The genome of the fish pathogen Aeromonas salmonicida subsp salmonicida harbors a large number of insertion sequences (ISs), many of which are located on plasmids. In the present study, we analyzed the small plasmid profile of A. salmonicida strains to identify evidences of plasmid alterations. Ten out of 78 strains analyzed displayed an unconventional plasmid profile. However the HER1104 strain was unique, having a positive PCR signal for pAsal1 plasmid despite not carrying this plasmid. Instead, HER1104 was bearing a plasmid at higher molecular weight than pAsal1. We characterized this new larger plasmid, which we called pAsal1B since it is a derivative of pAsal1 containing one more complete IS (ISAS5) than the parental plasmid. An additional 96 bp relic of ISAS5 was also present in pAsal1B. These results propose that ISAS5 is another active mobile genetic element in A. salmonicida subsp salmonicida and provided further proof of the genomic plasticity of this bacterium.
furunculosis; Aeromonas salmonicida; plasmid; DNA rearrangement; insertion sequence
Horizontal gene transfer (HGT) introduces advantageous genetic elements into pathogenic bacteria using tools such as class1 integrons. This study aimed at investigating the distribution of these integrons among uropathogenic E. coli (UPEC) isolated from patients in Aleppo, Syria. It also set to uncover the frequencies of the clinically relevant DfrA1 and DfrA17,7, as well as various associations leading to reduced susceptibility. This study involved 75 Trimethoprim-resistant E. coli isolates from in- and outpatients with urinary tract infections (UTIs) from 3 major hospitals in Aleppo. Bacterial identification, resistance and extended-spectrum-β-lactamase (ESBL) production testing were performed according to Clinical Laboratory Standards Institute guidelines. Detection of integrons and DfrA genes was done using PCR and statistical significance was inferred through χ2 (Fisher’s) test. Class1 integrons were detected in 54.6% of isolates while DfrA1 and DfrA17,7 were found in 16% and 70.6% of tested samples respectively. Furthermore, only DfrA17,7 were strongly associated with class1 integrons, as were reduced susceptibility to the majority of individual antibiotics, multidrug resistance and ESBL production. This study demonstrated the high prevalence of class1 integrons among UPEC strains in Aleppo, Syria, as well as their significant associations with MDR. This data give information for local healthcare provision using antibiotic chemotherapy.
DfrA17,7; DfrA1; antibiotic resistance; urinary tract infections
The unprecedented wealth of databases that have become available in the era of next-generation sequencing has considerably increased our knowledge of bacterial genetic elements (GEs). At the same time, the advent of single-cell based approaches has brought realization that unsuspected heterogeneity may occur in the bacterial population from a single colony. The increasing use of PCR-based techniques to study new GEs requires careful consideration of the possible different PCR targets associated with different subpopulations if incorrect or incomplete interpretations are to be avoided. In this commentary, confining ourselves to our direct experience, we illustrate some examples of PCR pitfalls that may be encountered while investigating GEs.
genetic elements; PCR mapping; PCR pitfalls; single-cell based approaches; Intra-colony heterogeneity
Transposons are mobile genetic elements that are capable of self-directed excision and subsequent reintegration within the host genome. Transposase such as piggyBac, Sleeping Beauty and Tol2 catalyze these reactions and have shown potential as tools for the stable integration of transgenes when used in the binary plasmid mode. Recent modifications to the transposase and/or the terminal repeats of the transposon have increased their integration efficiency and/or specificity. We recently described the development of a piggyBac transposase system, the helper independent, single construct self-inactivating plasmid called GENIE. Here we describe the structure, safety and function of these transpositional vectors and their use in animal transgenesis and cell transfection.
