacts with the cellular co-factor LEDGF/p75 and tethers the HIV preintegration complex to the host genome enabling integration. Recently a new class of IN inhibitors was described, the IN-LEDGF allosteric inhibitors (INLAIs). Designed to interfere with the IN-LEDGF interaction during integration, the major impact of these inhibitors was surprisingly found on virus maturation, causing a reverse transcription defect in target cells. Results: Here we describe the MUT-A compound as a genuine INLAI with an original chemical structure based on a new type of scaffold, a thiophene ring. MUT-A has all characteristics of INLAI compounds such as inhibition of IN-LEDGF/p75 interaction, IN multimerization, dual antiretroviral (ARV) activities, normal packaging of genomic viral RNA and complete Gag protein maturation. MUT-A has more potent ARV activity compared to other INLAIs previously reported, but similar profile of resistance mutations and absence of ARV activity on SIV. HIV-1 virions produced in the presence of MUT-A were non-infectious with the formation of eccentric condensates outside of the core. In studying the immunoreactivity of these non-infectious virions, we found that inactivated HIV-1 particles were captured by anti-HIV-specific neutralizing and non-neutralizing antibodies (b12, 2G12, PGT121, 4D4, 10-1074, 10E8, VRC01) with efficiencies comparable to non-treated virus. Autologous CD4+ T lymphocyte proliferation and cytokine induction by monocyte-derived dendritic cells (MDDC) pulsed either with MUT-A-inactivated HIV or non-treated HIV were also comparable. Conclusions: Although strongly defective in infectivity, HIV-1 virions produced in the presence of the MUT-A INLAI have a normal protein and genomic RNA content as well as B and T cell immunoreactivities comparable to nontreated HIV-1. These inactivated viruses might form an attractive new approach in vaccine research in an attempt to study if this new type of kkimmunogen could elicit an immune response against HIV-1 in animal models. [ABSTRACT FROM AUTHOR]

The article presents a study which identified certain vulnerabilities in cancers that express PGBD5, a DNA transposase. The study found that expression of PGBD5 is not tolerated by cells in mice and human beings which lack nonhomologous end joining DNA repair. Examination of cell survival and DNA damage was done using Western immunoblotting.

Gene regulatory networks (GRNs) describe the interactions for a developmental process at a given time and space. Historically, perturbation experiments represent one of the key methods for analyzing and reconstructing a GRN, and the GRN governing early development in the sea urchin embryo stands as one of the more deeply dissected so far. As technology progresses, so do the methods used to address different biological questions. Next-generation sequencing (NGS) has become a standard experimental technique for genome and transcriptome sequencing and studies of protein-DNA interactions and DNA accessibility. While several efforts have been made toward the integration of different omics approaches for the study of the regulatory genome in many animals, in a few cases, these are applied with the purpose of reconstructing and experimentally testing developmental GRNs. Here, we review emerging approaches integrating multiple NGS technologies for the prediction and validation of gene interactions within echinoderm GRNs. These approaches can be applied to both 'model' and 'non-model' organisms. Although a number of issues still need to be addressed, advances in NGS applications, such as assay for transposase-accessible chromatin sequencing, combined with the availability of embryos belonging to different species, all separated by various evolutionary distances and accessible to experimental regulatory biology, place echinoderms in an unprecedented position for the reconstruction and evolutionary comparison of developmental GRNs. We conclude that sequencing technologies and integrated omics approaches allow the examination of GRNs on a genome-wide scale only if biological perturbation and cis-regulatory analyses are experimentally accessible, as in the case of echinoderm embryos. [ABSTRACT FROM AUTHOR]

Selfish genetic elements, suchas insertion sequences and transposons are found inmost genomes. Transposons are usually identifiable by their high copy number within genomes. In contrast, REP-associated tyrosine transposases (RAYTs), a recently described class of bacterial transposase, are typically present at just one copy per genome. This suggests that RAYTs no longer copy themselves and thus they no longer function as a typical transposase. Motivated by this possibility we interrogated thousands of fully sequenced bacterial genomes in order to determine patterns of RAYT diversity, their distribution across chromosomes and accessory elements, and rate of duplication. RAYTs encompass exceptional diversity and are divisible into at least five distinct groups. They possess features more similar to housekeeping genes than insertion sequences, are predominantly vertically transmitted and have persisted through evolutionary time to the point where they are now found in 24% of all species for which at least onefully sequenced genome is available. Overall, the genomic distribution of RAYTs suggests that they have been coopted by host genomes to perform a function that benefits the host cell. [ABSTRACT FROM AUTHOR]

