Mobile DNA in the Pathogenic Neisseria

Abstract Background Trypanosomatid genomes are colonized by active and inactive mobile DNA elements, such as LINE, SINE-like, SIDER and DIRE retrotransposons. These elements all share a 77-nucleotide-long sequence at their 5′ ends, known as Pr77, which activates transcription, thereby generating abundant unspliced and translatable transcripts. However, transcription factors that mediates this process have still not been reported. Methods TATA-binding protein (TBP) and small nuclear RNA-activating protein 50 kDa (SNAP50) recombinant proteins and specific antibodies raised against them were generated. Protein capture assay, electrophoretic mobility-shift assays (EMSA) and EMSA competition assays carried out using these proteins and nuclear proteins of the parasite together to specific DNA sequences used as probes allowed detecting direct interaction of these transcription factors to Pr77 sequence. Results This study identified TBP and SNAP50 as part of the DNA-protein complex formed by the Pr77 promoter sequence and nuclear proteins of Trypanosoma cruzi. TBP establishes direct and specific contact with the Pr77 sequence, where the DPE and DPE downstream regions are docking sites with preferential binding. TBP binds cooperatively (Hill coefficient = 1.67) to Pr77 and to both strands of the Pr77 sequence, while the conformation of this highly structured sequence is not involved in TBP binding. Direct binding of SNAP50 to the Pr77 sequence is weak and may be mediated by protein–protein interactions through other trypanosomatid nuclear proteins. Conclusions Identification of the transcription factors that mediate Pr77 transcription may help to elucidate how these retrotransposons are mobilized within the trypanosomatid genomes and their roles in gene regulation processes in this human parasite. Graphic abstract

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Multidrug-Resistant Enterococci Lack CRISPR-cas

mBio ◽

10.1128/mbio.00227-10 ◽

2010 ◽

Vol 1 (4) ◽

Cited By ~ 227

Author(s):

Kelli L. Palmer ◽

Michael S. Gilmore

Keyword(s):

Antibiotic Resistance ◽

Draft Genome ◽

Inverse Correlation ◽

Multidrug Resistant ◽

Mobile Elements ◽

Mobile Dna ◽

Data Set ◽

Content Type ◽

Genome Defense ◽

Acquired Antibiotic Resistance

ABSTRACT Clustered, regularly interspaced short palindromic repeats (CRISPR) provide bacteria and archaea with sequence-specific, acquired defense against plasmids and phage. Because mobile elements constitute up to 25% of the genome of multidrug-resistant (MDR) enterococci, it was of interest to examine the codistribution of CRISPR and acquired antibiotic resistance in enterococcal lineages. A database was built from 16 Enterococcus faecalis draft genome sequences to identify commonalities and polymorphisms in the location and content of CRISPR loci. With this data set, we were able to detect identities between CRISPR spacers and sequences from mobile elements, including pheromone-responsive plasmids and phage, suggesting that CRISPR regulates the flux of these elements through the E. faecalis species. Based on conserved locations of CRISPR and CRISPR-cas loci and the discovery of a new CRISPR locus with associated functional genes, CRISPR3-cas, we screened additional E. faecalis strains for CRISPR content, including isolates predating the use of antibiotics. We found a highly significant inverse correlation between the presence of a CRISPR-cas locus and acquired antibiotic resistance in E. faecalis, and examination of an additional eight E. faecium genomes yielded similar results for that species. A mechanism for CRISPR-cas loss in E. faecalis was identified. The inverse relationship between CRISPR-cas and antibiotic resistance suggests that antibiotic use inadvertently selects for enterococcal strains with compromised genome defense. IMPORTANCE For many bacteria, including the opportunistically pathogenic enterococci, antibiotic resistance is mediated by acquisition of new DNA and is frequently encoded on mobile DNA elements such as plasmids and transposons. Certain enterococcal lineages have recently emerged that are characterized by abundant mobile DNA, including numerous viruses (phage), and plasmids and transposons encoding multiple antibiotic resistances. These lineages cause hospital infection outbreaks around the world. The striking influx of mobile DNA into these lineages is in contrast to what would be expected if a self (genome)-defense system was present. Clustered, regularly interspaced short palindromic repeat (CRISPR) defense is a recently discovered mechanism of prokaryotic self-defense that provides a type of acquired immunity. Here, we find that antibiotic resistance and possession of complete CRISPR loci are inversely related and that members of recently emerged high-risk enterococcal lineages lack complete CRISPR loci. Our results suggest that antibiotic therapy inadvertently selects for enterococci with compromised genome defense.