transposon; transposase; transgenesis; transgene; piggyBac; cell transfections
In addition to their natural role in eukaryotic genome evolution, transposons can be powerful tools for functional genomics in diverse taxa. The piggyBac transposon has been applied as such in eukaryotic parasites, both protozoa and helminths, and in several important vector mosquitoes. piggyBac is advantageous for functional genomics because of its ability to transduce a wide range of taxa, its capacity to integrate large DNA ‘cargoes’ relative to other mobile genetic elements, its propensity to target transcriptional units and its ability to re-mobilize without leaving a pattern of non-excised sequences or ‘footprint’ in the genome. We recently demonstrated that piggyBac can integrate transgenes into the genome of the parasitic nematode Strongyloides ratti, an important model for parasitic nematode biology and a close relative of the significant human pathogen S. stercoralis. Unlike transgenes encoded in conventional plasmid vectors, which we assume are assembled into multi-copy episomal arrays as they are in Caenorhabditis elegans, transgenes integrated via piggyBac are not only stably inherited in S. ratti, they are also continuously expressed. This has allowed derivation of the first stable transgene expressing lines in any parasitic nematode, a significant advance in the development of functional genomic tools for these important pathogens.
transposon; piggyBac; transgenesis; parasitic nematode; Strongyloides; chromosomal integration; gene silencing
Transposable elements (TEs) are ubiquitous residents in eukaryotic genomes. They can cause dramatic changes in gene expression and lead to gross rearrangements of chromosome structure, providing the basis for rapid evolution. The virilis species group of Drosophila contains certain species that can be crossed under experimental conditions and their phylogeny is thoroughly investigated. We have shown that Drosophila virilis, the most primitive karyotypically and probably the ancestral species of the group, is in the process of colonization by a very unusual retroelement Penelope which apparently repeatedly invaded the species of the group in the past. However, the molecular mechanisms and evolutionary consequences of such invasions are poorly understood. In this commentary, we discuss the implications of our recent investigation into the response of the RNA silencing system to Penelope invasion of a new host genome which can be achieved in different ways.
Drosophila; Penelope retroelement; evolution; invasion; small RNAs
We recently reported that acute stress causes a substantial upregulation of the epigenetic mark, Histone H3 Lysine 9 Trimethyl (H3K9me3) in the rat hippocampus within an hour of acute stress exposure. To determine the function of this change we used ChIP-sequencing to determine where this silencing mark was being localized. We found that it showed a strong bias toward localization at more active classes of retrotransposable elements and away from genes. Further, we showed that the change was functional in that it reduced transcription of some of these elements (notably the endogenous retrovirus IAP and the B2 SINE). In this commentary we examine these results, which appear to describe a selective genomic stress response and relate it to human health and disease, particularly stress related maladies such as Post-traumatic Stress Disorder, which have recently been shown to have both epigenetic elements in their causation as well as differences in epigenetic marking of retrotransposons in human patients.
epigenetic; hippocampus; post-traumatic stress disorder; corticosteroid; heterochromatin; chromatin
Class-1 integrons play an important role in the emergence and spread of antimicrobial resistance determinants. In a recent study we showed that host fitness was dramatically reduced following acquisition of these elements. These fitness costs were due to the presence of an active integrase and we suggested that the mechanistic explanation was due to reduced genetic stability through IntI1 mediated recombination events between attI/attC and non-canonical sites in the chromosome. Here we demonstrate that the attI degenerated target sequence is highly prevalent in our model organism Acinetobacter baylyi adding support to the hypothesis that IntI1 is costly due to genomic instability.
Class-1 integrons; fitness cost of resistance
The long-terminal repeat (LTR)-retrotransposon Ty1 is a mobile genetic element that replicates through an RNA intermediate. Retroelement genomic transcripts contain internal structures fundamental to gene expression and propagation. In addition, long non-coding antisense RNAs overlap the 5′-terminal region of the genomic RNA and confer post-translational copy number control. Although LTR- retrotransposons are functionally related to retroviruses, little is known about the structural determinants required for genomic RNA packaging or reverse transcription. This commentary summarizes two recent papers that provide the first snapshot of genomic RNA structures from the retrotransposon Ty1 involved in transposition. We combined structural approaches with functional and genetic assays to determine if antisense RNAs anneal with the genomic RNA. Analysis of various steps in the Ty1 life cycle showed that a novel RNA pseudoknot contributes to retrotransposon function. Comparing different RNA states provides additional information about regions potentially involved in Ty1 RNA dimerization or packaging.