Establishing stable Chinese Hamster Ovary (CHO) cells producing monoclonal antibodies (mAbs) usually pass through the random integration of vectors to the cell genome, which is sensitive to gene silencing. One approach to overcome this issue is to target a highly transcribed region in the genome. Transposons are useful devices to target active parts of genomes, and PiggyBac (PB) transposon can be considered as a good option. In the present study, three PB transposon donor vectors containing both heavy and light chains were constructed, one contained independent expression cassettes while the others utilized either an Internal Ribosome Entry Site (IRES) or 2A element to express mAb. Conventional cell pools were created by transferring donor vectors into the CHO cells, whereas transposon-based cells were generated by transfecting the cells with donor vectors with a companion of a transposase-encoding helper vector, with 1:2.5 helper/donor vectors ratio. To evaluate the influence of helper/donor vectors ratio on expression, the second transposon-based cell pools were generated with 1:5 helper/donor ratio. Expression levels in the transposon-based cells were two to five -folds more than those created by conventional method except for the IRES-mediated ones, in which the observed difference increased more than 100-fold. The results were dependent on both donor vector design and vectors ratios. [ABSTRACT FROM AUTHOR]

Objectives: Characterization of Proteus mirabilis isolates harbouring bla OXA-58 with emphasis on the genetic environment of this resistance determinant.Methods: Strains of P. mirabilis ( n  =   37) isolated from different patients were tested for the presence of bla OXA-58 . The genetic context of bla OXA-58 was determined by WGS of two strains and Sanger sequencing. Clonality of the strains was assessed by PFGE. Susceptibility testing was performed by microdilution according to EUCAST.Results: Four strains isolated in different geographical regions of Germany were positive for bla OXA-58 , and WGS showed that this resistance gene was harboured on a plasmid. Sanger sequencing confirmed the presence of two nearly identical plasmids, 6219 and 6208 bp in size, in all four strains. Upstream of bla OXA-58 an IS Aba 3-like transposase gene was located. The P. mirabilis strains were not clonally related according to PFGE. MICs of meropenem for three of the strains were only just above the EUCAST breakpoint and the Carba NP test was positive for only two of the strains.Conclusions: To our knowledge, this is the first description of bla OXA-58 in the species P. mirabilis . The resistance gene is harboured by almost identical plasmids in strains not clonally related and from different geographical regions. Apart from an IS Aba 3-like transposase gene upstream of bla OXA-58 the genetic context is different from bla OXA-58 harboured on plasmids in the genus Acinetobacter . With MICs of meropenem well below the EUCAST breakpoint or only just above it and equivocal or false negative results from the Carba NP test, bla OXA-58 can be easily overlooked in P. mirabilis . [ABSTRACT FROM AUTHOR]

Horizontal Gene Transfer (HGT) plays an important role in the physiology and evolution of microorganisms above all thermophilic prokaryotes. Some members of the Phylum Thermotogae (i.e., Thermotoga spp.) have been reported to present genomes constituted by a mosaic of genes from a variety of origins. This study presents a novel approach to search on the potential plasticity of Fervidobacterium genomes using putative transposase-encoding genes as the target of analysis. Transposases are key proteins involved in genomic DNA rearrangements. A comprehensive comparative analysis, including phylogeny, non-metric multidimensional scaling analysis of tetranucleotide frequencies, repetitive flanking sequences and divergence estimates, was performed on the transposase genes detected in four Fervidobacterium genomes: F. nodosum, F. pennivorans, F. islandicum and a new isolate (Fervidobacterium sp. FC2004). Transposase sequences were classified in different groups by their degree of similarity. The different methods used in this study pointed that over half of the transposase genes represented putative HGT events with closest relative sequences within the phylum Firmicutes, being Caldicellulosiruptor the genus showing highest gene sequence proximity. These results confirmed a direct evolutionary relationship through HGT between specific Fervidobacterium species and thermophilic Firmicutes leading to potential gene sequence and functionality sharing to thrive under similar environmental conditions. Transposase-encoding genes represent suitable targets to approach the plasticity and potential mosaicism of bacterial genomes. [ABSTRACT FROM AUTHOR]

