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In this article, a bioinformatics pipeline is proposed that focuses on two types of elements, namely the mobile genetic elements (MGE) and Ribonucleic acids (RNAs). The MGEs are called insertion sequences (ISs) in the prokaryotic domain. The objective of this research work is to study the behaviour of RNAs and MGEs genes, and the effects of their presence around inversions in genome sequences. The proposed pipeline finds the relation between the transposase gene types (e.g., DDE and DEDD) located within insertion sequences according to their IS family and sub-family, and RNAs (tRNA and rRNA) on the one hand, and genomic inversion on the other hand. More precisely, we wonder whether the insertion sequences and RNAs have any effects on the occurrence of inversions. To investigate this, we start by extracting all inversions between every two close isolates of P. aeruginosa from a high supported phylo- genetic tree, and we investigate inversion boundaries, to determine the impact of IS elements and RNAs around these inversions. For RNAs genes, we noticed that a high occurrence of these elements are found in what we called InBoundary, while few to zero occurrences have been recorded in OutBoundary. We remark that the NCGM2.S1 genome has the largest amount of tRNAs and inversions comparing to other genomes. For RNA genes, the tRNAs genes (Arg, Ala, Leu) recorded the highest occurrences around the inversions, while there was few occurrences of rRNA genes, within all genomes. For IS elements, we noticed that the family of IS3 has the highest impact on inversions, but sometimes other type of IS elements can also be found with IS3 around the inversions. In this case, these types of ISs shared the same type of transposase gene (DDE). We finally conclude that the distribution of RNA and IS genes has a big impact on the inversions in bacterial genomes.

The majority of DNA-based transposable elements comprise autonomous and nonautonomous copies, or only nonautonomous copies, where the autonomous copy contains an intact gene for a transposase protein and the nonautonomous copy does not. Even if autonomous copies coexist, they are generally less frequent. The Tol2 element of medaka fish is one of the few elements for which a nonautonomous copy has not yet been found. Here, we report the presence of a nonautonomous Tol2 copy that was identified by surveying the medaka genome sequence database. This copy contained three local sequence alterations that affected the deduced amino acid sequence of the transposase: a deletion of 15 nucleotides resulting in a deletion of 5 amino acids, a base substitution causing a single amino acid change, and another base substitution giving rise to a stop codon. Transposition assays using cultured human cells revealed that transposase activity was reduced by the 15-nucleotide deletion and abolished by the nonsense mutation. This is the first example of a nonautonomous Tol2 copy. Thus, Tol2 is in an early stage of decay in the medaka genome, and is therefore a unique element to observe an almost complete decay process that progresses in natural populations.

This work was supported by a Longer and Larger (LoLa) grant from the Biotechnology and Biological Sciences Research Council (BBSRC; grant numbers BB/G020744/1, BB/G019177/1, BB/G019274/1 and BB/G018553/1), the UK Department for Environment, Food and Rural Affairs and Zoetis awarded to the Bacterial Respiratory Diseases of Pigs-1 Technology (BRaDP1T) consortium. Funding for LZ was provided by the BBSRC (grant number BB/C508193/1). Comprehensive identification of conditionally essential genes requires efficient tools for generating high-density transposon libraries that, ideally, can be analysed using next-generation sequencing methods such as Transposon Directed Insertion-site Sequencing (TraDIS). The Himar1 (mariner) transposon is ideal for generating near-saturating mutant libraries, especially in AT-rich chromosomes, as the requirement for integration is a TA dinucleotide, and this transposon has been used for mutagenesis of a wide variety of bacteria. However, plasmids for mariner delivery do not necessarily work well in all bacteria. In particular, there are limited tools for functional genomic analysis of Pasteurellaceae species of major veterinary importance, such as swine and cattle pathogens, Actinobacillus pleuropneumoniae and Pasteurella multocida, respectively. Here, we developed plasmids, pTsodCPC9 and pTlacPC9 (differing only in the promoter driving expression of the transposase gene), that allow delivery of mariner into both these pathogens, but which should also be applicable to a wider range of bacteria. Using the pTlacPC9 vector, we have generated, for the first time, saturating mariner mutant libraries in both A. pleuropneumoniae and P. multocida that showed a near random distribution of insertions around the respective chromosomes as detected by TraDIS. A preliminary screen of 5000 mutants each identified 8 and 14 genes, respectively, that are required for growth under anaerobic conditions. Future high-throughput screening of the generated libraries will facilitate identification of mutants required for growth under different conditions, including in vivo, highlighting key virulence factors and pathways that can be exploited for development of novel therapeutics and vaccines. Publisher PDF

Transposition has a key role in reshaping genomes of all living organisms1. Insertion sequences of IS200/IS605 and IS607 families2 are among the simplest mobile genetic elements and contain only the genes that are required for their transposition and its regulation. These elements encode tnpA transposase, which is essential for mobilization, and often carry an accessory tnpB gene, which is dispensable for transposition. Although the role of TnpA in transposon mobilization of IS200/IS605 is well documented, the function of TnpB has remained largely unknown. It had been suggested that TnpB has a role in the regulation of transposition, although no mechanism for this has been established3–5. A bioinformatic analysis indicated that TnpB might be a predecessor of the CRISPR–Cas9/Cas12 nucleases6–8. However, no biochemical activities have been ascribed to TnpB. Here we show that TnpB of Deinococcus radiodurans ISDra2 is an RNA-directed nuclease that is guided by an RNA, derived from the right-end element of a transposon, to cleave DNA next to the 5′-TTGAT transposon-associated motif. We also show that TnpB could be reprogrammed to cleave DNA target sites in human cells. Together, this study expands our understanding of transposition mechanisms by highlighting the role of TnpB in transposition, experimentally confirms that TnpB is a functional progenitor of CRISPR–Cas nucleases and establishes TnpB as a prototype of a new system for genome editing.

Similar to other complex organisms, plants consist of diverse and specialized cell types. The gain of unique biological functions of these different cell types is the consequence of the establishment of cell-type-specific transcriptional programs. As a necessary step in gaining a deeper understanding of the regulatory mechanisms controlling plant gene expression, we report the use of single-nucleus RNA sequencing (sNucRNA-seq) and single-nucleus assay for transposase accessible chromatin sequencing (sNucATAC-seq) technologies on Arabidopsis roots. The comparison of our single-nucleus transcriptomes to the published protoplast transcriptomes validated the use of nuclei as biological entities to establish plant cell-type-specific transcriptomes. Furthermore, our sNucRNA-seq results uncovered the transcriptomes of additional cell subtypes not identified by single-cell RNA-seq. Similar to our transcriptomic approach, the sNucATAC-seq approach led to the distribution of the Arabidopsis nuclei into distinct clusters, suggesting the differential accessibility of chromatin between groups of cells according to their identity. To reveal the impact of chromatin accessibility on gene expression, we integrated sNucRNA-seq and sNucATAC-seq data and demonstrated that cell-type-specific marker genes display cell-type-specific patterns of chromatin accessibility. Our data suggest that the differential chromatin accessibility is a critical mechanism to regulate gene activity at the cell-type level.

Streptococcus thermophilus is one of the lactic acid bacteria applied as the main starter for dairy foods. A type strain of Streptococcus salivarius subsp. thermophilus ATCC 19258 has been used in the genetic and biochemical characterization of their genes or gene products. While the genome sequence of NCTC 12958 as an equivalent to ATCC 19258 is available, characterization of whether both collections are identical remains to be validated. Here, we report the complete genome sequence of ATCC 19258, which contains one 2.1 Mb chromosome with a 39.0% of G + C content, and includes 2255 protein-coding sequences, 77 RNAs, 4 riboswitches, and 3 CRISPRs. The data were further compared with NCTC 12958 and found that 54 mutations and 4 gaps occurred in NCTC 12958, resulted in both the mutations and insertions of nucleotides in the genome. Unlike ATCC 19258, pre-termination of three genes encoding IS981 transposase B, MltF, and FetB were detected in NCTC 12958. Our study highlights that type strains of Streptococcus thermophilus in two available independent strain collections are possibly different and therefore, the functions of previously identified or hitherto uncharacterized genes of Streptococcus thermophilus should be carefully assigned based on the genomic database of the strain.