Ty1 retrotransposon; RNA structure; pseudoknot; RNA packaging; reverse transcription
Long interspersed nuclear elements -1 (LINEs, L1s) are retroelements occupying almost 17% of the human genome. L1 retrotransposition can cause deleterious effects on the host-cell and it is generally inhibited by suppressive mechanisms, but it can occur in some specific cells during early development as well as in some tumor cells and in the presence of several environmental factors. In a recent publication we reported that extremely low frequency pulsed magnetic field can affect L1 retrotransposition in neuroblastoma cells. In this commentary we discuss the interaction between environment and L1 activity in the light of the new emerging paradigm of host-LINE relationship.
retrotransposition; LINE-1; environment; magnetic field; neuroblastoma
From their inception, Y chromosomes in plants and animals are subjected to the powerful effects of Müller’s ratchet, a process spurred by suppression of recombination that results in a rapid accumulation of mutations and repetitive elements. These mutations eventually lead to gene loss and degeneration of the Y chromosome. Y chromosomes in mammals are ancient, whereas most sex chromosomes in plants and many in insects and fish evolved recently. Sex type in papaya is controlled by a pair of nascent sex chromosomes that evolved around 7 million years ago. The papaya X and Yh were recently sequenced, providing valuable insight into the early stages of sex chromosome evolution. Here we discuss the fruits of this work with a focus on the repeat accumulation, gene trafficking and promiscuous DNA sequences found in the slowly degenerating Yh chromosome of papaya.
chloroplast genomic DNA; gene loss and gain; heterochromatin; retrotransposons; Y chromosome degeneration
Due to divergence, genetic variation is generally believed to be high among distantly related strains, low among closely related ones and little or none within the same classified clonal groups. Several recent genome-wide studies, however, revealed that significant genetic variation resides in a considerable number of genes among strains with identical MLST (Multilocus sequence typing) types and much of the variation was introduced by homologous recombination. Recognizing and understanding genomic variation within clonal bacterial groups could shed new light on the evolutionary path of infectious agents and the emergence of particularly pathogenic or virulent variants. This commentary presents our recent contributions to this line of work.
homologous recombination; horizontal gene transfer; prophage; multilocus sequence typing; pathogenic adaptation; phylogenomics
Insertion sequences (IS) are abundant in the bacterial fish pathogen Aeromonas salmonicida genome. IS are involved in rearrangement events that lead to the loss of virulence. In previous work, we studied a plasmid rearrangement that causes the deletion of the type three secretion system in A. salmonicida, resulting in a loss of virulence. We showed that the rearrangement is caused by the recombination of two IS (ISAS11) on an unstable plasmid (pAsa5). However, many rearrangements cannot be explained by our experimental approach and are thought to be the result of more complex or incomplete rearrangement events, as suggested by other plasmid loss profiles observed in various A. salmonicida strains. In this commentary, we examine the genetic instability of A. salmonicida indicating that its genome is rapidly evolving.
furunculosis; Aeromonas salmonicida; type three secretion system; plasmid; DNA rearrangement; insertion sequence; IS256
Microbes have several mechanisms that promote evolutionary adaptation in stressful environments. The corresponding molecular pathways promote diversity through modulating rates of recombination, mutation or influence the activity of transposable genetic elements. Recent experimental studies suggest an evolutionary conflict between these mechanisms. Specifically, presence of mismatch repair mutator alleles in a bacterial population dramatically reduced fixation of bacterial insertion sequence elements. When rare, these elements had only a limited impact on adaptive evolution compared with other mutation-generating pathways. IS elements may initially spread like molecular parasites, but once present in many copies in a given genome, they might become generators of novelty during bacterial evolution.
