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At least 19 records
All sourcesNASA NTRSDOE OSTI
DOE OSTI2026

Data for Transposon Signatures of Allopolyploid Genome Evolution

Hybridization brings together chromosome sets from two or more distinct progenitor species. Genome duplication associated with hybridization, or allopolyploidy, allows these chromosome sets to persist as distinct subgenomes during subsequent meioses. Here, we present a general method for identifying the subgenomes of a polyploid based on shared ancestry as revealed by the genomic distribution of repetitive elements that were active in the progenitors. This subgenome-enriched transposable element signal is intrinsic to the polyploid, allowing broader applicability than other approaches that depend on the availability of sequenced diploid relatives. We develop the statistical basis of the method, demonstrate its applicability in the well-studied cases of tobacco, cotton, and Brassica napus, and apply it to several cases: allotetraploid cyprinids, allohexaploid false flax, and allooctoploid strawberry. These analyses provide insight into the origins of these polyploids, revise the subgenome identities of strawberry, and provide perspective on subgenome dominance in higher polyploids.

Genomics↗
DOE OSTI2022

Functional characterization of diverse type I-F CRISPR-associated transposons

CRISPR-Cas systems generally provide adaptive immunity in prokaryotes through RNA-guided degradation of foreign genetic elements like bacteriophages and plasmids. Recently, however, transposon-encoded and nuclease-deficient CRISPR-Cas systems were characterized and shown to be co-opted by Tn7-like transposons for CRISPR RNA-guided DNA transposition. As a genome engineering tool, these CRISPR-Cas systems and their associated transposon proteins can be deployed for programmable, site-specific integration of sizable cargo DNA, circumventing the need for DNA cleavage and homology-directed repair involving endogenous repair machinery. Here, we selected a diverse set of type I-F3 CRISPR-associated transposon systems derived from Gammaproteobacteria, predicted all components essential for transposition activity, and deployed them for functionality testing within Escherichia coli. Our results demonstrate that these systems possess a significant range of integration efficiencies with regards to temperature, transposon size, and flexible PAM requirements. Additionally, our findings support the categorization of these systems into functional compatibility groups for efficient and orthogonal RNA-guided DNA integration. This work expands the CRISPR-based toolbox with new CRISPR RNA-guided DNA integrases that can be applied to complex and extensive genome engineering efforts.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Molecular and biochemical characterization of rice developed through conventional integration of nDart1-0 transposon gene

Mutations, the genetic variations in genomic sequences, play an important role in molecular biology and biotechnology. During DNA replication or meiosis, one of the mutations is transposons or jumping genes. An indigenous transposon nDart1-0 was successfully introduced into local indica cultivar Basmati-370 from transposon-tagged line viz., GR-7895 (japonica genotype) through conventional breeding technique, successive backcrossing. Plants from segregating populationsshowed variegated phenotypes were tagged as BM-37 mutants. Blast analysis of the sequence data revealed that the GTP-binding protein, located on the BAC clone OJ1781_H11 of chromosome 5, contained an insertion of DNA transposon nDart1-0. The nDart1-0 has “A” at position 254 bp, whereas nDart1 homologs have “G”, which efficiently distinguishes nDart1-0 from its homologs. The histological analysis revealed that the chloroplast of mesophyll cells in BM-37 was disrupted with reduction in size of starch granules and higher number of osmophillic plastoglobuli, which resulted in decreased chlorophyll contents and carotenoids, gas exchange parameters (Pn, g, E, Ci), and reduced expression level of genes associated with chlorophyll biosynthesis, photosynthesis and chloroplast development. Along with the rise of GTP protein, the salicylic acid (SA) and gibberellic acid (GA) and antioxidant contents(SOD) and MDA levels significantly enhanced, while, the cytokinins (CK), ascorbate peroxidase (APX), catalase (CAT), total flavanoid contents (TFC) and total phenolic contents (TPC) significantly reduced in BM-37 mutant plants as compared with WT plants. These results support the notion that GTP-binding proteins influence the process underlying chloroplast formation. Therefore, it is anticipated that to combat biotic or abiotic stress conditions, the nDart1-0 tagged mutant (BM-37) of Basmati-370 would be beneficial.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2025

