Pulsed field gel electrophoresis to rapidly detect the presence of IncJ conjugative transposon-like elements

AIMS: To develop a screening method to detect the presence of the IncJ group of integrating conjugative transposon-like elements upon transfer to Escherichia coli. METHODS AND RESULTS: The unique insertion site of known IncJ elements, the prfC gene, is located in a region of the E. coli chromosome between 98.5 and 100 min on the E. coli genetic map. Using pulsed field gel electrophoresis and the rare cutting restriction enzymes SfiI and XbaI insertions of IncJ elements and an estimate of their size could be determined physically. CONCLUSIONS: This method allows initial screening of putative IncJ conjugative transposon-like elements by physical determination of their integration. Significance and Impact of the Study: IncJ-like elements, which appear to be highly homologous to the prototype IncJ element R391, have been found associated with recent epidemic outbreaks of cholera in a number of locations worldwide. Because of their integrative biology this method provides the first initial screening method to physically determine their presence upon transfer to E. coli.     

Tn916-like genetic elements: a diverse group of modular mobile elements conferring antibiotic resistance

Antibiotic-resistant Gram-positive bacteria are responsible for morbidity and mortality in healthcare environments. Enterococcus faecium, Enterococcus faecalis, Staphylococcus aureus and Streptococcus pneumoniae can all exhibit clinically relevant multidrug resistance phenotypes due to acquired resistance genes on mobile genetic elements. It is possible that clinically relevant multidrug-resistant Clostridium difficile strains will appear in the future, as the organism is adept at acquiring mobile genetic elements (plasmids and transposons). Conjugative transposons of the Tn916/Tn1545 family, which carry major antibiotic resistance determinants, are transmissible between these different bacteria by a conjugative mechanism during which the elements are excised by a staggered cut from donor cells, converted to a circular form, transferred by cell-cell contact and inserted into recipient cells by a site-specific recombinase. The ability of these conjugative transposons to acquire additional, clinically relevant antibiotic resistance genes importantly contributes to the emergence of multidrug resistance.     

Use of mobile genetic elements as tools for molecular epidemiology

Trypanosomiasis is a complex zoonotic disease where human-infective and non-human-infective strains of Trypanosoma brucei interact in the same transmission cycles. Differentiating these strains is paramount to understanding disease epidemiology. Restriction fragment length polymorphism analysis of repetitive DNA has provided such a method for distinguishing human and non-human isolates. Unfortunately, this approach requires large amounts of material and a more rapid approach is required. We have developed a novel technique, mobile genetic element-PCR, for assaying for positional variation of the mobile genetic element, RIME. The trypanosome genome contains up to 400 copies of RIME. Using this approach we have observed considerable variation between strains of T. brucei. Such a technique may offer potential as a method for differentiating non-human- and human-infective trypanosomes and shows promise as a rapid sensitive tool for investigating the epidemiology of sleeping sickness.     

Gene flow, mobile genetic elements and the recruitment of antibiotic resistance genes into Gram-negative pathogens

Antibiotics were one of the great discoveries of the 20th century. However, resistance appeared even in the earliest years of the antibiotic era. Antibiotic resistance continues to become worse, despite the ever-increasing resources devoted to combat the problem. One of the most important factors in the development of resistance to antibiotics is the remarkable ability of bacteria to share genetic resources via Lateral Gene Transfer (LGT). LGT occurs on a global scale, such that in theory, any gene in any organism anywhere in the microbial biosphere might be mobilized and spread. With sufficiently strong selection, any gene may spread to a point where it establishes a global presence. From an antibiotic resistance perspective, this means that a resistance phenotype can appear in a diverse range of infections around the globe nearly simultaneously. We discuss the forces and agents that make this LGT possible and argue that the problem of resistance can ultimately only be managed by understanding the problem from a broad ecological and evolutionary perspective. We also argue that human activities are exacerbating the problem by increasing the tempo of LGT and bacterial evolution for many traits that are important to humans.     

Identification of antibiotic resistance genes and associated mobile genetic elements in permafrost

Science China Life Sciences -     

Landscape of mobile genetic elements and their antibiotic resistance cargo in prokaryotic genomes

Prokaryotic Mobile Genetic Elements (MGEs) such as transposons, integrons, phages and plasmids, play important roles in prokaryotic evolution and in the dispersal of cargo functions like antibiotic resistance. However, each of these MGE types is usually annotated and analysed individually, hampering a global understanding of phylogenetic and environmental patterns of MGE dispersal. We thus developed a computational framework that captures diverse MGE types, their cargos and MGE-mediated horizontal transfer events, using recombinases as ubiquitous MGE marker genes and pangenome information for MGE boundary estimation. Applied to ∼84k genomes with habitat annotation, we mapped 2.8 million MGE-specific recombinases to six operational MGE types, which together contain on average 13% of all the genes in a genome. Transposable elements (TEs) dominated across all taxa (∼1.7 million occurrences), outnumbering phages and phage-like elements (<0.4 million). We recorded numerous MGE-mediated horizontal transfer events across diverse phyla and habitats involving all MGE types, disentangled and quantified the extent of hitchhiking of TEs (17%) and integrons (63%) with other MGE categories, and established TEs as dominant carriers of antibiotic resistance genes. We integrated all these findings into a resource (proMGE.embl.de), which should facilitate future studies on the large mobile part of genomes and its horizontal dispersal.     