insertion sequence elements; evolution of mutation rate
Transposable elements have an ongoing, largely parasitic interaction with their hosts. We are interested in the timescale of this interaction. In a recent publication, we have examined the sequence divergence between class II DNA transposons from mammalian genomes. We asked whether these sequences undergo a continuing process of turnover, keeping a family as an integrated whole, as members of the family are continually created and lost. Alternatively, we envisaged that elements might have been involved in a burst of amplification, soon after they first occupied a mammalian genome, and the shared ancestry of present-day elements harks back to this initial amplification, a process that we termed a “life cycle.” We resolved between these processes by estimating the time to common ancestry predicted from the genetic diversity of sequences found in a transposon family, and also estimating, from the mammalian orders that currently possess copies of the family, the time when the family first entered the mammalian genome. These times are approximately the same, supporting the “life cycle” model. This casts light on how far we can infer genetic changes in the past through the study of DNA sequences from the present.
mammals; transposons; class II; molecular dating; evolution
Current methods used for phylogenetic classification of prokaryotes largely rely on the sequences of 16S rRNA genes that are ubiquitously present in the cell. Theoretical basis of this methodology is based on the assumption that 16S rRNA genes are only vertically inherited and are thus indigenous to each species. However, microbial genomic analysis has revealed the existence of prokaryotic species containing two types of rRNA (rrn) operons of seemingly different origins. It has also been reported that some bacteria contain 16S rRNA that are mosaics of sequences from multiple species. This suggests that horizontal gene transfer (HGT) occurred for 16S rRNA genes. In addition, a recent HGT experiment mimicking the natural HGT process has shown that a wide range of foreign 16S rRNA genes can be transferred into Escherichia coli, including those from different phylogenetic classes (with a minimum sequence identity of 80.9% to the Escherichia coli 16S rRNA gene). Thus, in contrast to the complexity hypothesis that states informational genes are rarely horizontally transferred between species, 16S rRNA is occasionally amenable to HGT. Results of the current method for rapid identification and classification of prokaryotes based on the 16S rRNA gene should thus be carefully analyzed and interpreted.
horizontal gene transfer; 16S ribosomal RNA; complexity hypothesis; phylogenetics
Phylogenetic reconstruction of three highly conserved proteins involved in bacterial conjugation (relaxase, coupling protein and a type IV secretion system ATPase) allowed the classification of transmissible elements in relaxase MOB families and mating pair formation MPF groups. These evolutionary studies point to the existence of a limited number of module combinations in transmissible elements, preferentially associated with specific genetic or environmental backgrounds. A practical protocol based on the MOB classification was implemented to detect and assort transmissible plasmids and integrative elements from γ-Proteobacteria. It was called “Degenerate Primer MOB Typing” or DPMT. It resulted in a powerful technique that discovers not only backbones related to previously classified elements (typically by PCR-based replicon typing or PBRT), but also distant new members sharing a common evolutionary ancestor. The DPMT method, conjointly with PBRT, promises to be useful to gain information on plasmid backbones and helpful to investigate the dissemination routes of transmissible elements in microbial ecosystems.
relaxase; plasmid classification; degenerate primer MOB typing; conjugation; ICE; transmissible elements
Natural transformation can lead to exchange of DNA between taxonomically diverse bacteria. In the case of chromosomal DNA, homology-based recombination with the recipient genome is usually necessary for heritable stability. In our recent study, we have shown that natural transformation can promote the transfer of transposons, IS elements, and integrons and gene cassettes, largely independent of the genetic relationship between the donor and recipient bacteria. Additional results from our study suggest that natural transformation with species-foreign DNA might result in the uptake of a wide range of DNA fragments; leading to changes in the antimicrobial susceptibility profile and contributing to the generation of antimicrobial resistance in bacteria.
