Family Of Mobile Genetic Elements Research Articles

Detection and classification of the integrative conjugative elements of Lactococcus lactis

Lactococcus lactis is widely applied by the dairy industry for the fermentation of milk into products such as cheese. Adaptation of L. lactis to the dairy environment often depends on functions encoded by mobile genetic elements (MGEs) such as plasmids. Other L. lactis MGEs that contribute to industrially relevant traits like antimicrobial production and carbohydrate utilization capacities belong to the integrative conjugative elements (ICE). Here we investigate the prevalence of ICEs in L. lactis using an automated search engine that detects colocalized, ICE-associated core-functions (involved in conjugation or mobilization) in lactococcal genomes. This approach enabled the detection of 36 candidate-ICEs in 69 L. lactis genomes. By phylogenetic analysis of conserved protein functions encoded in all lactococcal ICEs, these 36 ICEs could be classified in three main ICE-families that encompass 7 distinguishable ICE-integrases and are characterized by apparent modular-exchangeability and plasticity. Finally, we demonstrate that phylogenetic analysis of the conjugation-associated VirB4 ATPase function differentiates ICE- and plasmid-derived conjugation systems, indicating that conjugal transfer of lactococcal ICEs and plasmids involves genetically distinct machineries. Our genomic analysis and sequence-based classification of lactococcal ICEs creates a comprehensive overview of the conserved functional repertoires encoded by this family of MGEs in L. lactis, which can facilitate the future exploitation of the functional traits they encode by ICE mobilization to appropriate starter culture strains.

LINE-1 hypomethylation, increased retrotransposition and tumor-specific insertion in upper gastrointestinal cancer.

The long interspersed nuclear element-1 (LINE-1) retrotransposons are a major family of mobile genetic elements, comprising approximately 17% of the human genome. The methylation state of LINE-1 is often used as an indicator of global DNA methylation levels and it regulates the retrotransposition and somatic insertion of the genetic element. We have previously reported the significant relationship between LINE-1 hypomethylation and poor prognosis in upper gastrointestinal (GI) cancers. However, the causal relationships between LINE-1 hypomethylation, retrotransposition, and tumor-specific insertion in upper GI cancers remain unknown. We used bisulfite-pyrosequencing and quantitative real-time PCR to verify LINE-1 methylation and copy number in tissue samples of 101 patients with esophageal and 103 patients with gastric cancer. Furthermore, we analyzed the LINE-1 retrotransposition profile with an originally developed L1Hs-seq. In tumor samples, LINE-1 methylation levels were significantly lower than non-tumor controls, while LINE-1 copy numbers were markedly increased. As such, there was a significant inverse correlation between the LINE-1 methylation level and copy number in tumor tissues, with lower LINE-1 methylation levels corresponding to higher LINE-1 copy numbers. Of particular importance is that somatic LINE-1 insertions were more numerous in tumor than normal tissues. Furthermore, we observed that LINE-1 was inserted evenly across all chromosomes, and most often within genomic regions associated with tumor-suppressive genes. LINE-1 hypomethylation in upper GI cancers is related to increased LINE-1 retrotransposition and tumor-specific insertion events, which may collectively contribute to the acquisition of aggressive tumor features through the inactivation of tumor-suppressive genes.

To catch a hijacker: abundance, evolution and genetic diversity of P4-like bacteriophage satellites

Bacteriophages (phages) are bacterial parasites that can themselves be parasitized by phage satellites. The molecular mechanisms used by satellites to hijack phages are sometimes understood in great detail, but the origins, abundance, distribution and composition of these elements are poorly known. Here, we show that P4-like elements are present in more than 30% of the genomes of Enterobacterales, and in almost half of those of Escherichia coli, sometimes in multiple distinct copies. We identified over 1000 P4-like elements with very conserved genetic organization of the core genome and a few hotspots with highly variable genes. These elements are never found in plasmids and have very little homology to known phages, suggesting an independent evolutionary origin. Instead, they are scattered across chromosomes, possibly because their integrases are often exchanged with other elements. The rooted phylogenies of hijacking functions are correlated and suggest longstanding coevolution. They also reveal broad host ranges in P4-like elements, as almost identical elements can be found in distinct bacterial genera. Our results show that P4-like phage satellites constitute a very distinct, widespread and ancient family of mobile genetic elements. They pave the way for studying the molecular evolution of antagonistic interactions between phages and their satellites.This article is part of the theme issue ‘The secret lives of microbial mobile genetic elements’.