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 ~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.     

Isolation and analysis of a circular form of the IncJ conjugative transposon-like elements, R391 and R997: implications for IncJ incompatibility

The incompatibility between the chromosomally integrating, conjugative transposon-like, IncJ elements R997 (ampicillin resistant) and R391 (kanamycin resistant) was examined by constructing strains harbouring both elements. Unusually, recA(+) strains harbouring the resistance determinants of both elements could be isolated but all strains lacked detectable extrachromosomal DNA. The phenotypic characteristics and transfer patterns observed suggested the formation of recombinant hybrids rather than strains harbouring both elements independently. Formation of strains harbouring two IncJ elements in a recA background was thus examined and resulted in the visualisation of extrachromosomal DNA. When R391 was transferred to a recA strain containing integrated R997, both elements co-existed stably and resulted in the isolation of a plasmid of 93.9 kb. When R997 was transferred to a recA strain harbouring an integrated R391, a plasmid of 85 kb was isolated. Comparison of restriction patterns for both elements revealed many common and several distinct fragments indicating a close physical relationship. These data suggest that although IncJ elements normally integrate at a unique site in the Escherichia coli chromosome, they possess the ability for autonomous replication which becomes manifest in a recA background when this site is occupied. This observation has implications for the nature of the incompatibility associated with IncJ elements and also provides a reliable method for isolating IncJ elements for molecular characterisation.     

Human mobile genetic elements: Structure, distribution and functional role

In this review, we present the data about mobile genetic elements that comprise about 45% of human genome. Along with classification and localization of the human mobile genetic elements, their role in genome functioning became the center of exclusive attention; in particular, we discussed a role of the human mobile elements in recombination, regulation of gene expression and origin of new genes.     

Truncation family selection and nonsystematic inbreeding leads to a rapid fixation of a pattern of mobile genetic elements in a computer model

A computer simulation model of the population dynamics of a polygenic system and a pattern of mobile genetic elements (MGEs) under directional truncation selection for a quantitative trait was developed. Modifier MGEs were shown to be rapidly and adaptively fixed (or lost) together with the modified polygenes. Marker MGEs and independent MGE copies were fixed and lost just as rapidly but in a random manner. Using specific marking of initial haploid genomes and direct computing of the mean proportion of identical encounters at each locus in each generation, it was shown that the mean nonselective inbreeding coefficient F(n) dramatically increases in the course of selection, reaching values 0.7-0.9 in 15-20 generations. As a result, adaptive homozygotization of polygenes and modifier MGEs and random homozygotization of marker MGEs, independent MGE copies, and all other genes of the genome occurs. These results confirm the hypothesis on the "champion" polygene pattern advanced earlier to explain the data of selection experiments.     

Molecular tools to detect anatoxin-a genes in aquatic ecosystems: Toward a new nested PCR-based method

Over the last few decades, cyanobacterial mass occurrence has become a recurrent feature of aquatic ecosystems. This has led to ecosystem exposure and health hazards associated with cyanotoxin production. The neurotoxin anatoxin-a and its homologs can be synthesized by benthic cyanobacterial species in lotic systems, but also by planktonic lacustrine species such as Dolichospermum (also known as Anabaena). However, only a few studies have focused on anatoxin-a occurrence and its biosynthesis genes in freshwater lakes. The initial aim of this study was to evaluate the molecular tools available in the literature to detect anatoxin-a biosynthesis genes in lacustrine environments. Having tested different sets of PCR primers, we found that that some sets of primers, such as anxC, were too specific and did not amplify anatoxin-a biosynthesis genes in all producing strains. On the other hand, some sets of primers, such as atxoa, seemed not to be specific enough, amplifying numerous non-specific bands in environmental samples, especially those from sediments. Furthermore, anaC and anaF amplification exhibited different band intensities during electrophoresis, suggesting a high variation in number of gene copies between samples. As a result, we proposed a new nested PCR-based method which considerably improved the amplification of the anaC gene in our environmental samples, eliminating non-specific bands and weak detections. Using this tool, our study also highlighted that anatoxin-a genes are widely distributed throughout freshwater lakes. This suggests the need for further ecological investigations into anatoxin-a in these ecosystems.     