natural transformation; transposition; homologous recombination; class 1 integrons; gene cassettes; transposons; mosaic genes; Acinetobacter
Transfer of DNA between different compartments of the plant cell, i.e., plastid, mitochondrion and nucleus, is a well-known phenomenon in plant evolution. Six directions of inter-compartmental DNA migration are possible in theory, however only four of them have been previously reported. These include frequent cases of mitochondrion and plastid to nucleus transfer, plastid to mitochondrion transfer, and rare nucleus to mitochondrion migrations. The connection between the plastid and mitochondrial genomes in flowering plants has been viewed as a one way road. Contrary to these observations we found that a sequence widespread in the carrot mitochondrial genome, designated as DcMP, was transferred to the plastid genome of a carrot ancestor. Interestingly, DcMP was integrated into a tRNA promoter of the plastid trnV gene, replacing the original promoter sequence. The rearrangement of the plastid genome is specific for carrot and closely related species belonging Scandiceae clade. The structure of the sequence and the presence of a 6 nt target site duplication led us to speculate that the transfer was a result of a transposition event of a non-LTR retrotransposon. These findings open interesting questions about the evolution of organellar genomes and mobile genetic elements and provide a useful plastid marker to phylogenetically delineate species relationships within the Scandiceae clade.
Daucus carota; inter-compartmental DNA migration; plastid and mitochondrial genome; retrotransposon
We have recently shown that GAA repeats severely impede replication elongation during the first replication cycle of transfected DNA wherein the chromatin is still at the formation stage.1 Here we extend this study by showing that two GAA repeats located within the same plasmid in the direct orientation can form complexes upon transient transfection of mammalian Cos-1 cells. However, these complexes do not form in DNA that went through several replication rounds in mammalian cells. We suggest that formation of such complexes in mammalian genomes can contribute to genomic instability.
replication; GAA repeat; Friedreich ataxia; genome instability; chromatin
The resiliency and adaptive ability of microbial life in real time on Earth relies heavily upon horizontal gene transfer. Based on that knowledge, how likely is earth based microbial life to colonize extraterrestrial targets such as Mars? To address this question, we consider manned and unmanned space exploration, the resident microbiota that is likely to inhabit those vehicles, the adaptive potential of that microbiota in an extraterrestrial setting especially with regards to mobile genetic elements, and the likelihood that Mars like environments could initiate and sustain colonization.
Gaia theory; HGT; Mars; forward contamination; horizontal gene transfer; manned space flight; microbiome; mobile genetic elements
Anecdotal evidence shows promoters being reused separate from their downstream gene, thus providing a mechanism for the efficient and rapid rewiring of a gene’s transcriptional regulation. We have identified over 4000 groups of highly similar promoters using a conservative sequence similarity search in all fully sequenced prokaryotic genomes. About 6% of those groups are shared between bacteria from different taxonomic depth, including different genera, families, orders, classes and even phyla. Database searches against known mobile elements and RNA motifs have indicated that regulatory motifs such as riboswitches could be moved around on putative mobile promoters.
horizontal gene transfer; mobile elements; prokaryotes; promoters; rewiring
SINEs; ORF2 protein; retrotransposition; Alu; retroelement; LINE-1; TRAMP complex
The insertion of a retrotransposable element is usually associated with adverse or, at best, neutral effects on the host. Diversity-generating retroelements (DGRs) are the first elements that seem to offer a direct selective advantage to their phage or prokaryote host by exact replacement of a short, defined region of a host gene with a hypermutated variant. In a previous study, we presented the software DiGReF for identification of DGRs in genome sequences, and compiled the first comprehensive set of diversity-generating retroelements in public databases. We identified 155 elements in more than 6000 prokaryotic and phage genomes, which was a surprisingly low number. In this commentary, we will discuss the low incidence of these elements and speculate about the biological role of bacterial DGRs.
Diversity-generating retroelements; DiGReF; phages; bioinformatics; formylglycine-generating enzyme; database bias