Graphical abstract, Highlights • SETMAR/Metnase preferentially targets Hsmar1 transposon ends (ITRs) in living cells. • Sequence fidelity of the ITR motif determines the affinity of SETMAR/Metnase to chromosomes. • Higher ITR fidelity results in increased affinity for chromatin and stronger repression of SETMAR-bound gene loci., Throughout evolution, DNA transposons provide a recurrent supply of genetic information to give rise to novel gene functions by fusion of their transposase domain to various domains of host-encoded proteins. One of these “domesticated”, transposase-derived factors is SETMAR/Metnase which is a naturally occurring fusion protein that consists of a histone-lysine methyltransferase domain and an HsMar1 transposase. To elucidate the biological role of SETMAR, it is crucial to identify genomic targets to which SETMAR specifically binds and link these sites to the regulation of gene expression. Herein, we mapped the genomic landscape of SETMAR binding in a near-haploid human leukemia cell line (HAP1) in order to identify on-target and off-target binding sites at high resolution and to elucidate their role in terms of gene expression. Our analysis revealed a perfect correlation between SETMAR and inverted terminal repeats (ITRs) of HsMar1 transposon remnants, which are considered as natural target sites for SETMAR binding. However, we did not detect any untargeted events at non-ITR sequences, calling into question previously proposed off-target binding sites. We identified sequence fidelity of the ITR motif as a key factor for determining the binding affinity of SETMAR for chromosomes, as higher conservation of ITR sequences resulted in increased affinity for chromatin and stronger repression of SETMAR-bound gene loci. These associations highlight how SETMAR’s chromatin binding fine-tune gene regulatory networks in human tumour cells.

Objective Plasmids are key to antimicrobial resistance transmission among enteric bacteria. It is becoming increasingly clear that resistance genes alone do not account for the selective advantage of plasmids and bacterial strains that harbor them. Deletion of a 32 Kb fitness-conferring region of pMB2, a conjugative resistance plasmid, produced a hyper-autoaggregation phenotype in laboratory Escherichia coli. This study sought to determine the genetic basis for hyper-autoaggregation conferred by the pMB2-derived mini-plasmid. Results The 32 Kb fragment deleted from pMB2 included previously characterized nutrient acquisition genes as well as putative transposase and integrase genes, a 272 bp papB/ pefB-like gene, and several open-reading frames of unknown function. We cloned the papB/ pefB paralogue and found it sufficient to temper the hyper-autoaggregation phenotype. Hyper-autoaggregation conferred by the mini-plasmid did not occur in a fim-negative background. This study has identified and characterized a gene capable of down-regulating host adhesins and has shown that trans-acting papB/pefB paralogues can occur outside the context of an adhesin cluster. This plasmid-mediated modification of a bacterial host’s colonization program may optimize horizontal transfer of the mobile element bearing the genes.

The medaka is a small freshwater fish found in ponds and rivers in Japan and has been a familiar pet since the 17th century. It has also been used as a model of basic biology for more than 100 years. Recently, because of the development of molecular genetic tools, the medaka has drawn attention because of its amenability to genetic modification. In this chapter, we will introduce a brief history, biological features, and application techniques for genetic modification, including disruption and foreign gene introduction. In addition to such basic approaches involving the generation of transgenic and knockout medaka, we will introduce several techniques for increasing the efficiency and range of possible applications: bacterial artificial chromosome/Fosmid-based vector construction, the dual promoter method, transposase, and phiC31 integrase. Lastly, we will introduce several gene knock-in techniques that involve Cas9/clustered regularly interspaced short palindromic repeats-mediated genome cleavage and the following of various DNA repair pathways.

Sequence-indexed insertional libraries in maize (Zea mays) are fundamental resources for functional genetics studies. Here, we constructed a Mutator (Mu) insertional library in the B73 inbred background designated BonnMu. A total of 1,152 Mu-tagged F(2)-families were sequenced using the Mu-seq approach. We detected 225,936 genomic Mu insertion sites and 41,086 high quality germinal Mu insertions covering 16,392 of the annotated maize genes (37% of the B73v4 genome). On average, each F(2)-family of the BonnMu libraries captured 37 germinal Mu insertions in genes of the Filtered Gene Set (FGS). All BonnMu insertions and phenotypic seedling photographs of Mu-tagged F(2)-families can be accessed via MaizeGDB.org. Downstream examination of 137,410 somatic and germinal insertion sites revealed that 50% of the tagged genes have a single hotspot, targeted by Mu. By comparing our BonnMu (B73) data to the UniformMu (W22) library, we identified conserved insertion hotspots between different genetic backgrounds. Finally, the vast majority of BonnMu and UniformMu transposons was inserted near the transcription start site of genes. Remarkably, 75% of all BonnMu insertions were in closer proximity to the transcription start site (distance: 542 bp) than to the start codon (distance: 704 bp), which corresponds to open chromatin, especially in the 5′ region of genes. Our European sequence-indexed library of Mu insertions provides an important resource for functional genetics studies of maize.

Aims The goal was to explore the effects of subinhibitory concentration (SIC) (0·5 MIC = 20 µg l-1 ) of ciprofloxacin on the transcriptome of enterohaemorrhagic Escherichia coli O26:H11 isolate by 60 minutes of exposure. Materials and results We used a combination of comparative genomic and transcriptomic (RNAseq) analyses. The whole genome of the E. coli O26:H11 #30934 strain of bovine origin was sequenced and assembled. This genome was next used as reference for the differential gene expression analysis. A whole-genome-based analysis of 36 publicly available E. coli O26:H11 genomes was performed to define the core and the accessory transcriptome of E. coli O26:H11. Using RNAseq and RT-qPCR analysis we observed overexpression of the SOS response and of T3SS effectors, together with the inhibition of specific motility-associated genes. Among the large set of transposases present, only three were activated, suggesting moderate transposition of genes with low doses of ciprofloxacin. Our results illustrated that transcriptional repressors, such as the CopG family protein, belonging to the core genome of E. coli O26:H11, are altered in response to fluoroquinolone exposure. The gene ontology enrichment analysis showed SIC of ciprofloxacin induced binding functions and catalytic activities, including mostly transferase and hydrolase proteins. The amino acid pathways involved in metabolic processes were significantly enhanced after the treatment. Conclusions Although the core genome of E. coli O26:H11 constituted only 54·5% of the whole genome, we demonstrated that most differentially expressed genes were associated with the core genome of E. coli O26:H11, and that effects on the mobile genetic element, phage, and plasmid-related genes were rare. Significance and impact of the study For the first time the effect of low dose of ciprofloxacin on the core transcriptome of E. coli O26:H11 was described. The effects on the main biological functions and protein classes including transcriptional regulators were illustrated.

The CRISPR-Cas system has undoubtedly revolutionized the genome editing field, enabling targeted gene disruption, regulation, and recovery in a guide RNA-specific manner. In this review, we focus on currently available gene recovery strategies that use CRISPR nucleases, particularly for the treatment of genetic disorders. Through the action of DNA repair mechanisms, CRISPR-mediated DNA cleavage at a genomic target can shift the reading frame to correct abnormal frameshifts, whereas DNA cleavage at two sites, which can induce large deletions or inversions, can correct structural abnormalities in DNA. Homology-mediated or homology-independent gene recovery strategies that require donor DNAs have been developed and widely applied to precisely correct mutated sequences in genes of interest. In contrast to the DNA cleavage-mediated gene correction methods listed above, base-editing tools enable base conversion in the absence of donor DNAs. In addition, CRISPR-associated transposases have been harnessed to generate a targeted knockin, and prime editors have been developed to edit tens of nucleotides in cells. Here, we introduce currently developed gene recovery strategies and discuss the pros and cons of each., Experimental & Molecular Medicine: Genetic disease: Gene editing for recovery The CRISPR-Cas gene editing system, which relies on small RNA molecules to guide a gene-editing enzyme to specific locations on DNA, is being developed as an effective tool for correcting genetic disorders. Researchers in South Korea led by Sangsu Bae at Hanyang University in South Korea, review recent progress towards such “gene recovery” procedures. The possibilities range from correcting mutations at the level of a single base in the base sequence of DNA, to deleting, inverting or inserting large sections of DNA to correct major structural abnormalities. The authors discuss the pros and cons of different procedures, including CRISPR-Cas nucleases, base editors, and prime editors. They expect current laboratory animal investigations will lead to a new era in human genetic medicine, yielding treatments for genetic diseases that cannot currently be treated with drugs.

Transposable elements (TEs) are DNA sequences capable of transpositions within the genome and thus exerting a considerable influence on the genome functioning and structure and serving as a source of new genes. TE biodiversity studies in previously unexplored species are important for the fundamental understanding of the TE influence on eukaryotic genomes. TEs are classified into retrotransposons and DNA transposons. IS630/Tc1/mariner (ITm) superfamily of DNA transposons is one of the most diverse groups broadly represented among the eukaryotes. The study of 19 mollusk genomes revealed a new group of ITm superfamily elements, which we henceforth refer to as TLEWI. These TEs are characterized by the low copy number, the lack of terminal inverted repeats, the catalytic domain with DD36E signature and the presence of spliceosomal introns in transposase coding sequence. Their prevalence among the mollusks is limited to the class Bivalvia. Since TLEWI possess the features of domesticated TE and structures similar to the eukaryotic genes which are not typical for the DNA transposons, we consider the hypothesis of co-optation of TLEWI gene by the bivalves. The results of our study will fill the gap of knowledge about the prevalence, activity, and evolution of the ITm DNA transposons in multicellular genomes and will facilitate our understanding of the mechanisms of TE domestication by the host genome.