Cognate restriction of transposition by piggyBac-like proteins.

Mobile genetic elements have been harnessed for gene transfer for a wide variety of applications including generation of stable cell lines, recombinant protein production, creation of transgenic animals, and engineering cell and gene therapy products. The piggyBac transposon family includes transposase or transposase-like proteins from a variety of species including insect, bat and human. Recently, human piggyBac transposable element derived 5 (PGBD5) protein was reported to be able to transpose piggyBac transposons in human cells raising possible safety concerns for piggyBac-mediated gene transfer applications. We evaluated three piggyBac-like proteins across species including piggyBac (insect), piggyBat (bat)and PGBD5 (human) for their ability to mobilize piggyBac transposons in human cells. We observed a lack of cross-species transposition activity. piggyBac and piggyBat activity was restricted to their cognate transposons. PGBD5 was unable to mobilize piggyBac transposons based on excision, colony count and plasmid rescue analysis, and it was unable to bind piggyBac terminal repeats. Within the piggyBac family, we observed a lack of cross-species activity and found that PGBD5 was unable to bind, excise or integrate piggyBac transposons in human cells. Transposition activity appears restricted within species within the piggyBac family of mobile genetic elements.

Tn6603, a Carrier of Tn5053 Family Transposons, Occurs in the Chromosome and in a Genomic Island of Pseudomonas aeruginosa Clinical Strains.

Transposons of the Pseudomonas aeruginosa accessory gene pool contribute to phenotype and to genome plasticity. We studied local P. aeruginosa strains to ascertain the encroachment of mer-type res site hunter transposons into clinical settings and their associations with other functional modules. Five different Tn5053 family transposons were detected, all chromosomal. Some were solitary elements; one was in res of Tn1013#, a relative of a reported carrier of int-type res site hunters (class 1 integrons), but most were in res of Tn6603, a new Tn501-related transposon of unknown phenotype. Most of the Tn6603::Tn elements, and some Tn6603 and Tn6603::Tn elements found in GenBank sequences, were at identical sites in an hypothetical gene of P. aeruginosa genomic island PAGI-5v. The island in clonally differing strains was at either of two tRNALys loci, suggesting lateral transfer to these sites. This observation is consistent with the membership of the prototype PAGI-5 island to the ICE family of mobile genetic elements. Additionally, the res site hunters in the nested transposons occupied different positions in the Tn6603 carrier. This suggested independent insertion events on five occasions at least. Tn5053 family members that were mer-/tni-defective were found in Tn6603- and Tn501-like carriers in GenBank sequences of non-clinical Pseudomonas spp. The transposition events in these cases presumably utilized tni functions in trans, as can occur with class 1 integrons. We suggest that in the clinical context, P. aeruginosa strains that carry Tn6603 alone or in PAGI-5v can serve to disseminate functional res site hunters; these in turn can provide the requisite trans-acting tni functions to assist in the dissemination of class 1 integrons, and hence of their associated antibiotic resistance determinants.

Rebooting Synthetic Phage-Inducible Chromosomal Islands: One Method to Forge Them All.

Phage-inducible chromosomal islands (PICIs) are a widespread family of mobile genetic elements, which have an important role in bacterial pathogenesis. These elements mobilize among bacterial species at extremely high frequencies, representing an attractive tool for the delivery of synthetic genes. However, tools for their genetic manipulation are limited and timing consuming. Here, we have adapted a synthetic biology approach for rapidly editing of PICIs in Saccharomyces cerevisiae based on their ability to excise and integrate into the bacterial chromosome of their cognate host species. As proof of concept, we engineered several PICIs from Staphylococcus aureus and Escherichia coli and validated this methodology for the study of the biology of these elements by generating multiple and simultaneous mutations in different PICI genes. For biotechnological purposes, we also synthetically constructed PICIs as Trojan horses to deliver different CRISPR-Cas9 systems designed to either cure plasmids or eliminate cells carrying the targeted genes. Our results demonstrate that the strategy developed here can be employed universally to study PICIs and enable new approaches for diagnosis and treatment of bacterial diseases.

Detection of Retrotransposition Activity of Hot LINE-1s by Long-Distance Inverse PCR.