Dual transposon sequencing profiles the genetic interaction landscape in bacteria

Gene redundancy complicates systematic characterization of gene function as single-gene deletions may not produce discernible phenotypes. We report dual transposon sequencing (dual Tn-seq), a platform for assaying the fitness of a comprehensive double mutant pool in parallel. Dual Tn-seq couples random barcode transposon site sequencing with the Cre-lox system, enabling deep sampling of 73% of the 1.3 million possible double gene deletions in Streptococcus pneumoniae. The genetic interactions identified span a wide range of biochemical processes, revealing new factors in presumably well-studied pathways, exemplified by a cytidine triphosphate synthase PyrJ. Moreover, this approach should permit further investigation of growth condition–specific genetic interactions. Because dual Tn-seq does not require the construction of a large array of single mutants, it should be readily adaptable to various microorganisms.

CTP synthesis↗
DOE OSTI2024

A novel conjugative transposon carrying an autonomously amplified plasmid

Tetracyclines serve as broad-spectrum antibiotics to treat bacterial infections. The discovery of new tetracycline resistance genes has led to new questions about the underlying mechanisms of resistance, gene transfer, and their relevance to human health. We tracked changes in the abundance of a 55-kbp conjugative transposon (CTn214) carrying tetQ, a tetracycline resistance gene, within a Bacteroides fragilis metagenome-assembled genome derived from shotgun sequencing of microbial DNA extracted from the ileal pouch of a patient with ulcerative colitis. The mapping of metagenomic reads to CTn214 revealed the multi-copy nature of a 17,044-nt region containing tetQ in samples collected during inflammation and uninflamed visits. B. fragilis cultivars isolated from the same patient during periods of inflammation harbored CTn214 integrated into the chromosome or both a circular, multi-copy, extrachromosomal region of the CTn214 containing tetQ and the corresponding integrated form. The tetracycline-dependent mechanism for the transmission of CTn214 is nearly identical to a common conjugative transposon found in the genome of B. fragilis (CTnDOT), but the autonomously amplified nature of a circular 17,044-nt region of CTn214 that codes for tetQ and the integration of the same sequence in the linear chromosome within the same cell is a novel observation. Genome and transcriptome sequencing of B. fragilis cultivars grown under different concentrations of tetracycline and ciprofloxacin indicates that tetQ in strains containing the circular form remains actively expressed regardless of treatment, while the expression of tetQ in strains containing the linear form increases only in the presence of tetracycline.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Transposon sequencing analysis of Bradyrhizobium diazoefficiens 110 spc4

Bradyrhizobium diazoefficiens USDA110 is one of the most effective nitrogen-fixing symbionts of soybeans. Here we carried out a large-scale transposon insertion sequencing (Tn-seq) analysis of strain Bd110spc4, which is derived from USDA110, with the goal of increasing available resources for identifying genes crucial for the survival of this plant symbiont under diverse conditions. We prepared two transposon (Tn) insertion libraries of Bd110spc4 with 155,042 unique Tn insertions when the libraries were combined, which is an average of one insertion every 58.7 bp of the reference USDA110 genome. Application of bioinformatic filtering steps to remove genes too small to be expected to have Tn insertions, resulted in a list of genes that were classified as putatively essential. Comparison of this gene set with genes putatively essential for the growth of the closely related alpha-proteobacterium, Rhodopseudomonas palustris, revealed a small set of five genes that may be collectively essential for closely related members of the family Bradyrhizobiaceae. This group includes bacteria with diverse lifestyles ranging from plant symbionts to animal-associated species to free-living species.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