Transposable elements represent the largest components of many eukaryotic genomes and different genomes harbor different combinations of elements. Here, we discovered a novel DNA transposon in the genome of the clubmoss Selaginella lepidophylla. Further searching for related sequences to the conserved DDE region uncovered the presence of this superfamily of elements in fish, coral, sea anemone, and other animal species. However, this element appears restricted to Bryophytes and Lycophytes in plants. This transposon, named GingerRoot, is associated with a 6 bp (base pair) target site duplication, and 100–150 bp terminal inverted repeats. Analysis of transposase sequences identified the DDE motif, a catalytic domain, which shows similarity to the integrase of Gypsy-like long terminal repeat retrotransposons, the most abundant component in plant genomes. A total of 77 intact and several hundred truncated copies of GingerRoot elements were identified in S. lepidophylla. Like Gypsy retrotransposons, GingerRoots show a lack of insertion preference near genes, which contrasts to the compact genome size of about 100 Mb. Nevertheless, a considerable portion of GingerRoot elements was found to carry gene fragments, suggesting the capacity of duplicating gene sequences is unlikely attributed to the proximity to genes. Elements carrying gene fragments appear to be less methylated, more diverged, and more distal to genes than those without gene fragments, indicating they are preferentially retained in gene-poor regions. This study has identified a broadly dispersed, novel DNA transposon, and the first plant DNA transposon with an integrase-related transposase, suggesting the possibility of de novo formation of Gypsy-like elements in plants.

While multiple technologies for small allele genome editing exist, robust technologies for targeted integration of large DNA fragments in mammalian genomes are still missing. Here we develop a gene delivery tool (FiCAT) combining the precision of a CRISPR-Cas9 (find module), and the payload transfer efficiency of an engineered piggyBac transposase (cut-and-transfer module). FiCAT combines the functionality of Cas9 DNA scanning and targeting DNA, with piggyBac donor DNA processing and transfer capacity. PiggyBac functional domains are engineered providing increased on-target integration while reducing off-target events. We demonstrate efficient delivery and programmable insertion of small and large payloads in cellulo (human (Hek293T, K-562) and mouse (C2C12)) and in vivo in mouse liver. Finally, we evolve more efficient versions of FiCAT by generating a targeted diversity of 394,000 variants and undergoing 4 rounds of evolution. In this work, we develop a precise and efficient targeted insertion of multi kilobase DNA fragments in mammalian genomes., Mammalian genome engineering has advanced tremendously over the last decade, however there is still a need for robust gene writing with size scaling capacity. Here the authors present Find Cut-and-Transfer (FiCAT) technology to delivery large targeted payload insertion in cell lines and in vivo in mouse models.

Helitrons are the only group of rolling-circle transposons that encode a transposase with a helicase domain (Hel), which belongs to the Pif1 family. Because Pif1 helicases are important components of eukaryotic genomes, it has been suggested that Hel domains probably originated after a host eukaryotic Pif1 gene was captured by a Helitron ancestor. However, the few analyses exploring the evolution of Helitron transposases (RepHel) have focused on its Rep domain, which is also present in other mobile genetic elements. Here, we used phylogenetic and nonmetric multidimensional scaling analyses to investigate the relationship between Hel domains and Pif1-like helicases from a variety of organisms. Our results reveal that Hel domains are only distantly related to genomic helicases from eukaryotes and prokaryotes, and thus are unlikely to have originated from a captured Pif1 gene. Based on this evidence, and on recent studies indicating that Rep domains are more closely related to rolling-circle plasmids and phages, we suggest that Helitrons are descendants of a RepHel-encoding prokaryotic plasmid element that invaded eukaryotic genomes before the radiation of its major groups. We discuss how a Pif1-like helicase domain might have favored the transposition of Helitrons in eukaryotes beyond simply unwinding DNA intermediates. Finally, we demonstrate that some examples in the literature describing genomic helicases from eukaryotes actually consist of Hel domains from Helitrons, a finding that underscores how transposons can hamper the analysis of eukaryotic genes. This investigation also revealed that two groups of land plants appear to have lost genomic Pif1 helicases independently.

BackgroundTransposons are mobile genetic elements that colonize genomes and drive their plasticity in all organisms. DNA transposon-encoded transposases bind to the ends of their cognate transposons and catalyze their movement. In some cases, exaptation of transposon genes has allowed novel cellular functions to emerge. The PiggyMac (Pgm) endonuclease of the ciliateParamecium tetraureliais a domesticated transposase from the PiggyBac family. It carries a core catalytic domain typical of PiggyBac-related transposases and a short cysteine-rich domain (CRD), flanked by N- and C-terminal extensions. During sexual processes Pgm catalyzes programmed genome rearrangements (PGR) that eliminate ~ 30% of germline DNA from the somatic genome at each generation. How Pgm recognizes its DNA cleavage sites in chromatin is unclear and the structure-function relationships of its different domains have remained elusive.ResultsWe provide insight into Pgm structure by determining the fold adopted by its CRD, an essential domain required for PGR. Using Nuclear Magnetic Resonance, we show that the Pgm CRD binds two Zn2+ions and forms an unusual binuclear cross-brace zinc finger, with a circularly permutated treble-clef fold flanked by two flexible arms. The Pgm CRD structure clearly differs from that of several other PiggyBac-related transposases, among which is the well-studied PB transposase fromTrichoplusia ni. Instead, the arrangement of cysteines and histidines in the primary sequence of the Pgm CRD resembles that of active transposases frompiggyBac-like elements found in other species and of human PiggyBac-derived domesticated transposases. We show that, unlike the PB CRD, the Pgm CRD does not bind DNA. Instead, it interacts weakly with the N-terminus of histone H3, whatever its lysine methylation state.ConclusionsThe present study points to the structural diversity of the CRD among transposases from the PiggyBac family and their domesticated derivatives, and highlights the diverse interactions this domain may establish with chromatin, from sequence-specific DNA binding to contacts with histone tails. Our data suggest that the Pgm CRD fold, whose unusual arrangement of cysteines and histidines is found in all PiggyBac-related domesticated transposases fromParameciumandTetrahymena, was already present in the ancestral active transposase that gave rise to ciliate domesticated proteins.

International audience; Four serine/threonine kinases are present in all mycobacteria: PknA, PknB, PknG and PknL. PknA and PknB are essential for growth and replication, PknG regulates metabolism, but little is known about PknL. Inactivation of pknL and adjacent regulator MSMEG_4242 in rough colony M. smegmatis mc2155 produced both smooth and rough colonies. Upon restreaking rough colonies, smooth colonies appeared at a frequency of ~ 1/250. Smooth mutants did not form biofilms, showed increased sliding motility and anomalous lipids on thin-layer chromatography, identified by mass spectrometry as lipooligosaccharides and perhaps also glycopeptidolipids. RNA-seq and Sanger sequencing revealed that all smooth mutants had inactivated lsr2 genes due to mutations and different IS1096 insertions. When complemented with lsr2, the colonies became rough, anomalous lipids disappeared and sliding motility decreased. Smooth mutants showed increased expression of IS1096 transposase TnpA and MSMEG_4727, which encodes a protein similar to PKS5. When MSMEG_4727 was deleted, smooth pknL/MSMEG_4242/lsr2 mutants reverted to rough, formed good biofilms, their motility decreased slightly and their anomalous lipids disappeared. Rough delpknL/del4242 mutants formed poor biofilms and showed decreased, aberrant sliding motility and both phenotypes were complemented with the two deleted genes. Inactivation of lsr2 changes colony morphology from rough to smooth, augments sliding motility and increases expression of MSMEG_4727 and other enzymes synthesizing lipooligosaccharides, apparently preventing biofilm formation. Similar morphological phase changes occur in other mycobacteria, likely reflecting environmental adaptations. PknL and MSMEG_4242 regulate lipid components of the outer cell envelope and their absence selects for lsr2 inactivation. A regulatory, phosphorylation cascade model is proposed.