Long interspersed nuclear elements 1 (LINE-1s) are the only family of mobile genetic elements in the human genome that can move autonomously. They do so by a process called retrotransposition wherein they transcribe to form an mRNA intermediate which is then consequently inserted into the genome by reverse transcription. Despite being silent in normal cells, LINE-1s are highly active in different epithelial tumors. De novo LINE-1 insertions can potentially drive tumorigenesis, and hence it is important to systematically study LINE-1 retrotransposition in cancer. Out of ~150 retrotransposition-competent LINE-1s present in the human genome, only a handful of LINE-1 loci, also referred to as "hot" LINE-1s, account for the majority of de novo LINE-1 insertion in different cancer types. We have developed a simple polymerase chain reaction (PCR)-based method to monitor retrotransposition activity of these hot LINE-1s. This method, based on long-distance inverse (LDI)-PCR, takes advantage of 3´ transduction, a mechanism by which a LINE-1 mobilizes its flanking non-repetitive region, which can subsequently be used to identify de novo LINE-1 3´ transduction events stemming from a particular hot LINE-1.

LINE-1 retrotransposon-mediated DNA transductions in endometriosis associated ovarian cancers.

Endometrioid (ENOC) and clear cell ovarian carcinoma (CCOC) share a common precursor lesion, endometriosis, hence the designation endometriosis associated ovarian cancers (EAOC). Long interspersed nuclear element 1 (LINE-1 or L1), is a family of mobile genetic elements activated in many cancers capable of moving neighboring DNA through 3' transductions. Here we investigated the involvement of specific L1-mediated transductions in EAOCs. Through whole genome sequencing, we identified active L1-mediated transductions originating within the TTC28 gene in 34% (10/29) of ENOC and 31% (11/35) of CCOC cases. We used PCR and capillary sequencing to assess the presence of specific TTC28-L1 transductions in formalin-fixed paraffin-embedded (FFPE) blocks from six different anatomical sites (five tumors and one normal control) for four ENOC and three CCOC cases, and compared the results to the presence of single nucleotide variations (SNVs)/frame shift (fs) mutations detected using multiplex PCR and next generation sequencing. TTC28-L1 mediated transductions were identified in at least three tumor samplings in all cases, and were present in all five tumor samplings in 5/7 (71%) cases. In these cases, KRAS, PIK3CA, CTNNB1, ARID1A, and PTEN mutations were found across all tumor sites while other selected SNV/fs mutations of unknown significance were present at varying allelic frequencies. The TTC28-L1 transductions along with classical driver mutations were near ubiquitous across the tumors, suggesting that L1 activation likely occurred early in the development of EAOCs. TTC28-L1 transductions could potentially be used to determine clonal relationships and to track ovarian cancer progression.

Flipping chromosomes in deep-sea archaea.

One of the major mechanisms driving the evolution of all organisms is genomic rearrangement. In hyperthermophilic Archaea of the order Thermococcales, large chromosomal inversions occur so frequently that even closely related genomes are difficult to align. Clearly not resulting from the native homologous recombination machinery, the causative agent of these inversions has remained elusive. We present a model in which genomic inversions are catalyzed by the integrase enzyme encoded by a family of mobile genetic elements. We characterized the integrase from Thermococcus nautili plasmid pTN3 and showed that besides canonical site-specific reactions, it catalyzes low sequence specificity recombination reactions with the same outcome as homologous recombination events on DNA segments as short as 104bp both in vitro and in vivo, in contrast to other known tyrosine recombinases. Through serial culturing, we showed that the integrase-mediated divergence of T. nautili strains occurs at an astonishing rate, with at least four large-scale genomic inversions appearing within 60 generations. Our results and the ubiquitous distribution of pTN3-like integrated elements suggest that a major mechanism of evolution of an entire order of Archaea results from the activity of a selfish mobile genetic element.

Transposase-Mediated Excision, Conjugative Transfer, and Diversity of ICE6013 Elements in Staphylococcus aureus.