The Enterprise , a massive transposon carrying Spok meiotic drive genes

The genomes of eukaryotes are full of parasitic sequences known as transposable elements (TEs). Here, we report the discovery of a putative giant tyrosine-recombinase-mobilized DNA transposon, Enterprise, from the model fungus Podospora anserina. Previously, we described a large genomic feature called the Spok block which is notable due to the presence of meiotic drive genes of the Spok gene family. The Spok block ranges from 110 kb to 247 kb and can be present in at least four different genomic locations within P. anserina, despite what is an otherwise highly conserved genome structure. We propose that the reason for its varying positions is that the Spok block is not only capable of meiotic drive but is also capable of transposition. More precisely, the Spok block represents a unique case where the Enterprise has captured the Spoks, thereby parasitizing a resident genomic parasite to become a genomic hyperparasite. Furthermore, we demonstrate that Enterprise (without the Spoks) is found in other fungal lineages, where it can be as large as 70 kb. Lastly, we provide experimental evidence that the Spok block is deleterious, with detrimental effects on spore production in strains which carry it. This union of meiotic drivers and a transposon has created a selfish element of impressive size in Podospora, challenging our perception of how TEs influence genome evolution and broadening the horizons in terms of what the upper limit of transposition may be.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Identification of recG genetic interactions in Escherichia coli by transposon sequencing

ABSTRACT Maintaining the integrity of the genome is of utmost importance for cell division and propagation. In Escherichia coli , the RecG protein has been implicated in processing branched recombination intermediates during DNA repair processes, but the primary cellular role(s) of RecG and the repair pathways in which it acts have been difficult to define. To gain additional insight into RecG function, we employed transposon sequencing to identify recG genetic interactions and reveal complementary or redundant functions. The strongest hits from the screen were the dam , uvrD , rnhA , radA , and rep genes. The conditional importance of these five genes in cells lacking recG was confirmed using a plasmid-based assay, revealing synthetic lethal interactions for most double deletion strains. Several of the synthetic lethal gene combinations (with uvrD, rep, and radA , but not rnhA or dam ) were suppressed by deletion of recF or recO , indicating that their genetic relationships involved roles in post-replication gap repair. Additionally, loss of the RecG/SSB interaction phenocopied a recG deletion when combined with dam , uvrD , radA , or rnhA deletions but not with rep . The results reinforce the idea of RecG as a general genome guardian. RecG has at least two functions. It plays an important role in the resolution of joint molecules behind the fork, formed during post-replication gap repair. RecG is also required to suppress genome over-replication caused by unscheduled replication initiation at R-loops, at double-strand breaks caused by dam inactivation, and during replication termination. IMPORTANCE DNA damage and subsequent DNA repair processes are mutagenic in nature and an important driver of evolution in prokaryotes, including antibiotic resistance development. Genetic screening approaches, such as transposon sequencing (Tn-seq), have provided important new insights into gene function and genetic relationships. Here, we employed Tn-seq to gain insight into the function of the recG gene, which renders Escherichia coli cells moderately sensitive to a variety of DNA-damaging agents when they are absent. The reported recG genetic interactions can be used in combination with future screens to aid in a more complete reconstruction of DNA repair pathways in bacteria.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Characterization and Diversification of AraC/XylS Family Regulators Guided by Transposon Sequencing

In this study, we explored the development of engineered inducible systems. Publicly available data from previous transposon sequencing assays were used to identify regulators of metabolism in Pseudomonas putida KT2440. For AraC family regulators (AFRs) represented in these data, we posited AFR/promoter/inducer groupings. Twelve promoters were characterized for a response to their proposed inducers in P. putida, and the resultant data were used to create and test nine two-plasmid sensor systems in Escherichia coli. Several of these were further developed into a palette of single-plasmid inducible systems. From these experiments, we observed an unreported inducer response from a previously characterized AFR, demonstrated that the addition of a P. putida transporter improved the sensor dynamics of an AFR in E. coli, and identified an uncharacterized AFR with a novel potential inducer specificity. Finally, targeted mutations in an AFR, informed by structural predictions, enabled the further diversification of these inducible plasmids.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2022

Widespread transposon co-option in the Caenorhabditis germline regulatory network