Bacteriophage evolves to control the population of fast-growing bacterial cells, without which explosion in bacterial population may induce unimaginable harm to diverse ecosystems. But, bacteriophage also “hide” in bacterial genomes when nutritional and environmental circumstances are unfavourable. This involves the integration of phage genome into the host genome at appropriate genomic loci in a process known as lysogeny. This work sought to delineate the prophages present in the annotated genome of Bacillus subtilis 168, and assess their relative importance through RNA-seq expression analysis. Firstly, examination of the annotated genome of the model Gram-positive bacterium revealed five distinct prophage regions: SPBeta, prophage 6, PBSX, prophage 3 region, and prophage 1 region. All prophage regions contain host genes, which suggests that host transposase activity have swapped in host genes for phage genes in the prophage genome. Given the significant number of phage genes that have been swapped into each of the prophage genome, all prophage regions are deemed to be defective. BLAST analysis further highlighted that many of the prophages in B. subtilis are extinct given that they do not have ancestral or daughter brethren. However, RNA-seq transcriptome analysis of B. subtilis turned out an interesting paradox indicative of the important role that host transposase have in swapping in host promoters for prophage genes. Specifically, a significant number of prophage genes are highly expressed, which is implausible given that phage genes should be transcriptionally silent. The result and phenomenon further suggests the relative facile nature in which host promoters could be swapped in for phage genes, which is indicative of presence of genomic motifs in prophage genome recognizable by host transposase. Existence of such sequence motifs is thus indicative of possible co-evolution of transposase and phages where transposases were originally a part of the phage genome, which latter “jumped out” into the host genome to aid the swapping in of host genes into the prophage genome for augmenting prophage genetic repertoire in the face of changing environmental conditions. Overall, it is not uncommon for bacterial species to harbour multiple prophages. But, lysogeny may not be a viable option for long-term preservation of prophage genetic repertoire given that host transposase would inevitable swap in host genes at random locations in the prophage genome.

DDE transposons are widespread selfish genetic elements, often comprising a large proportion of eukaryotic genomic content. DDE transposons have also made important contributions to varied host functions during eukaryotic evolution, and their transposases may be the most abundant and ubiquitous genes in nature. Yet much remains unknown about their basic biology. We employ a broadscale screen of DDE transposase diversity to characterise major evolutionary patterns for all 19 DDE transposon superfamilies. We identify considerable variation in DDE transposon superfamily size, and find a dominant association with animal hosts. While few DDE transposon superfamilies specialise in plants or fungi, the four largest superfamilies contain major plant-associated clades, at least partially underlying their relative success. We recover a pattern of host conservation among DDE transposon lineages, punctuated by occasional horizontal transfer to distantly related hosts. Host range and horizontal transfer are strongly positively correlated with DDE transposon superfamily size, arguing against variation in the capacity for generalism. We find that rates of horizontal transfer decrease sharply with increasing levels of host taxonomy, supporting the existence of host-associated barriers to DDE transposon spread. Overall, despite their relatively simple genetic structure, our results imply that trade-offs in host adaptation are important in defining DDE transposon-host relationships and evolution. In addition, our study provides a phylogenetic framework to facilitate the identification and further analysis of DDE transposons.

Clostridium beijerinckii is a well-known anaerobic solventogenic bacterium which inhabits a wide range of different niches. Previously, we isolated five butyrate-producing C. beijerinckii strains from pit mud (PM) of strong-flavor baijiu (SFB) ecosystems. Genome annotation of the five strains showed that they could assimilate various carbon sources as well as ammonium to produce acetate, butyrate, lactate, hydrogen, and esters but did not produce the undesirable flavors isopropanol and acetone, making them useful for further exploration in SFB production. Our analysis of the genomes of an additional 233 C. beijerinckii strains revealed an open pangenome based on current sampling and will likely change with additional genomes. The core genome, accessory genome, and strain-specific genes comprised 1567, 8851, and 2154 genes, respectively. A total of 298 genes were found only in the five C. beijerinckii strains from PM, among which only 77 genes were assigned to Clusters of Orthologous Genes categories. In addition, 15 transposase and 12 phage integrase families were found in all five C. beijerinckii strains from PM. Between 18 and 21 genome islands were predicted for the five C. beijerinckii genomes. The existence of a large number of mobile genetic elements indicated that the genomes of the five C. beijerinckii strains evolved with the loss or insertion of DNA fragments in the PM of SFB ecosystems. This study presents a genomic framework of C. beijerinckii strains from PM that could be used for genetic diversification studies and further exploration of these strains.

CRISPR-associated transposons (CASTs) co-opt Cas genes for RNA-guided transposition. CASTs are exceedingly rare in genomic databases; recent surveys have reported Tn7-like transposons that co-opt Type I-F, I-B, and V-K CRISPR effectors. Here, we expand the diversity of reported CAST systems via a bioinformatic search of metagenomic databases. We discover new architectures for all known CASTs, including novel arrangements of the Cascade effectors, new self-targeting modalities, and minimal V-K systems. We also describe new families of CASTs that have co-opted the Type I-C and Type IV CRISPR-Cas systems. Our search for non-Tn7 CASTs identifies putative candidates that co-opt Cas12a for horizontal gene transfer. These new systems shed light on how CRISPR systems have co-evolved with transposases and expand the programmable gene editing toolkit.

THAP9 is a transposable element-derived gene that encodes the THAP9 protein. It is a homolog of Drosophila P-element transposase (DmTNP). hTHAP9 belongs to the THAP (Thanatos-associated protein) protein family in humans, which has twelve proteins (hTHAP0-hTHAP11). Human THAP proteins have a [~]90 amino acid long conserved THAP domain located at the N-terminal end of the protein. The THAP domain is a C2CH type Zinc Finger domain that binds specific DNA sequences and has a conserved secondary structure, namely a characteristic {beta}--{beta} fold. THAP family proteins are known to be involved in various human diseases like torsional dystonia, hemophilia, heart diseases, and multiple cancers. Also, the THAP9-AS1 gene, which is the neighboring gene in the 5 upstream region of THAP9, is overexpressed in pancreatic cancer. But the exact functional role of THAP9 and its association with various diseases remains a significant gap in our understanding. Here, we present the evolutionary analysis and extensive in silico characterization, including predicting domains and putative post-translational modification sites for THAP9 to understand its function better. Our studies identified some previously unreported functional features in the THAP9 protein sequence, including four adjacent motifs following the THAP domain: N-glycosylation site, Protein kinase C (PKC) phosphorylation site, Leucine zipper domain, and Bipartite nuclear localization signal (NLS). These motifs, which are highly conserved in mammals, may play a role in the subcellular localization of THAP9. The study also revealed two N-myristoylation sites within the THAP domain, pointing towards the post-translational modification of THAP9. We also observed that the THAP9 has evolved under a strong pervasive purifying selection, yielding high conservation of THAP9. Furthermore, investigation of THAP9 expression profiles in various cancer and matched normal datasets demonstrated overexpression and underexpression in Testicular cancers and Thymic Epithelial Tumors respectively. These findings make Testicular cancer and Thymic Epithelial Tumor good candidates for further examination on the role of THAP9 in cancer.

While it is well recognized that the environmental resistome is global, diverse, and augmented by human activities, it has been difficult to assess risk because of the inability to culture many environmental organisms, and it is difficult to evaluate risk from current sequence-based environmental methods. The four most important criteria to determine risk are whether the antibiotic-resistance genes (ARGs) are a complete, potentially functional complement; if they are linked with other resistances; whether they are mobile; and the identity of their host. Long-read sequencing fills this important gap between culture and short sequence-based methods. To address these criteria, we collected feces from a ceftiofur-treated cow, enriched the samples in the presence of antibiotics to favor ARG functionality, and sequenced long reads using Nanopore and PacBio technologies. Multidrug-resistance genes comprised 58% of resistome abundance, but only 0.8% of them were plasmid associated; fluroquinolone-, aminoglycoside-, macrolide-lincosamide-streptogramin (MLS)-, and β-lactam–resistance genes accounted for 2.7 to 12.3% of resistome abundance but with 19 to 78% located on plasmids. A variety of plasmid types were assembled, some of which share low similarity to plasmids in current databases. Enterobacteriaceae were dominant hosts of antibiotic-resistant plasmids; physical linkage of extended-spectrum β-lactamase genes (CTX-M, TEM, CMY, and CARB) was largely found with aminoglycoside-, MLS-, tetracycline-, trimethoprim-, phenicol-, sulfonamide-, and mercury-resistance genes. A draft circular chromosome of Vagococcus lutrae was assembled; it carries MLS-, tetracycline- (including tetM and tetL on an integrative conjugative element), and trimethoprim-resistance genes flanked by many transposase genes and insertion sequences, implying that they remain transferrable.