ICE6013 represents one of two families of integrative conjugative elements (ICEs) identified in the pan-genome of the human and animal pathogen Staphylococcus aureus Here we investigated the excision and conjugation functions of ICE6013 and further characterized the diversity of this element. ICE6013 excision was not significantly affected by growth, temperature, pH, or UV exposure and did not depend on recA The IS30-like DDE transposase (Tpase; encoded by orf1 and orf2) of ICE6013 must be uninterrupted for excision to occur, whereas disrupting three of the other open reading frames (ORFs) on the element significantly affects the level of excision. We demonstrate that ICE6013 conjugatively transfers to different S. aureus backgrounds at frequencies approaching that of the conjugative plasmid pGO1. We found that excision is required for conjugation, that not all S. aureus backgrounds are successful recipients, and that transconjugants acquire the ability to transfer ICE6013 Sequencing of chromosomal integration sites in serially passaged transconjugants revealed a significant integration site preference for a 15-bp AT-rich palindromic consensus sequence, which surrounds the 3-bp target site that is duplicated upon integration. A sequence analysis of ICE6013 from different host strains of S. aureus and from eight other species of staphylococci identified seven divergent subfamilies of ICE6013 that include sequences previously classified as a transposon, a plasmid, and various ICEs. In summary, these results indicate that the IS30-like Tpase functions as the ICE6013 recombinase and that ICE6013 represents a diverse family of mobile genetic elements that mediate conjugation in staphylococci.IMPORTANCE Integrative conjugative elements (ICEs) encode the abilities to integrate into and excise from bacterial chromosomes and plasmids and mediate conjugation between bacteria. As agents of horizontal gene transfer, ICEs may affect bacterial evolution. ICE6013 represents one of two known families of ICEs in the pathogen Staphylococcus aureus, but its core functions of excision and conjugation are not well studied. Here, we show that ICE6013 depends on its IS30-like DDE transposase for excision, which is unique among ICEs, and we demonstrate the conjugative transfer and integration site preference of ICE6013 A sequence analysis revealed that ICE6013 has diverged into seven subfamilies that are dispersed among staphylococci.

Variation on a theme; an overview of the Tn916/Tn1545 family of mobile genetic elements in the oral and nasopharyngeal streptococci.

The oral and nasopharyngeal streptococci are a major part of the normal microbiota in humans. Most human associated streptococci are considered commensals, however, a small number of them are pathogenic, causing a wide range of diseases including oral infections such as dental caries and periodontitis and diseases at other body sites including sinusitis and endocarditis, and in the case of Streptococcus pneumoniae, meningitis. Both phenotypic and sequence based studies have shown that the human associated streptococci from the mouth and nasopharynx harbor a large number of antibiotic resistance genes and these are often located on mobile genetic elements (MGEs) known as conjugative transposons or integrative and conjugative elements of the Tn916/Tn1545 family. These MGEs are responsible for the spread of the resistance genes between streptococci and also between streptococci and other bacteria. In this review we describe the resistances conferred by, and the genetic variations between the many different Tn916-like elements found in recent studies of oral and nasopharyngeal streptococci and show that Tn916-like elements are important mediators of antibiotic resistance genes within this genus. We will also discuss the role of the oral environment and how this is conducive to the transfer of these elements and discuss the contribution of both transformation and conjugation on the transfer and evolution of these elements in different streptococci.

Movement of IS26-associated antibiotic resistance genes occurs via a translocatable unit that includes a single IS26 and preferentially inserts adjacent to another IS26.

ABSTRACTThe insertion sequence IS26 plays a key role in disseminating antibiotic resistance genes in Gram-negative bacteria, forming regions containing more than one antibiotic resistance gene that are flanked by and interspersed with copies of IS26. A model presented for a second mode of IS26 movement that explains the structure of these regions involves a translocatable unit consisting of a unique DNA segment carrying an antibiotic resistance (or other) gene and a single IS copy. Structures resembling class I transposons are generated via RecA-independent incorporation of a translocatable unit next to a second IS26 such that the ISs are in direct orientation. Repeating this process would lead to arrays of resistance genes with directly oriented copies of IS26 at each end and between each unique segment. This model requires that IS26 recognizes another IS26 as a target, and in transposition experiments, the frequency of cointegrate formation was 60-fold higher when the target plasmid contained IS26. This reaction was conservative, with no additional IS26 or target site duplication generated, and orientation specific as the IS26s in the cointegrates were always in the same orientation. Consequently, the cointegrates were identical to those formed via the known mode of IS26 movement when a target IS26 was not present. Intact transposase genes in both IS26s were required for high-frequency cointegrate formation as inactivation of either one reduced the frequency 30-fold. However, the IS26 target specificity was retained. Conversion of each residue in the DDE motif of the Tnp26 transposase also reduced the cointegration frequency.

