scholarly journals CRISPR-interference based modulation of mobile genetic elements in bacteria

2018 ◽  
Author(s):  
Ákos Nyerges ◽  
Balázs Bálint ◽  
Judit Cseklye ◽  
István Nagy ◽  
Csaba Pál ◽  
...  
Keyword(s):  
Complete Removal ◽  
Mobile Genetic Elements ◽  
Industrial Biotechnology ◽  
Chromosome Engineering ◽  
Crispr Interference ◽  
Genetic Elements ◽  
Promising Tool ◽  
Single Plasmid ◽  
Bacterial Chromosomes

ABSTRACTSpontaneous mutagenesis of synthetic genetic constructs by mobile genetic elements frequently results in the rapid loss of advantageous functions. Previous efforts to minimize such mutations required the exceedingly time-consuming manipulation of bacterial chromosomes and the complete removal of insertional sequences (ISes). To this aim, we developed a single plasmid-based system (pCRIS) that applies CRISPR-interference to inhibit the transposition of bacterial ISes. pCRIS expresses multiple guide RNAs to direct inactivated Cas9 (dCas9) to simultaneously silence IS1, IS3, IS5, and IS150at up to 38 chromosomal loci inEscherichia coli,in vivo. As a result, the transposition rate of all four targeted ISes dropped to negligible levels at both chromosomal and episomal targets, increasing the half-life of exogenous protein expression. Most notably, pCRIS, while requiring only a single plasmid delivery performed within a single day, provided a reduction of IS-mobility comparable to that seen in genome-scale chromosome engineering projects. Global transcriptomics analysis revealed nevertheless only minute alterations in the expression of untargeted genes. Finally, the transposition-silencing effect of pCRIS was easily transferable across multipleE. colistrains. The plasticity and robustness of our IS-silencing system make it a promising tool to stabilize bacterial genomes for synthetic biology and industrial biotechnology applications.

scholarly journals CRISPR-interference-based modulation of mobile genetic elements in bacteria

2019 ◽  
Vol 4 (1) ◽  
Author(s):  
Ákos Nyerges ◽  
Balázs Bálint ◽  
Judit Cseklye ◽  
István Nagy ◽  
Csaba Pál ◽  
...  
Keyword(s):  
Fluorescent Protein ◽  
Complete Removal ◽  
Mobile Genetic Elements ◽  
Industrial Biotechnology ◽  
Chromosome Engineering ◽  
Crispr Interference ◽  
Genetic Elements ◽  
Promising Tool ◽  
Single Plasmid

Abstract Spontaneous mutagenesis of synthetic genetic constructs by mobile genetic elements frequently results in the rapid loss of engineered functions. Previous efforts to minimize such mutations required the exceedingly time-consuming manipulation of bacterial chromosomes and the complete removal of insertional sequences (ISes). To this aim, we developed a single plasmid-based system (pCRIS) that applies CRISPR-interference to inhibit the transposition of bacterial ISes. pCRIS expresses multiple guide RNAs to direct inactivated Cas9 (dCas9) to simultaneously silence IS1, IS3, IS5 and IS150 at up to 38 chromosomal loci in Escherichia coli, in vivo. As a result, the transposition rate of all four targeted ISes dropped to negligible levels at both chromosomal and episomal targets. Most notably, pCRIS, while requiring only a single plasmid delivery performed within a single day, provided a reduction of IS-mobility comparable to that seen in genome-scale chromosome engineering projects. The fitness cost of multiple IS-knockdown, detectable in flask-and-shaker systems was readily outweighed by the less frequent inactivation of the transgene, as observed in green fluorescent protein (GFP)-overexpression experiments. In addition, global transcriptomics analysis revealed only minute alterations in the expression of untargeted genes. Finally, the transposition-silencing effect of pCRIS was easily transferable across multiple E. coli strains. The plasticity and robustness of our IS-silencing system make it a promising tool to stabilize bacterial genomes for synthetic biology and industrial biotechnology applications.

scholarly journals Mobile genetic elements: in silico,in vitro,in vivo

2016 ◽  
Vol 25 (5) ◽  
pp. 1027-1031 ◽  
Author(s):  
Irina R. Arkhipova ◽  
Phoebe A. Rice
Keyword(s):  
In Silico ◽  
Mobile Genetic Elements ◽  
Genetic Elements

scholarly journals Characterization of mcr-5-Harboring Salmonella enterica subsp. enterica Serovar Typhimurium Isolates from Animal and Food Origin in Germany

2019 ◽  
Vol 63 (6) ◽  
Author(s):  
Maria Borowiak ◽  
Jens A. Hammerl ◽  
Carlus Deneke ◽  
Jennie Fischer ◽  
Istvan Szabo ◽  
...  
Keyword(s):  
Salmonella Enterica ◽  
Mobile Genetic Elements ◽  
Sequence Type ◽  
Content Type ◽  
Genetic Elements ◽  
Bacterial Chromosomes ◽  
Serovar Typhimurium