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Mobile DNA and evolution in the 21st century

Mobile DNA ◽

10.1186/1759-8753-1-4 ◽

2010 ◽

Vol 1 (1) ◽

pp. 4 ◽

Cited By ~ 56

Author(s):

James A Shapiro

Keyword(s):

21St Century ◽

Mobile Dna

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Mobile DNA: Mechanisms, Utility, and Consequences

Molecular Life Sciences ◽

10.1007/978-1-4614-6436-5_157-1 ◽

2014 ◽

pp. 1-23

Author(s):

Adam R. Parks ◽

Joseph E. Peters

Keyword(s):

Mobile Dna

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A Novel and Direct Metamobilome Approach improves the Detection of Larger-sized Circular Elements across Kingdoms

10.1101/761098 ◽

2019 ◽

Cited By ~ 1

Author(s):

Katrine Skov Alanin ◽

Tue Sparholt Jørgensen ◽

Patrick Browne ◽

Bent Petersen ◽

Leise Riber ◽

...

Keyword(s):

Multiple Displacement Amplification ◽

Bacterial Species ◽

Human Health Risk ◽

Prokaryotic Genome ◽

Mobile Genetic Elements ◽

Wastewater Sample ◽

Mobile Dna ◽

Complex Samples ◽

Genetic Elements ◽

Dna Elements

AbstractMobile genetic elements (MGEs) are instrumental in natural prokaryotic genome editing, permitting genome plasticity and allowing microbes to accumulate immense genetic diversity. MGEs include DNA elements such as plasmids, transposons and Insertion Sequences (IS-elements), as well as bacteriophages (phages), and they serve as a vast communal gene pool. These mobile DNA elements represent a human health risk as they can add new traits, such as antibiotic resistance or virulence, to a bacterial strain. Sequencing libraries targeting circular MGEs, referred to as mobilomes, allows the expansion of our current understanding of the mechanisms behind the mobility, prevalence and content of these elements. However, metamobilomes from bacterial communities are not studied to the same extent as metagenomics, partly because of methodological biases arising from multiple displacement amplification (MDA), often used in previous metamobilome publications. In this study, we show that MDA is detrimental to the detection of larger-sized plasmids if small plasmids are present by comparing the abundances of reads mapping to plasmids in a wastewater sample spiked with a mock community of selected plasmids with and without MDA. Furthermore, we show that it is possible to produce samples consisting almost exclusively of circular MGEs and obtain a catalog of larger, complete, circular MGEs from complex samples without the use of MDA.ImportanceMobile genetic elements (MGEs) can transport genetic information between genomes in different bacterial species, adding new traits, potentially generating dangerous multidrug-resistant pathogens. In fact, plasmids and circular MGEs can encode bacterial genetic specializations such as virulence, resistance to metals, antimicrobial compounds, and bacteriophages, as well as the degradation of xenobiotics. For this reason, circular MGEs are crucial to investigate, but they are often missed in metagenomics and ecological studies. In this study, we present, for the first time, an improved method, which reduces the bias towards small MGEs and we demonstrate that this method can unveil larger, complete circular MGEs from complex samples without the use of multiple displacement amplification. This method may result in the detection of larger-sized plasmids that have hitherto remained unnoticed and therefore has the potential to reveal novel accessory genes, acting as possible targets in the development of preventive strategies directed at pathogens.