The movement of selfish DNA elements can lead to widespread genomic alterations with potential to create novel functions. We show that transposon expansions in Caenorhabditis nematodes led to extensive rewiring of germline transcriptional regulation. We find that about one-third of Caenorhabditis elegans germline-specific promoters have been co-opted from two related miniature inverted repeat transposable elements (TEs), CERP2 and CELE2. These promoters are regulated by HIM-17, a THAP domain–containing transcription factor related to a transposase. Expansion of CERP2 occurred before radiation of the Caenorhabditis genus, as did fixation of mutations in HIM-17 through positive selection, whereas CELE2 expanded only in C. elegans. Through comparative analyses in Caenorhabditis briggsae, we find not only evolutionary conservation of most CERP2 co-opted promoters but also a substantial fraction that are species-specific. Our work reveals the emergence and evolutionary conservation of a novel transcriptional network driven by TE co-option with a major impact on regulatory evolution.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

The widespread IS200/IS605 transposon family encodes diverse programmable RNA-guided endonucleases

Tracing the origin of CRISPR-Cas CRISPR-Cas systems have transformed genome editing and other biotechnologies; however, the broader origins and diversity of RNA-guided nucleases have largely remained unexplored. Altae-Tran et al . show that three distinct transposon-encoded proteins, IscB, IsrB, and TnpB, are naturally occurring, reprogrammable RNA-guided DNA nucleases (see the Perspective by Rousset and Sorek). In addition to identifying diverse guide-encoding mechanisms, the authors elucidate the evolutionary relationship between IsrB, IscB, and CRISPR-Cas9. Overall, these newly characterized systems, called OMEGA (for obligate mobile element–guided activity) systems, are found in all domains of life and may be harnessed for biotechnology development. —DJ

Science & Technology - Other Topics↗
DOE OSTI2022

Nitrogen Metabolism in Pseudomonas putida : Functional Analysis Using Random Barcode Transposon Sequencing

Pseudomonas putida KT2440 has long been studied for its diverse and robust metabolisms, yet many genes and proteins imparting these growth capacities remain uncharacterized. Using pooled mutant fitness assays, we identified genes and proteins involved in the assimilation of 52 different nitrogen containing compounds. To assay amino acid biosynthesis, 19 amino acid drop-out conditions were also tested. From these 71 conditions, significant fitness phenotypes were elicited in 672 different genes including 100 transcriptional regulators and 112 transport-related proteins. We divide these conditions into 6 classes, and propose assimilatory pathways for the compounds based on this wealth of genetic data. To complement these data, we characterize the substrate range of three promiscuous aminotransferases relevant to metabolic engineering efforts in vitro. So we examine the specificity of five transcriptional regulators, explaining some fitness data results and exploring their potential to be developed into useful synthetic biology tools. In addition, we use manifold learning to create an interactive visualization tool for interpreting our BarSeq data, which will improve the accessibility and utility of this work to other researchers.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Transposon signatures of allopolyploid genome evolution

Hybridization brings together chromosome sets from two or more distinct progenitor species. Genome duplication associated with hybridization, or allopolyploidy, allows these chromosome sets to persist as distinct subgenomes during subsequent meioses. Here, we present a general method for identifying the subgenomes of a polyploid based on shared ancestry as revealed by the genomic distribution of repetitive elements that were active in the progenitors. This subgenome-enriched transposable element signal is intrinsic to the polyploid, allowing broader applicability than other approaches that depend on the availability of sequenced diploid relatives. We develop the statistical basis of the method, demonstrate its applicability in the well-studied cases of tobacco, cotton, and Brassica napus, and apply it to several cases: allotetraploid cyprinids, allohexaploid false flax, and allooctoploid strawberry. These analyses provide insight into the origins of these polyploids, revise the subgenome identities of strawberry, and provide perspective on subgenome dominance in higher polyploids.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Correction for Thompson et al., “Fatty Acid and Alcohol Metabolism in Pseudomonas putida : Functional Analysis Using Random Barcode Transposon Sequencing”

Volume 86, no. 21, 2020, e01665-20, https://doi.org/10.1128/AEM.01665-20. Readers should note that when the authors created the deletion mutant of PP_2675, they deleted the entire region between the gene’s start and stop codons. However, this also deleted the DNA that codes for the first 14 amino acids of the overlapping ORF PP_2676. Additionally, the originally published URL for the JBEI public strain repository was incorrect. The correct link to all strains and plasmids in this study is https://public-registry.jbei .org/folders/641.

59 BASIC BIOLOGICAL SCIENCES↗