Casposons: mobile genetic elements that gave rise to the CRISPR-Cas adaptation machinery

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Transgenesis of schistosomes: approaches employing mobile genetic elements

Draft genome sequences for Schistosoma mansoni and Schistosoma japonicum are now available. However, the identity and importance of most schistosome genes have yet to be determined. Recently, progress has been made towards the genetic manipulation and transgenesis of schistosomes. Both loss-of-function and gain-of-function approaches appear to be feasible in schistosomes based on findings described in the past 5 years. This review focuses on reports of schistosome transgenesis, specifically those dealing with the transformation of schistosomes with exogenous mobile genetic elements and/or their endogenous relatives for the genetic manipulation of schistosomes. Transgenesis mediated by mobile genetic elements offers a potentially tractable route to introduce foreign genes to schistosomes, a means to determine the importance of schistosome genes, including those that could be targeted in novel interventions and the potential to undertake large-scale forward genetics by insertional mutagenesis.     

MGE-PCR: a novel approach to the analysis of Toxoplasma gondii strain differentiation using mobile genetic elements

The position of mobile genetic elements (MGE) within eukaryotic genomes is often highly variable and we have exploited this phenomenon to develop a novel approach to strain differentiation in Toxoplasma gondii. Two PCR based strategies were designed in which specific primers were used to amplify T. gondii MGE's revealing information on element size and positional variation. The first PCR strategy involved the use of a standard two primer PCR while the second strategy used a single specific primer in a step-up PCR protocol. This approach was applied to T. gondii reference strains which were either acute virulent or avirulent to mice. The use of a standard two primer PCR reaction revealed the presence of a virulence related marker in which all avirulent strains possessed an additional 688 bp band. The single primer PCR strategy demonstrated that all virulent strains had identical banding patterns suggesting invariance within this group of strains. However, all avirulent strains had different banding patterns indicating the presence of a number of individual lineages within this group. The applicability and sensitivity of MGE-PCR in epidemiological studies was demonstrated by direct amplification of T. gondii from sheep tissue samples. All sheep isolates, tested in this way, gave identical banding patterns suggesting the presence of an endemic Toxoplasma strain on this farm.     

Contribution of mobile genetic elements to Desulfovibrio vulgaris genome plasticity

The genome of Desulfovibrio vulgaris strain DePue, a sulfate-reducing Deltaproteobacterium isolated from heavy metal-impacted lake sediment, was completely sequenced and compared with the type strain D. vulgaris Hildenborough. The two genomes share a high degree of relatedness and synteny, but harbour distinct prophage and signatures of past phage encounters. In addition to a highly variable phage contribution, the genome of strain DePue contains a cluster of open-reading frames not found in strain Hildenborough coding for the production and export of a capsule exopolysaccharide, possibly of relevance to heavy metal resistance. Comparative whole-genome microarray analysis on four additional D. vulgaris strains established greater interstrain variation within regions associated with phage insertion and exopolysaccharide biosynthesis.     

Plasmid metagenome reveals high levels of antibiotic resistance genes and mobile genetic elements in activated sludge

The overuse or misuse of antibiotics has accelerated antibiotic resistance, creating a major challenge for the public health in the world. Sewage treatment plants (STPs) are considered as important reservoirs for antibiotic resistance genes (ARGs) and activated sludge characterized with high microbial density and diversity facilitates ARG horizontal gene transfer (HGT) via mobile genetic elements (MGEs). However, little is known regarding the pool of ARGs and MGEs in sludge microbiome. In this study, the transposon aided capture (TRACA) system was employed to isolate novel plasmids from activated sludge of one STP in Hong Kong, China. We also used Illumina Hiseq 2000 high-throughput sequencing and metagenomics analysis to investigate the plasmid metagenome. Two novel plasmids were acquired from the sludge microbiome by using TRACA system and one novel plasmid was identified through metagenomics analysis. Our results revealed high levels of various ARGs as well as MGEs for HGT, including integrons, transposons and plasmids. The application of the TRACA system to isolate novel plasmids from the environmental metagenome, coupled with subsequent high-throughput sequencing and metagenomic analysis, highlighted the prevalence of ARGs and MGEs in microbial community of STPs.     

Fungal transposable elements: Generators of diversity and genetic tools

Representatives of several classes of transposable elements (TEs) have been characterized in a broad range of fungal species. The studies indicate that these elements are ancient and ubiquitous components of fungal genomes. Some of these elements have been shown to actively affect gene structure and function in several ways: inactivation of gene expression upon insertion, modification of the nucleotide sequence through excision, and probably by inducing extensive chromosomal rearrangements. The ability of TEs to generate a high degree of genetic diversity may therefore be important in the evolution of the fungal genome. TEs also have many potential applications in genetic research, including insertional mutagenesis and population fingerprinting, as well as gene transfer within and between species. All these genetic approaches are important as tools in studies of molecular biology and evolution of fungal species, many of which lack a functional sexual cycle.