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Fis negatively affects binding of Tn4652 transposase by out-competing IHF from the left end of Tn4652

Microbiology ◽

10.1099/mic.0.022830-0 ◽

2009 ◽

Vol 155 (4) ◽

pp. 1203-1214 ◽

Cited By ~ 10

Author(s):

Riho Teras ◽

Julia Jakovleva ◽

Maia Kivisaar

Keyword(s):

Dnase I ◽

Integration Host Factor ◽

Mobile Dna ◽

Mobility Shift ◽

Transposase Gene ◽

Transposition Activity ◽

Global Regulators ◽

Factor For Inversion Stimulation ◽

Dna Elements ◽

Gel Mobility Shift

Transposition activity in bacteria is generally maintained at a low level. The activity of mobile DNA elements can be controlled by bacterially encoded global regulators. Regulation of transposition of Tn4652 in Pseudomonas putida is one such example. Activation of transposition of Tn4652 in starving bacteria requires the stationary-phase sigma factor RpoS and integration host factor (IHF). IHF plays a dual role in Tn4652 translocation by activating transcription of the transposase gene tnpA of the transposon and facilitating TnpA binding to the inverted repeats of the transposon. Our previous results have indicated that besides IHF some other P. putida-encoded global regulator(s) might bind to the ends of Tn4652 and regulate transposition activity. In this study, employing a DNase I footprint assay we have identified a binding site of P. putida Fis (factor for inversion stimulation) centred 135 bp inside the left end of Tn4652. Our results of gel mobility shift and DNase I footprint studies revealed that Fis out-competes IHF from the left end of Tn4652, thereby abolishing the binding of TnpA. Thus, the results obtained in this study indicate that the transposition of Tn4652 is regulated by the cellular amount of P. putida global regulators Fis and IHF.

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Five months of voluntary wheel running downregulates skeletal muscle LINE-1 gene expression in rats

AJP Cell Physiology ◽

10.1152/ajpcell.00301.2019 ◽

2019 ◽

Vol 317 (6) ◽

pp. C1313-C1323 ◽

Cited By ~ 1

Author(s):

Matthew A. Romero ◽

Petey W. Mumford ◽

Paul A. Roberson ◽

Shelby C. Osburn ◽

Hailey A. Parry ◽

...

Keyword(s):

Skeletal Muscle ◽

Dna Methylation ◽

Exercise Training ◽

Mrna Expression ◽

Wheel Running ◽

Mobile Dna ◽

Voluntary Wheel Running ◽

L6 Myotubes ◽

Voluntary Wheel

Transposable elements (TEs) are mobile DNA and constitute approximately half of the human genome. LINE-1 (L1) is the only active autonomous TE in the mammalian genome and has been implicated in a number of diseases as well as aging. We have previously reported that skeletal muscle L1 expression is lower following acute and chronic exercise training in humans. Herein, we used a rodent model of voluntary wheel running to determine whether long-term exercise training affects markers of skeletal muscle L1 regulation. Selectively bred high-running female Wistar rats ( n = 11 per group) were either given access to a running wheel (EX) or not (SED) at 5 wk of age, and these conditions were maintained until 27 wk of age. Thereafter, mixed gastrocnemius tissue was harvested and analyzed for L1 mRNA expression and DNA content along with other L1 regulation markers. We observed significantly ( P < 0.05) lower L1 mRNA expression, higher L1 DNA methylation, and less L1 DNA in accessible chromatin regions in EX versus SED rats. We followed these experiments with 3-h in vitro drug treatments in L6 myotubes to mimic transient exercise-specific signaling events. The AMP-activated protein kinase (AMPK) agonist 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR; 4 mM) significantly decreased L1 mRNA expression in L6 myotubes. However, this effect was not facilitated through increased L1 DNA methylation. Collectively, these data suggest that long-term voluntary wheel running downregulates skeletal muscle L1 mRNA, and this may occur through chromatin modifications. Enhanced AMPK signaling with repetitive exercise bouts may also decrease L1 mRNA expression, although the mechanism of action remains unknown.