Compartmentalization of interactions between genomic regulatory elements and potential target genes is influenced by the binding of insulator proteins such as CTCF, which act as potent enhancer blockers when interposed between an enhancer and a promoter in a reporter assay. But only a minority of CTCF sites genome-wide function as boundary elements, depending on cellular and genomic context. To dissect the influence of genomic context on enhancer blocker activity, we integrated reporter constructs with promoter-only, promoter and enhancer, and enhancer blocker configurations at hundreds of thousands of genomic sites using the Sleeping Beauty transposase. Deconvolution of reporter activity by genomic position revealed strikingly different patterns of reporter function, including a compartment of enhancer blocker reporter integrations with robust expression. The high density of integration sites permits quantitative delineation of characteristic genomic context sensitivity profiles, and their decomposition into sensitivity to both local and distant DNaseI hypersensitive sites. Furthermore, a single-cell expression approach permits direct linkage of reporters integrated into the same clonal lineage with differential endogenous gene expression, revealing that CTCF insulator activity does not completely abrogate reporter effects on endogenous gene expression. Collectively, our results lend new insight to genomic regulatory compartmentalization and its influence on the determinants of promoter-enhancer specificity.

Coagulation is a critical pathogenic pattern in Staphylococcus aureus (S. aureus). Although the agglutination of S. aureus has been studied for a long time, the genes involved in this are not completely clear. We adopted tube agglutination and dynamic turbidimetry tests to identify novel genes involving reduced plasma coagulation. There were 15 genes had been identified, including coa, clfA, vwbp, saeS, agrA, trpC, spdC, mroQ, cydA, qoxC, sucC, pyrP, menH, threonine aldolase and truncated transposase for IS1272. These genes have different functions and map onto functional agglutination, two-component regulatory, membrane transfer, energy metabolism and biosynthesis, etc. cydA, spdC and mroQ genes were further studied by gene knockout and complementation. Results of gene knockout and complement and RT-qPCR proved that cydA, spdC and mroQ genes were necessary in plasma coagulation. Furthermore, 7-day mice's survival ability decreased significantly when cydA, spdC and mroQ genes had been knocked out. This article is protected by copyright. All rights reserved.

Cell line development (CLD) by random integration (RI) can be labor intensive, inconsistent, and unpredictable due to uncontrolled gene integration after transfection. Unlike RI, targeted integration (TI) based CLD introduces the antibody-expressing cassette to a predetermined site by recombinase-mediated cassette exchange (RMCE). The key to success for the development of a TI host for therapeutic antibody production is to identify a transcriptionally active hotspot that enables highly efficient RMCE and antibody expression with good stability. In this study, a genome wide search for hotspots in the Chinese hamster ovary (CHO)-K1-M genome by either RI or PiggyBac (PB) transposase-based integration has been described. Two CHO-K1-M derived TI host cells were established with the Cre/Lox RMCE system and are described here. Both TI hosts contain a GFP-expressing landing pad flanked by two incompatible LoxP recombination sites (L3 and 2L). In addition, a third incompatible LoxP site (LoxFAS) is inserted in the GFP landing pad to enable an innovative two-plasmid based RMCE strategy, in which two separate vectors can be targeted to a single locus simultaneously. Cell lines generated by the TI system exhibit comparable or higher productivity, better stability and fewer sequence variant (SV) occurrences than the RI cell lines.

The catalytic domain of most ‘cut and paste’ DNA transposases have the canonical RNase-H fold which is also shared by other polynucleotidyl transferases like retroviral integrases and the RAG1 subunit of V(D)J recombinase. The RNase-H fold is a mixture of beta sheets and alpha helices with three acidic residues (Asp, Asp, Glu/Asp - DDE/D) that are involved in metal-mediated cleavage and subsequent integration of DNA. Human THAP9 (hTHAP9), homologous to the well-studied Drosophila P-element transposase (DmTNP), is an active DNA transposase that, although domesticated, still retains the catalytic activity to mobilize transposons. In this study we have modelled the structure of hTHAP9 using the recently available cryo-EM structure of DmTNP as a template to identify an RNase-H like fold along with important acidic residues in its catalytic domain. Site-directed mutagenesis of the predicted catalytic residues followed by screening for DNA excision and integration activity, led to the identification of candidate Ds and Es in the RNaseH fold that appear to constitute the catalytic triad of hTHAP9.Significance statementMany DNA transposases execute DNA excision via a catalytic domain, which has a canonical RNase-H fold. Despite the similar nature of the catalytic domain, these transposases exhibit mechanistically different strategies of transposition. We identify a potential RNase-H fold in hTHAP9 with a conserved DDE motif required for cutting DNA. Additionally, we have found a residue, which when mutated, leads to an increase in hTHAP9’s transposition activity. Such hyperactive transposase mutants can be exploited as tools in genome engineering and gene therapy. This study has helped widen our knowledge about the catalytic activity of a functionally uncharacterized transposon-derived gene in the human genome.

International audience; The vertebrate piggyBac derived transposase 5 (PGBD5) encodes a domesticated transposase, which is active and able to transpose its distantly related piggyBac-like element (pble), Ifp2. This raised the question whether PGBD5 would be more effective at mobilizing a phylogenetically closely related pble element. We aimed to identify the pble most closely related to the pgbd5 gene. We updated the landscape of vertebrate pgbd genes to develop efficient filters and identify the most closely related pble to each of these genes. We found that Tcr-pble is phylogenetically the closest pble to the pgbd5 gene. Furthermore, we evaluated the capacity of two murine and human PGBD5 isoforms, Mm523 and Hs524, to transpose both Tcr-pble and Ifp2 elements. We found that both pbles could be transposed by Mm523 with similar efficiency. However, integrations of both pbles occurred through both proper transposition and improper PGBD5-dependent recombination. This suggested that the ability of PGBD5 to bind both pbles may not be based on the primary sequence of element ends, but may involve recognition of inner DNA motifs, possibly related to palindromic repeats. In agreement with this hypothesis, we identified internal palindromic repeats near the end of 24 pble sequences, which display distinct sequences.

Objectives: Repetitive elements are ubiquitous in eukaryotes and have contributed in expression of genes and maintaining the architecture of genome. Fungi although being a eukaryote, repeat sequences are typically limited due to small genome size and typical genome defense mechanisms which fights against expansion of repeats and maintains streamlined genome. Methods/Findings: Despite, the presence of such mechanisms Tad element was found to be active in Adiopodoume strain of N. crassa. This is one of the most enigmatic discoveries made in Neurospora. From this perspective, we have analyzed the genome of N. crassa OR74A (NC12) by in silico tools for the identification of repeats. The comprehensive analysis of these elements suggests that about six (PuntRIP) DNA transposons and 2 Tad1 (Non-Long Terminal Repeat) elements share highest homology with known repeats from RepBase. The presence of DNA binding and transposase domain in the protein sequence of NCU02991 indicates that the element identified is a DNA transposon. Application: The results also suggest that among the identified six PuntRIP sequences four of them share homology, which indicates that similar sequence is repeated four times within the genome. The promoters identified in the dataset of identified repeats in the upstream and downstream regions indicate that these repeats may play certain role in the regulation of genes. Keywords: DNA Transposons, Non-LTR, Repetitive Elements, Tad1

Background: Insertion sequence is a short DNA sequence encode for proteins implicated in the transposition activity. Transposase catalyzes the enzymatic reaction allowing the insertion sequence to +9*lo2 move. ;qqa;. Objective: To study the sequencing of transposase gene, tnp, IS1216V of S. aureus isolated from food and then compared with that documented in National Center for Biotechnology Information (NCBI). Methods: Food samples of animal and plant origin were collected, and screened for presence of S. aureus, IS1216V was identified in the Tn1546-like elements in the genomes of all Staphylococcus aureus isolates. Results: About 75% of total food samples were positive to S. aureus especially in the food of animal origin. tnp amplification showed that, 85% of isolates gave positive result. Sequencing of amplified part of IS1216V tnp of S. aureus isolates showed that, tnp gene had high identity (78-79%) with the reference strains of NCBI. Conclusion: High percentage of local food samples were contaminated with S. aureus especially of animal origin. Most of the S. aureus isolates showed the presence of transposase gene (tnp) of IS1216V. Sequencing showed some dissimilarity between the sequence of transposase gene (tnp) of IS1216V S. aureus isolated from local foods and strains recorded in database of NCBI.