Mariner-like elements (MLEs) are Class-II transposons that are widely present in diverse organisms and encode a D,D34D transposase motif. MLE sequences from two coleopteran species, Bruchus pisorum and B. rufimanus were obtained using the terminal-inverted repeats (TIRs) of mariner elements belonging to the mauritiana subfamily as primer. The characterized elements were between 1073 and 1302 bp in length and are likely to be inactive, based on the presence of multiple stop codons and/or frameshifts. A single consensus of MLE was detected in B. pisorum and was named Bpmar1. This element exhibited several conserved amino acid blocks as well as the specific D,D(34)D signature. As for B. rufimanus, two MLE consensuses, designated Brmar1 and Brmar2, were isolated, both containing deletions overlapping the internal region of the transposase. Structural and phylogenetic analysis of these sequences suggested a relatively recent origin of Bpmar1 versus a more ancient invasion of Brmar1 and Brmar2 in their respective host genomes. Given that MLEs are potential mediators of insect resistance and have been used as vectors to transfer genes into host genomes, the MLEs characterized in this study will have valuable implications for selecting appropriate transposable elements in transgenesis. [ABSTRACT FROM AUTHOR]

The moso bamboo genome contains the two structurally intact and thus potentially functional mariner -like elements Ppmar1 and Ppmar2 . Both elements contain perfect terminal inverted repeats (TIRs) and a full-length intact transposase gene. Here we investigated whether Ppmar1 is functional in yeast ( Saccharomyces cerevisiae ). We have designed a two-component system consisting of a transposase expression cassette and a non-autonomous transposon on two separate plasmids. We demonstrate that the Ppmar1 transposase Pptpase1 catalyses excision of the non-autonomous Ppmar1NA element from the plasmid and reintegration at TA dinucleotide sequences in the yeast chromosomes. In addition, we generated 14 hyperactive Ppmar1 transposase variants by systematic single amino acid substitutions. The most active transposase variant, S171A, induces 10-fold more frequent Ppmar1NA excisions in yeast than the wild type transposase. The Ppmar1 transposon is a promising tool for insertion mutagenesis in moso bamboo and may be used in other plants as an alternative to the established transposon tagging systems. [ABSTRACT FROM AUTHOR]

Activation of transcription requires alteration of chromatin by complexes that increase the accessibility of nucleosomal DNA. Removing nucleosomes from regulatory sequences has been proposed to play a significant role in activation. Wetested whether changes in nucleosome occupancy occurred on the set of genes that is activated by the unfolded protein response (UPR). We observed no decrease in occupancy on most promoters, gene bodies, and enhancers. Instead, there was an increase in the accessibility of nucleosomes, as measured by micrococcal nuclease (MNase) digestion and ATAC-seq (assay for transposase-accessible chromatin [ATAC] using sequencing), that did not result fromremoval of the nucleosome. Thus, changes in nucleosome accessibility predominate over changes in nucleosome occupancy during rapid transcriptional induction during the UPR. [ABSTRACT FROM AUTHOR]

The transposition of bacteriophage Mu serves as a model system for understanding DDE transposases and integrases. All available structures of these enzymes at the end of the transposition reaction, including Mu, exhibit significant bends in the transposition target site DNA. Here we use Mu to investigate the ramifications of target DNA bending on the transposition reaction. Enhancing the flexibility of the target DNA or prebending it increases its affinity for transpososomes by over an order of magnitude and increases the overall reaction rate. This and FRET confirm that flexibility is interrogated early during the interaction between the transposase and a potential target site, which may be how other DNA binding proteins can steer selection of advantageous target sites. We also find that the conformation of the target DNA after strand transfer is involved in preventing accidental catalysis of the reverse reaction, as conditions that destabilize this conformation also trigger reversal. [ABSTRACT FROM AUTHOR]

Over the last years, the number Coxiella burnetii infections increased throughout the wourld. A molecular detection and phylogenetic analysis were performed in Al-Diwaniayh city in which the first phylogenetic documentation of C.burnetii reported in this area with accession numbers KY576802, KY576803, KY576804 and KY576805. Molecular detection and phylogenetic analysis of C.burnetii targeting the transposase gene partial sequence was done on the milk samples from 50 apperently healthy goats. DNA sequence was amplified in 6 out of 50 (12%) milk samples collected from different regions of Al Diwaniya city. Four of these amplicons were submitted to sequences analysis and gave 99% nucleotides sequence similarity. Also the amplicon sequences of local strain were compared with global C.burnetti sequences in NCBI which revealed close related to NCBI-Blast C.burnetii transposase gene of Indian (AB84899.1) and Brazilian (JF970261.1) strains, whereas there was genetic variation to NCBI-Blast C.burnetii transposase gene for USA (DQ379976.1), Portugal (EU009657.1) and Taiwan (EU000273.1). This work confirm goats infection and shedding the C. burnetii in Al-Diwaniayh, Iraq and determined the phylogenetic tree of local strain of this bacterium. [ABSTRACT FROM AUTHOR]