ABSTRACT We characterized eight mcr-5-positive Salmonella enterica subsp. enterica serovar Typhimurium sequence type 34 (ST34) isolates obtained from pigs and meat in Germany. Five plasmid types were identified harboring mcr-5 on Tn6452 or putative mobile insertion cassettes. The mobility of mcr-5 was confirmed by integration of Tn6452 into the bacterial chromosomes of two strains and the detection of conjugative mcr-5 plasmids. The association with mobile genetic elements might further enhance mcr-5 distribution.

scholarly journals Type II Toxin-Antitoxin Systems: Evolution and Revolutions

2020 ◽  
Vol 202 (7) ◽  
Author(s):  
Nathan Fraikin ◽  
Frédéric Goormaghtigh ◽  
Laurence Van Melderen
Keyword(s):  
Mobile Genetic Elements ◽  
Phage Infection ◽  
Type Ii ◽  
Toxic Protein ◽  
Genetic Elements ◽  
The Poor ◽  
Poor Understanding ◽  
Bacterial Chromosomes ◽  
Postsegregational Killing

ABSTRACT Type II toxin-antitoxin (TA) systems are small genetic elements composed of a toxic protein and its cognate antitoxin protein, the latter counteracting the toxicity of the former. While TA systems were initially discovered on plasmids, functioning as addiction modules through a phenomenon called postsegregational killing, they were later shown to be massively present in bacterial chromosomes, often in association with mobile genetic elements. Extensive research has been conducted in recent decades to better understand the physiological roles of these chromosomally encoded modules and to characterize the conditions leading to their activation. The diversity of their proposed roles, ranging from genomic stabilization and abortive phage infection to stress modulation and antibiotic persistence, in conjunction with the poor understanding of TA system regulation, resulted in the generation of simplistic models, often refuted by contradictory results. This review provides an epistemological and critical retrospective on TA modules and highlights fundamental questions concerning their roles and regulations that still remain unanswered.

scholarly journals Type I toxin-antitoxin systems contribute to the maintenance of mobile genetic elements in Clostridioides difficile

2020 ◽  
Vol 3 (1) ◽  
Author(s):  
Johann Peltier ◽  
Audrey Hamiot ◽  
Julian R. Garneau ◽  
Pierre Boudry ◽  
Anna Maikova ◽  
...  
Keyword(s):  
Ectopic Expression ◽  
Mobile Genetic Elements ◽  
Toxin Gene ◽  
Type I ◽  
Genetic Elements ◽  
Clostridioides Difficile ◽  
Bacterial Chromosomes ◽  
Toxin Protein

AbstractToxin-antitoxin (TA) systems are widespread on mobile genetic elements and in bacterial chromosomes. In type I TA, synthesis of the toxin protein is prevented by the transcription of an antitoxin RNA. The first type I TA were recently identified in the human enteropathogen Clostridioides difficile. Here we report the characterization of five additional type I TA within phiCD630-1 (CD0977.1-RCd11, CD0904.1-RCd13 and CD0956.3-RCd14) and phiCD630-2 (CD2889-RCd12 and CD2907.2-RCd15) prophages of C. difficile strain 630. Toxin genes encode 34 to 47 amino acid peptides and their ectopic expression in C. difficile induces growth arrest that is neutralized by antitoxin RNA co-expression. We show that type I TA located within the phiCD630-1 prophage contribute to its stability and heritability. We have made use of a type I TA toxin gene to generate an efficient mutagenesis tool for this bacterium that allowed investigation of the role of these widespread TA in prophage maintenance.

Mobile genetic elements in Drosophila melanogaster (recent experiments)

Genome ◽  
1989 ◽  
Vol 31 (2) ◽  
pp. 920-928 ◽  
Author(s):  
Georgii P. Georgiev ◽  
Nickolai A. Tchurikov ◽  
Yurii V. Ilyin ◽  
Sofia G. Georgieva ◽  
Lev J. Mizrokhi ◽  
...  
Keyword(s):  
Drosophila Melanogaster ◽  
Rna Polymerase Ii ◽  
Mobile Element ◽  
Complete Removal ◽  
Mobile Genetic Elements ◽  
Internal Promoter ◽  
Genetic Elements ◽  
First Case ◽  
Large Numbers ◽  
White Locus