Homology-directed genome engineering is limited by transgene size. Although DNA transposons are more efficient with large transgenes, random integrations are potentially mutagenic. Here we present an in vitro mechanistic study that demonstrates efficient Cas9 targeting of the mariner transposon Hsmar1. Integrations were unidirectional and tightly constrained to one side of the sgRNA binding site. Further analysis of the nucleoprotein intermediates demonstrated that the transposase and Cas9 moieties can bind their respective substrates independently or in concert. Kinetic analysis of the reaction in the presence of the Cas9 target–DNA revealed a delay between first and second strand cleavage at the transposon end. This step involves a significant conformational change that may be hindered by the properties of the interdomainal linker. Otherwise, the transposase moiety behaved normally and was proficient for integration in vitro and in Escherichia coli. Specific integration into the lacZ gene in E. coli was obscured by a high background of random integrations. Nevertheless, Cas9 is an attractive candidate for transposon-targeting because it has a high affinity and long dwell-time at its target site. This will facilitate a future optogenetic strategy for the temporal control of integration, which will increase the ratio of targeted to untargeted events.

Although chromatin condensation is a well-known hallmark of apoptosis, the generation mechanism has not been clarified. Histone H1, a positively-charged abundant nuclear protein, is located in the linker region of chromatin. There are several Histone H1 subtypes that are encoded by variant genes. Using serial histone H1-deletion mutant cells established from the chicken B-cell leukemia line DT40, we found that apoptotic chromatin condensation was decreased in relation to histone H1 protein level and that the chromatin in nuclei prepared from the live null mutant cells had a high accessibility of DNases and transposase. This indicated that linker histone H1 was the general chromatin condensation factor and that the loss of histone H1 generated open chromatin in both apoptotic and live cells.

Mosaic plasmids, plasmids composed of genetic elements from distinct sources, are associated with the spread of antibiotic resistance genes. Transposons are considered the primary mechanism for mosaic plasmid formation, though other mechanisms have been observed in specific instances. The frequency with which mosaic plasmids have been described suggests they may play an important role in plasmid population dynamics. Our survey of the confirmed plasmid sequences available from complete and draft genomes in the RefSeq database shows that 46% of them fit a strict definition of mosaic. Mosaic plasmids are also not evenly distributed over the taxa represented in the database. Plasmids from some genera, including Piscirickettsia and Yersinia, are almost all mosaic, while plasmids from other genera, including Borrelia, are rarely mosaic. While some mosaic plasmids share identical regions with hundreds of others, the median mosaic plasmid only shares with 8 other plasmids.When considering only plasmids from finished genomes (51.6% of the total), mosaic plasmids have significantly higher proportions of transposases and antibiotic resistance genes. Conversely, only 56.6% of mosaic fragments (DNA fragments shared between mosaic plasmids) contain a recognizable transposase, and only 1.2% of mosaic fragments are flanked by inverted repeats. Mosaic fragments associated with the IS26 transposase are 3.8-fold more abundant than any other sequence shared between mosaic plasmids in the database, though this is at least partly due to overrepresentation of Enterobacteriaceae plasmids.Mosaic plasmids are a complicated trait of some plasmid populations, only partly explained by transposition. Though antibiotic resistance genes led to the identification of many mosaic plasmids, mosaic plasmids are a broad phenomenon encompassing many more traits than just antibiotic resistance. Further research will be required to determine the influence of ecology, host repair mechanisms, conjugation, and plasmid host range on the formation and influence of mosaic plasmids.Author SummaryPlasmids are extrachromosomal genetic entities that are found in many prokaryotes. They serve as flexible storage for genes, and individual cells can make substantial changes to their characteristics by acquiring, losing, or modifying a plasmid. In some pathogenic bacteria, such as Escherichia coli, antibiotic resistance genes are known to spread primarily on plasmids. By analyzing a database of 8,592 plasmid sequences we determined that many of these plasmids have exchanged genes with each other, becoming mosaics of genes from different sources. We next separated these plasmids into groups based on the organism they were isolated from and found that different groups had different fractions of mosaic plasmids. This result was unexpected and suggests that the mechanisms and selective pressures causing mosaic plasmids do not occur evenly over all species. It also suggests that plasmids may provide different levels of potential variation to different species. This work uncovers a previously unrecognized pattern in plasmids across prokaryotes, that could lead to new insights into the evolutionary role that plasmids play.

Insertions of transposable elements in the S-haplotype-specific F-box gene were shown many times to confer self-compatibility in Prunus species. In apricot, we identified a miniature inverted repeat transposable element (MITE) with structural features (its 349-bp size, 82-bp terminal inverted repeats and 9-bp target site duplications) that are consistent with this MITE being a putative member of the Mutator-like transposase superfamily. This non-autonomous element was labelled as FaSt (Falling Stones). FaSt showed a preferential accumulation in short AT-rich segments within euchromatin regions of the peach genome. A bioinformatics based survey of the known Rosaceae and other genomes and a newly designed PCR-assay verified the Prunoideae-specific occurrence of FaSt elements. In addition, FaSt copies in diploid apricot and hexaploid plum showed considerable sequence variations. Several copies were found in promoter regions and introns of certain genes. This is the first study to report on a presumably active MITE in the Prunus genus that might have exhibited direct and indirect genome shaping forces. FaSts and other transposable elements are compared in their contribution to the breakdown of self-incompatibility in Prunus species.

The phytohormone cytokinin regulates diverse aspects of plant growth and development, probably through context-dependent transcriptional regulation that relies on a dynamic interplay between regulatory proteins and chromatin. We employed the assay for transposase accessible chromatin with sequencing to profile changes in the chromatin landscape of Arabidopsis roots and shoots in response to cytokinin. Our results reveal differentially accessible chromatin regions indicative of dynamic regulation in response to cytokinin. These changes in chromatin occur preferentially upstream of cytokinin-regulated genes. The changes also largely overlap with binding sites for the type-B ARABIDOPSIS RESPONSE REGULATORS (ARRs), transcription factors that mediate the primary response to cytokinin. Furthermore, the type-B ARRs were found to be necessary for the changes in chromatin state in response to cytokinin. Last, we identified context-dependent responses by comparing root and shoot profiles. This study provides new insight into the dynamics between cytokinin and chromatin with regard to directing transcriptional programmes and how cytokinin mediates its pleiotropic effects.

The emergence of carbapenem resistance (CR) caused by hydrolytic enzymes called carbapenemases has become a major concern worldwide. So far, CR genes have been widely detected in various bacteria. However, there is no report of CR gene harboring Comamonas thiooxydans. We first isolated a strain of an IMP-8-producing C. thiooxydans from a patient with urinary tract infection in China. Species identity was determined using MALDI-TOF MS analysis and carbapenemase-encoding genes were detected using PCR. The complete genomic sequence of C. thiooxydans was identified using Illumina Novaseq and Oxford Nanopore PromethION. Antimicrobial susceptibility analysis indicated that the C. thiooxydans strain ZDHYF418 was susceptible to imipenem, intermediate to meropenem, and was resistant to aztreonam, fluoroquinolones, and aminoglycosides. The blaIMP–8 gene was chromosomally located, and was part of a Tn402-like class 1 integron characterized by the following structure: DDE-type integrase/transposase/recombinase-tniB-tniQ-recombinase family protein-aac(6′)-Ib-cr-blaIMP–8-intI1. Phylogenetic analysis demonstrated that the closest relative of ZDHYF418 is C. thiooxydans QYY (accession number: CP053920.1). We detected 330 SNP differences between ZDHYF418 and C. thiooxydans QYY. Strain QYY was isolated from activated sludge in Jilin province, China in 2015. In summary, we isolated a strain of C. thiooxydans that is able to produce IMP-8 and a novel blaOXA. This is the first time that a CR gene has been identified in C. thiooxydans. The occurrence of the strain needs to be closely monitored.

Background: The resistance to colistin and carbapenems in Klebsiella pneumoniae infections have been associated with increased morbidity and mortality worldwide. A retrospective observational study was conducted to determine the prevalence and molecular events contributing to colistin resistance.Methods: Clinical samples were screened for colistin resistance and underlying mechanisms were studied by PCR-based amplification and sequence analysis of genes of two-component regulatory system (phoPQ and pmrAB), regulatory transmembrane protein-coding mgrB, and mobilized colistin resistance genes (mcr-1-8). Gene expression of pmrC and pmrK was analyzed by qRT-PCR, and the genetic relationship was assessed by MLST. The putative effect of amino-acid substitutions was predicted by a combination of bioinformatics tools.Results: Of 335 Klebsiella spp. screened, 11 (3.2%) were identified as colistin-resistant (MIC range, 8 to >128 μg/ml). K. pneumoniae isolates belonged to clonal complex-11 (CC11) with sequence types (STs): 14, 16, 43, 54, 147 and 395, whereby four isolates conferred three novel STs (3986, 3987 and 3988) profiles. Sequence analysis revealed non-synonymous potentially deleterious mutations in phoP (T151A), phoQ (del87–90, del263–264, L30Q, and A351D), pmrA (G53S), pmrB (D150V, T157P, L237R, G250C, A252G, R315P, and Q331H), and mgrB (C28G) genes. The mgrB gene in three strains was disrupted by insertion sequences encoding IS1-like and IS5/IS1182 family-like transposase genes. All 11 isolates showed an elevation in the transcription level of pmrC gene. Mobilized colistin-resistance (mcr) genes were not detected. All but one of the colistin-resistant isolates was also resistant to carbapenems; β-lactamase genes blaNDM-1-like, blaOXA-48-like, and blaCTX-M-like were detected in eight, five, and nine isolates, respectively.Conclusion: All the studied colistin- and carbapenem-resistant K. pneumoniae isolates were genetically distinct, and various mechanisms of colistin resistance were detected, indicating its spontaneous emergence in this bacterial species.