A novel comprehensive analysis method for Staphylococcus aureus pathogenicity islands

Staphylococcus aureus pathogenicity islands (SaPIs) form a growing family of mobile genetic elements (MGEs) in Staphylococci. Horizontal genetic transfer by MGEs plays an important role in the evolution of S. aureus. Several SaPIs carry staphylococcal enterotoxin and SE-like toxin genes. To comprehensively investigate the diversity of SaPIs, a series of primers corresponding to sequences flanking six SaPI insertion sites in S. aureus genome were designed and a long and accurate (LA)-PCR analysis method established. LA-PCR products of 13-17 kbp were observed in strains with seb, selk or selq genes. Restriction fragment length polymorphism (RFLP) analysis showed that the products have different RFLP characteristics than do previously described SaPIs; they were therefore predicted to include new SaPIs. Nucleotide sequencing analysis revealed seven novel SaPIs: seb-harboring SaPIivm10, SaPishikawa11, SaPIivm60, SaPIno10 and SaPIhirosaki4, selk and selq-harboring SaPIj11 and non-superantigen-harboring SaPIhhms2. These SaPIs have mosaic structures containing components of known SaPIs and other unknown genes. Strains carrying different SaPIs were found to have significantly different production of superantigen toxins. The present results show that the LA-PCR approach can comprehensively identify SaPI diversity and is useful for investigating the evolution of S. aureus pathogenicity.

Characterization of the conjugative transposon Tn6000 from Enterococcus casseliflavus 664.1H1 (formerly Enterococcus faecium 664.1H1)

Tn6000 (formerly EfcTn1) from Enterococcus casseliflavus strain 664.1H1 (previously Enterococcus faecium 664.1H1) is a tetracycline resistance-encoding conjugative transposon of the Tn916-like family of mobile genetic elements. Sequence analysis of Tn6000 shows that it has a novel modular structure, comprising fragments of diverse proven and putative mobile elements including plasmids, conjugative transposons and virulence and pathogenicity islands.

Mammalian Small Nucleolar RNAs Are Mobile Genetic Elements

Small nucleolar RNAs (snoRNAs) of the H/ACA box and C/D box categories guide the pseudouridylation and the 2′-O-ribose methylation of ribosomal RNAs by forming short duplexes with their target. Similarly, small Cajal body–specific RNAs (scaRNAs) guide modifications of spliceosomal RNAs. The vast majority of vertebrate sno/scaRNAs are located in introns of genes transcribed by RNA polymerase II and processed by exonucleolytic trimming after splicing. A bioinformatic search for orthologues of human sno/scaRNAs in sequenced mammalian genomes reveals the presence of species- or lineage-specific sno/scaRNA retroposons (sno/scaRTs) characterized by an A-rich tail and an ∼14-bp target site duplication that corresponds to their insertion site, as determined by interspecific genomic alignments. Three classes of snoRTs are defined based on the extent of intron and exon sequences from the snoRNA parental host gene they contain. SnoRTs frequently insert in gene introns in the sense orientation at genomic hot spots shared with other genetic mobile elements. Previously characterized human snoRNAs are encoded in retroposons whose parental copies can be identified by phylogenic analysis, showing that snoRTs can be faithfully processed. These results identify snoRNAs as a new family of mobile genetic elements. The insertion of new snoRNA copies might constitute a safeguard mechanism by which the biological activity of snoRNAs is maintained in spite of the risk of mutations in the parental copy. I furthermore propose that retroposition followed by genetic drift is a mechanism that increased snoRNA diversity during vertebrate evolution to eventually acquire new RNA-modification functions.

A novel family of mobile genetic elements is limited to the germline genome in Tetrahymena thermophila

In the ciliated protozoan Tetrahymena thermophila, extensive DNA elimination is associated with differentiation of the somatic macronucleus from the germline micronucleus. This study describes the isolation and complete characterization of Tlr elements, a family of approximately 30 micronuclear DNA sequences that are efficiently eliminated from the developing macronucleus. The data indicate that Tlr elements are comprised of an approximately 22 kb internal region flanked by complex and variable termini. The Tlr internal region is highly conserved among family members and contains 15 open reading frames, some of which resemble genes encoded by transposons and viruses. The Tlr termini appear to be long inverted repeats consisting of (i) a variable region containing multiple direct repeats which differ in number and sequence from element to element and (ii) a conserved terminal 47 bp sequence. Taken together, these results suggest that Tlr elements comprise a novel family of mobile genetic elements that are confined to the Tetrahymena germline genome. Possible mechanisms of developmentally programmed Tlr elimination are discussed.