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Restriction Enzyme Based Enriched L1Hs Sequencing (REBELseq): A Scalable Technique for Detection of Ta Subfamily L1Hs in the Human Genome

G3 Genes|Genome|Genetics ◽

10.1534/g3.119.400613 ◽

2020 ◽

Vol 10 (5) ◽

pp. 1647-1655

Author(s):

Benjamin C. Reiner ◽

Glenn A. Doyle ◽

Andrew E. Weller ◽

Rachel N. Levinson ◽

Esin Namoglu ◽

...

Keyword(s):

Next Generation Sequencing ◽

Restriction Enzyme ◽

Postmortem Brain ◽

Whole Genome ◽

Mobile Dna ◽

Neuronal Nuclei ◽

Dna Elements ◽

Long Interspersed Element ◽

Generation Sequencing ◽

Human Specific

Long interspersed element-1 retrotransposons (LINE-1 or L1) are ∼6 kb mobile DNA elements implicated in the origins of many Mendelian and complex diseases. The actively retrotransposing L1s are mostly limited to the L1 human specific (L1Hs) transcriptional active (Ta) subfamily. In this manuscript, we present REBELseq as a method for the construction of Ta subfamily L1Hs-enriched next-generation sequencing libraries and bioinformatic identification. REBELseq was performed on DNA isolated from NeuN+ neuronal nuclei from postmortem brain samples of 177 individuals and empirically-driven bioinformatic and experimental cutoffs were established. Putative L1Hs insertions passing bioinformatics cutoffs were experimentally validated. REBELseq reliably identified both known and novel Ta subfamily L1Hs insertions distributed throughout the genome. Differences in the proportion of individuals possessing a given reference or non-reference retrotransposon insertion were identified. We conclude that REBELseq is an unbiased, whole genome approach to the amplification and detection of Ta subfamily L1Hs retrotransposons.

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Identification of a Novel Streptococcal Gene Cassette Mediating SOS Mutagenesis in Streptococcus uberis

Journal of Bacteriology ◽

10.1128/jb.00473-07 ◽

2007 ◽

Vol 189 (14) ◽

pp. 5210-5222 ◽

Cited By ~ 23

Author(s):

Emilia Varhimo ◽

Kirsi Savijoki ◽

Jari Jalava ◽

Oscar P. Kuipers ◽

Pekka Varmanen

Keyword(s):

Uv Light ◽

Streptococcus Thermophilus ◽

Gene Cassette ◽

Repeat Sequence ◽

Mobile Dna ◽

Mobility Shift ◽

Gene Encoding ◽

Sos Mutagenesis ◽

Streptococcus Uberis ◽

Y Family

ABSTRACT Streptococci have been considered to lack the classical SOS response, defined by increased mutation after UV exposure and regulation by LexA. Here we report the identification of a potential self-regulated SOS mutagenesis gene cassette in the Streptococcaceae family. Exposure to UV light was found to increase mutations to antibiotic resistance in Streptococcus uberis cultures. The mutational spectra revealed mainly G:C→A:T transitions, and Northern analyses demonstrated increased expression of a Y-family DNA polymerase resembling UmuC under DNA-damaging conditions. In the absence of the Y-family polymerase, S. uberis cells were sensitive to UV light and to mitomycin C. Furthermore, the UV-induced mutagenesis was almost completely abolished in cells deficient in the Y-family polymerase. The gene encoding the Y-family polymerase was localized in a four-gene operon including two hypothetical genes and a gene encoding a HdiR homolog. Electrophoretic mobility shift assays demonstrated that S. uberis HdiR binds specifically to an inverted repeat sequence in the promoter region of the four-gene operon. Database searches revealed conservation of the gene cassette in several Streptococcus species, including at least one genome each of Streptococcus pyogenes, Streptococcus agalactiae, Streptococcus mitis, Streptococcus sanguinis, and Streptococcus thermophilus strains. In addition, the umuC operon was localized in several mobile DNA elements of Streptococcus and Lactococcus species. We conclude that the hdiR-umuC-ORF3-ORF4 operon represents a novel gene cassette capable of mediating SOS mutagenesis among members of the Streptococcaceae.

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