Oxford Nanopore Technologies (ONT) sequencing platforms currently offer two approaches to whole-genome native-DNA library preparation: ligation and rapid. In this study, we compared these two approaches for bacterial whole-genome sequencing, with a specific aim of assessing their ability to recover small plasmid sequences. To do so, we sequenced DNA from seven plasmid-rich bacterial isolates in three different ways: ONT ligation, ONT rapid and Illumina. Using the Illumina read depths to approximate true plasmid abundance, we found that small plasmids (Impact statementResearchers who use Oxford Nanopore Technologies (ONT) platforms to sequence bacterial genomes can currently choose from two library preparation methods. The first is a ligation-based approach, which uses ligase to attach sequencing adapters to the ends of DNA molecules. The second is a rapid approach, which uses a transposase enzyme to cleave DNA and attach adapters in a single step. There are advantages to each preparation, for example ligation can produce better yields but rapid is a simpler procedure. Our study reveals another advantage of rapid preparations: they are more effective at sequencing small plasmids. We show that sequencing of ligation-based libraries yields fewer reads derived from small plasmids, making such plasmids harder to detect in bacterial genomes. Since small plasmids can contain clinically relevant genes, including antimicrobial resistance (AMR) or virulence determinants, their exclusion could lead to unreliable conclusions that have serious consequences for AMR surveillance and prediction. We therefore recommend that researchers performing ONT-only sequencing of bacterial genomes should consider using rapid preparations whenever small plasmid recovery is important.Data summarySupplementary figures, tables, data and code can be found at: github.com/rrwick/Small-plasmid-Nanopore

The human transcription factor (TF) CGGBP1 (CGG-binding protein) is conserved only in amniotes and is believed to derive from the zf-BED and Hermes transposase DNA-binding domains (DBDs) of a hAT DNA transposon. Here, we show that sequence-specific DNA-binding proteins with this bipartite domain structure have resulted from dozens of independent hAT domestications in different eukaryotic lineages. CGGBPs display a wide range of sequence specificity, usually including preferences for CGG or CGC trinucleotides, whereas some bind AT-rich motifs. The CGGBPs are almost entirely nonsyntenic, and their protein sequences, DNA-binding motifs, and patterns of presence or absence in genomes are uncharacteristic of ancestry via speciation. At least eight CGGBPs in the coelacanth Latimeria chalumnae bind distinct motifs, and the expression of the corresponding genes varies considerably across tissues, suggesting tissue-restricted function.

The IS26 family includes the ISs that are commonly found associated with antibiotic resistance genes in multiply resistant Gram-negative and Gram-positive bacteria. IS26 is most prevalent in Gram-negative species and can generate the clusters of antibiotic resistance genes interspersed with directly oriented IS26 seen in multiply resistant pathogens., IS26 forms cointegrates using two distinct routes, a copy-in mechanism involving one insertion sequence (IS) and a target and a targeted conservative mechanism involving two ISs in different DNA molecules. In this study, the ability of IS26 and some close relatives, IS1006, IS1008, and a natural hybrid, IS1006/IS1008, which are found predominantly in Acinetobacter spp., to interact was examined. IS1006/1008 consists of 175 bp from IS1006 at the left end, with the remainder from IS1008. These ISs all have the same 14-bp terminal inverted repeats, and the Tnp26, Tnp1006, and Tnp1008 transposases, with pairwise identities of 83.7% to 93.1%, should be able to recognize each other’s ends. In a recA-negative Escherichia coli strain, IS1006, IS1008, and IS1006/1008 each formed cointegrates via the copy-in route and via the targeted conservative route, albeit at frequencies for the targeted reaction at least 10-fold lower than for IS26. However, using mixed pairs, targeted cointegration was detected only when IS1008 was paired with the IS1006/1008 hybrid, which also encodes Tnp1008, and the targeted cointegrates formed all arose from a reaction occurring at the end where the DNA sequences are identical. The reaction also occurred at the end with extended DNA identity using IS26 paired with IS26::catA1, an artificially constructed IS26 derivative that includes the catA1 gene. Thus, both identical transposases and identical DNA sequences at the reacting end were required. These features indicate that the targeted conservative pathway proceeds via a single transposase-catalyzed strand transfer, followed by migration and resolution of the Holliday junction formed. IMPORTANCE The IS26 family includes the ISs that are commonly found associated with antibiotic resistance genes in multiply resistant Gram-negative and Gram-positive bacteria. IS26 is most prevalent in Gram-negative species and can generate the clusters of antibiotic resistance genes interspersed with directly oriented IS26 seen in multiply resistant pathogens. This ability relies on the novel dual mechanistic capabilities of IS26 family members. However, the mechanism underlying the recently discovered targeted conservative mode of cointegrate formation mediated by IS26, IS257/IS431, and IS1216, which is unlike any previously studied IS movement mechanism, is not well understood. An important question is what features of the IS and the transposase are key to allowing IS26 family members to undertake targeted conservative reaction. In this study, this question was addressed using mixed-partner crosses involving IS26 and naturally occurring close relatives of IS26 that are found near resistance genes in Acinetobacter baumannii and are widespread in Acinetobacter species.

Bacillus pumilus64-1, a bacterial strain isolated from the marine spongePlakina cyanorosea, which exhibits antimicrobial activity against both pathogenic and drug-resistant Gram-positive and Gram-negative bacteria. This study aimed to conduct an in-depth genomic analysis of this bioactive sponge-derived strain. The nearly complete genome of strain 64-1 consists of 3.6 Mbp (41.5% GC), which includes 3,705 coding sequences (CDS). An open pangenome was observed when limiting to the type strains of theB. pumilusgroup and aquatic-derivedB. pumilusrepresentatives. The genome appears to encode for at least 12 potential biosynthetic gene clusters (BGCs), including both types I and III polyketide synthases (PKS), non-ribosomal peptide synthetases (NRPS), and one NRPS-T1PKS hybrid, among others. In particular, bacilysin and other bacteriocin-coding genes were found and may be associated with the detected antimicrobial activity. Strain 64-1 also appears to possess a broad repertoire of genes encoding for plant cell wall-degrading carbohydrate-active enzymes (CAZymes). A myriad of genes which may be involved in various process required by the strain in its marine habitat, such as those encoding for osmoprotectory transport systems and the biosynthesis of compatible solutes were also present. Several heavy metal tolerance genes are also present, together with various mobile elements including a region encoding for a type III-B Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) region, four prophage segments and transposase elements. This is the first report on the genomic characterization of a cultivable bacterial member of thePlakina cyanoroseaholobiont.

This study describes a novel transposable bacteriophage, ɸSHP3, continuously released by Stenotrophomonas maltophilia strain c31. Morphological observation and genomic analysis revealed that ɸSHP3 is a siphovirus with a 37,611-bp genome that encodes 51 putative proteins. Genomic comparisons indicated that ɸSHP3 is a B3-like transposable phage. Its genome configuration is similar to that of Pseudomonas phage B3, except for the DNA modification module. Similar to B3-like phages, the putative transposase B of ɸSHP3 is a homolog of the type two secretion component ExeA, which is proposed to serve as a potential virulence factor. Moreover, most proteins of ɸSHP3 have homologs in transposable phages, but only ɸSHP3 carries an RdgC-like protein encoded by gene 3, which exhibits exonuclease activity in vitro. Two genes and their promoters coding for ɸSHP3 regulatory proteins were identified and appear to control the lytic-lysogenic switch. One of the proteins represses one promoter activity and confers immunity to ɸSHP3 superinfection in vivo. The short regulatory region, in addition to the canonical bacterial promoter sequences, displays one LexA and two CpxR recognition sequences. This suggests that LexA and the CpxR/CpxA two-component system might be involved in the control of the ɸSHP3 genetic switch. IMPORTANCE S. maltophilia is an emerging global pathogenic bacterium that displays genetic diversity in both environmental and clinical strains. Transposable phages have long been known to improve the genetic diversity of bacterial strains by transposition. More than a dozen phages of S. maltophilia have been characterized. However, no transposable phage infecting S. maltophilia has been reported to date. Characterization of the first transposable phage, ɸSHP3, from S. maltophilia will contribute to our understanding of host-phage interactions and genetic diversity, especially the interchange of genetic materials among S. maltophilia.