Metagenomics and other metaomics approaches (including metatranscriptomics) provide insights into the composition and function of microbial communities living in different environments or animal hosts. Metatranscriptomics research provides an unprecedented opportunity to examine gene regulation for many microbial species simultaneously, and more importantly, for the majority that are unculturable microbial species, in their natural environments (or hosts). Current analyses of metatranscriptomic datasets focus on the detection of gene expression levels and the study of the relationship between changes of gene expression and changes of environment. As a demonstration of utilizing metatranscriptomics beyond these common analyses, we developed a computational and statistical procedure to analyze the antisense transcripts in strand-specific metatranscriptomic datasets. Antisense RNAs encoded on the DNA strand opposite a gene’s CDS have the potential to form extensive base-pairing interactions with the corresponding sense RNA, and can have important regulatory functions. Most studies of antisense RNAs in bacteria are rather recent, are mostly based on transcriptome analysis, and have been applied mainly to single bacterial species. Application of our approaches to human gut-associated metatranscriptomic datasets allowed us to survey antisense transcription for a large number of bacterial species associated with human beings. The ratio of protein coding genes with antisense transcription ranges from zero to 35.8% (median = 10.0%) among 47 species. Our results show that antisense transcription is dynamic, varying between human individuals. Functional enrichment analysis revealed a preference of certain gene functions for antisense transcription, and transposase genes are among the most prominent ones (but we also observed antisense transcription in bacterial house-keeping genes).

A major fraction of Earth's prokaryotic biomass dwells in the deep subsurface, where cellular abundances per volume of sample are lower, metabolism is slower, and generation times are longer than those in surface terrestrial and marine environments. How these conditions impact biotic interactions and evolutionary processes is largely unknown. Here we employed single cell genomics to analyze cell-to-cell genome content variability and signatures of horizontal gene transfer (HGT) and viral infections in five cells of Candidatus Desulforudis audaxviator, which were collected from a three km-deep fracture water in the 2.9 Ga-old Witwatersrand Basin of South Africa. Between 0 and 32 % of genes recovered from single cells were not present in the original, metagenomic assembly of Desulforudis, which was obtained from a neighboring subsurface fracture. We found a transposable prophage, a retron, multiple clustered regularly interspaced short palindromic repeats (CRISPRs) and restriction-modification systems, and an unusually high frequency of transposases in the analyzed single cell genomes. This indicates that recombination, HGT and viral infections are prevalent evolutionary events in the studied population of microorganisms inhabiting a highly stable deep subsurface environment.

Transposable elements are able to move along eukaryotic genomes. They are divided into two classes according to their transposition intermediate: RNA (class I or retrotransposons) or DNA (class II or DNA transposons). Most of these sequences are inactive or non-autonomous in eukaryotic genomes. Inactivate transposons can accumulate mutations at neutral rates until losing their molecular identity. They may either be eliminated from the genome or take on different molecular functions. Transposable elements may also participate in the differentiation of sex chromosomes. Therefore, the structural variations and nucleotide similarity of Tc1/Mariner sequences were analyzed along with their potential participation in the differentiation processes of sex chromosomes in the genomes of Parodontidae fish. All Parodontidae species presented non-autonomous copies of Tc1/Mariner with structural variation, different levels of deterioration (genetic distance), and variations in insertion and deletion patterns. The physical mapping of Tc1/Mariner on chromosomes revealed dispersed signals in euchromatins, with small accumulations in terminal regions and in the sex chromosomes. The gene dosage ratios indicated copy number variations of Tc1/Mariner among the genomes and high transposase open reading frame deterioration in Parodon hilarii and Parodon pongoensis genomes. This transposon presented transcriptional activity in gonads, but there was no significant difference between sexes. This may indicate non-functional protein expression or may correspond to DNA binding proteins derived from Tc1/Mariner . Thus, our results show Tc1/Mariner inactivation along with a diversity in Parodontidae genomes and its participation in the differentiation of the W sex chromosome. [ABSTRACT FROM AUTHOR]