Antibiotic resistance genes (ARGs) are emerging contaminants causing serious global health concern. Interventions to address this concern include improving our understanding of methods for treating waste material of human and animal origin that are known to harbor ARGs. Anaerobic digestion is a commonly used process for treating dairy manure, and although effective in reducing ARGs, its mechanism of action is not clear. In this study, we used three ARGs to conducted a longitudinal bench scale anaerobic digestion experiment with various temperatures (28, 36, 44, and 52°C) in triplicate using fresh dairy manure for 30 days to evaluate the reduction of gene abundance. Three ARGs and two mobile genetic elements (MGEs) were studied: sulfonamide resistance gene (sulII), tetracycline resistance genes (tetW), macrolide-lincosamide-streptogramin B (MLSB) superfamily resistance genes (ermF), class 1 integrase gene (intI1), and transposase gene (tnpA). Genes were quantified by real-time quantitative PCR. Results show that the thermophilic anaerobic digestion (52°C) significantly reduced (p < 0.05) the absolute abundance of sulII (95%), intI1 (95%), tnpA (77%) and 16S rRNA gene (76%) after 30 days of digestion. A modified Collins–Selleck model was used to fit the decay curve, and results suggest that the gene reduction during the startup phase of anaerobic digestion (first 5 days) was faster than the later stage, and reductions in the first five days were more than 50% for most genes.

Modern plant breeding increasingly relies on genomic information to guide crop improvement. Although some genes are characterized, additional tools are needed to effectively identify and characterize genes associated with crop traits. To address this need, the mPing element from rice was modified to serve as an activation tag to induce expression of nearby genes. Embedding promoter sequences in mPing resulted in a decrease in overall transposition rate; however, this effect was negated by using a hyperactive version of mPing called mmPing20. Transgenic soybean events carrying mPing‐based activation tags and the appropriate transposase expression cassettes showed evidence of transposition. Expression analysis of a line that contained a heritable insertion of the mmPing20F activation tag indicated that the activation tag induced overexpression of the nearby soybean genes. This represents a significant advance in gene discovery technology as activation tags have the potential to induce more phenotypes than the original mPing element, improving the overall effectiveness of the mutagenesis system.

Peer reviewed: True, P elements were first discovered in the fruit fly Drosophila melanogaster as the causative agents of a syndrome of aberrant genetic traits called hybrid dysgenesis. This occurs when P element-carrying -males mate with females that lack P elements and results in progeny displaying sterility, mutations and chromosomal rearrangements. Since then numerous genetic, developmental, biochemical and structural studies have culminated in a deep understanding of P element transposition: from the cellular regulation and repression of transposition to the mechanistic details of the transposase nucleoprotein complex. Recent studies have revealed how piwi-interacting small RNA pathways can act to control splicing of the P element pre-mRNA to modulate transposase production in the germline. A recent cryo-electron microscopy structure of the P element transpososome reveals an unusual DNA architecture at the transposon termini and shows that the bound GTP cofactor functions to position the transposon ends within the transposase active site. Genome sequencing efforts have shown that there are P element transposase-homologous genes (called THAP9) in other animal genomes, including humans). This review highlights recent and previous studies, which together have led to new insights, and surveys our current understanding of the biology, biochemistry, mechanism and regulation of P element transposition.

Cold-water corals (CWCs) are important habitats for creatures in the deep-sea environment, but they have been degraded by anthropogenic activity. So far, no genome for any CWC has been reported. Here, we report a draft genome of Trachythela sp., which represents the first genome of CWCs to date. In total, 56 and 65 Gb of raw reads were generated from Illumina and Nanopore sequencing platforms, respectively. The final assembled genome was 578.26 Mb, which consisted of 396 contigs with a contig N50 of 3.56 Mb, and the genome captured 90.1% of the metazoan Benchmarking Universal Single-Copy Orthologs. We identified 335 Mb (57.88% of the genome) of repetitive elements, which is a higher proportion compared with others in the Cnidarians, along with 35,305 protein-coding genes. We also detected 483 expanded and 51 contracted gene families, and many of them were associated with longevity, ion transposase, heme-binding nicotinamide adenine dinucleotide, and metabolic regulators of transcription. Overall, we believe this genome will serve as an important resource for studies on community protection for CWCs.

Plants have evolved regulatory mechanisms at multiple levels to regulate gene expression in order to improve their cold adaptability. However, limited information is available regarding the stress response at the chromatin and translational levels. Here, we characterize the chromatin accessibility, transcriptional, and translational landscapes of tea plants in vivo under chilling stress for the first time. Chilling stress significantly affected both the transcription and translation levels as well as the translation efficiency of tea plants. A total of 3010 genes that underwent rapid and independent translation under chilling stress were observed, and they were significantly enriched in the photosynthesis-antenna protein and phenylpropanoid biosynthesis pathways. A set of genes that were significantly responsive to cold at the transcription and translation levels, including four (+)-neomenthol dehydrogenases (MNDs) and two (E)-nerolidol synthases (NESs) arranged in tandem on the chromosomes, were also found. We detected potential upstream open reading frames (uORFs) on 3082 genes and found that tea plants may inhibit the overall expression of genes by enhancing the translation of uORFs under chilling stress. In addition, we identified distal transposase hypersensitive sites (THSs) and proximal THSs and constructed a transcriptional regulatory network for tea plants under chilling stress. We also identified 13 high-confidence transcription factors (TFs) that may play a crucial role in cold regulation. These results provide valuable information regarding the potential transcriptional regulatory network in plants and help to clarify how plants exhibit flexible responses to chilling stress.

Background Anaerobic ammonium oxidizing bacteria (anammox bacteria) are contributing significantly to the nitrogen cycle and are successfully used in wastewater treatment. Due to the lack of complete genomes in the databases, little is known about the stability and variability of their genomes and how the genomes evolve in response to changing environments. Results Here we report the complete genome of the anammox bacterium “Candidatus Kuenenia stuttgartiensis” strain CSTR1 which was enriched planktonically in a semi-continuous stirred-tank reactor. A comparison of the genome of strain CSTR1 with the genome of “Ca. Kuenenia stuttgartiensis” MBR1 and the draft genome of KUST showed > 99% average nucleotide identity among all. Rearrangements of large genomic regions were observed, most of which were associated with transposase genes. Phylogenetic analysis suggests that strain MBR1 is more distantly related to the other two strains. Proteomic analysis of actively growing cells of strain CSTR1 (growth rate ~ 0.33 d− 1) failed to detect the annotated cytochrome cd1-type nitrite reductase (NirS) although in total 1189 proteins were found in the proteome. Yet, this NirS was expressed when strain CSTR1 was under stress or starvation (growth rate − 1). We also observed large sequence shifts in the strongly expressed S-layer protein compared to other “Ca. Kuenenia” strains, indicating the formation of hybrids of genes encoding the surface proteins. Conclusions “Ca. Kuenenia” strains appear to be relatively stable in their basic physiological traits, but show high variability in overall genome structure and surface proteins.

International audience; DDE Transposons have been recruited at least four times as site-specific recombination activating gene allowing programmed DNA elimination in eukaryotes. The described cases are RAG in jawed vertebrates, Kat 1 and Alpha 3 in the Kluyveromyces lactis yeast and Piggymac/TPB1 TPB2 and TPB6 in ciliates. The domesticated RAG is the most known case. It constitutes the enzymatic core of the Jawed vertebrates V(D)J recombination machinery. It directs random assembly and joining of gene segments during the development of B and T cells helping in the generation of the enormous gene diversity encoding antibodies or T cell receptors. It was shown in the case of RAG that the shift from DDE transposon to site-specific recombination activating gene is an evolutionary phenomenon that did not require dramatic changes. This explains why the co-option of DDE transposon as site-specific recombination activating gene can occur in a convergent manner. As numerous genes coding for DDE transposases are widespread through numerous members of the life tree, it is expected that several of them might correspond to domesticated transposons involved in programmed DNA elimination and maybe in the generation of receptor diversity. The domestication of DDE transposon could have been and still be of an extreme importance for organisms' evolution.