Recent data obtained in the authors' laboratories concerning the behaviour of mobile genetic elements of Drosophila melanogaster are reviewed. It was found that the mobile element jockey represents the typical LINE element. It is efficiently transcribed in D. melanogaster cells in flies and in culture. Transcription is initiated from the +1 nucleotide of jockey and depends on an internal promoter. This is the first case of an internal promoter being used by RNA polymerase II. Several events which take place during the transposition bursts in ctMR2 family of strains were described. Among them are the removal of mobile dispersed genetics (mdg) elements (with solo long terminal repeat (LTR) remaining at the site of excision), complete removal of an mdg element, and reinsertion of the same mdg to the same place either in the presence or in absence of solo LTR sequence. Finally, the formation of deletions was observed. A 462-bp deletion destroying the white locus can be further repaired (w+ reversion). Thus, transposition bursts include many different genetic events. A novel system of prolonged genome destabilization was described. It depends on mobilization of a new mobile element called Stalker. After certain crosses Stalker actively moves for dozens of generations giving rise to large numbers of insertion mutations. Several novel genes were detected using mobilized Stalker. They include a modifier of mdg4 and six enhancers of yellow mutations.Key words: Drosophila melanogaster, mobile elements, transcription.

scholarly journals The Superantigen Gene ypm Is Located in an Unstable Chromosomal Locus of Yersinia pseudotuberculosis

2002 ◽  
Vol 184 (16) ◽  
pp. 4489-4499 ◽  
Author(s):  
Christophe Carnoy ◽  
Stephanie Floquet ◽  
Michael Marceau ◽  
Florent Sebbane ◽  
Stephanie Haentjens-Herwegh ◽  
...  
Keyword(s):  
Insertion Sequence ◽  
Yersinia Pseudotuberculosis ◽  
Gc Content ◽  
Mobile Genetic Elements ◽  
Open Reading Frames ◽  
Chromosomal Locus ◽  
Genetic Elements ◽  
Encoding Genes ◽  
Reading Frames

ABSTRACT Yersinia pseudotuberculosis produces YPM (Y. pseudotuberculosis-derived mitogen), a superantigenic toxin that exacerbates the virulence of the bacterium in vivo. To date, three alleles of the superantigen gene (ypmA, ypmB, and ypmC) have been described. These genes are not found in all Y. pseudotuberculosis strains and have a low GC content, suggesting their location on mobile genetic elements. To elucidate this question, the genetic environment of the superantigen-encoding genes was characterized and 11 open reading frames (ORFs) were defined. Sequence analysis revealed that the ypm genes were not associated with plasmids, phages, transposons, or pathogenicity islands and that the superantigen genes were always located in the chromosome between ORF3 and ORF4. Nonsuperantigenic strains exhibited the same genetic organization of the locus but lacked the ypm gene between ORF3 and ORF4. A new insertion sequence, designated IS1398, which displays features of the Tn3 family, was characterized downstream of the ypmA and ypmC genes. A 13.3-kb region containing the ypm genes was not found in the genome of Y. pestis (CO92 and KIM 5 strains). We experimentally induced deletion of the ypm gene from a superantigen-expressing Y. pseudotuberculosis: using the association of aph(3′)-IIIa and sacB genes, we demonstrated that when these reporter genes were present in the ypm locus, deletion of these genes was about 250 times more frequent than when they were located in another region of the Y. pseudotuberculosis chromosome. These results indicate that unlike other superantigenic toxin genes, the Yersinia ypm genes are not associated with mobile genetic elements but are inserted in an unstable locus of the genome.

scholarly journals Long-Term Persistence of Plasmids Targeted by CRISPR Interference in Bacterial Populations

2021 ◽  
Author(s):  
Viktor Mamontov ◽  
Alexander Martynov ◽  
Natalia Morozova ◽  
Anton Bukatin ◽  
Dmitry B. Staroverov ◽  
...  
Keyword(s):  
Complex Dynamics ◽  
Mobile Genetic Elements ◽  
Genetic Alterations ◽  
Common Mechanism ◽  
Type I ◽  
Bacterial Populations ◽  
Crispr Interference ◽  
Genetic Elements ◽  
Rapid Changes

CRISPR-Cas systems provide prokaryotes with an RNA-guided defense against foreign mobile genetic elements (MGEs) such as plasmids and viruses. A common mechanism by which MGEs avoid interference by CRISPR consists of acquisition of escape mutations in regions targeted by CRISPR. Here, using microbiological, live microscopy, and microfluidics analyses we demonstrated that plasmids can persist in Escherichia coli cells at conditions of continuous targeting by the type I-E CRISPR-Cas system without acquiring any genetic alterations. We used mathematical modeling to show how plasmid persistence in a subpopulation of cells mounting CRISPR interference is achieved due to the stochastic nature of CRISPR interference and plasmid replication events. We hypothesize that the observed complex dynamics provides bacterial populations with long-term benefits due to the presence of mobile genetic elements in some cells, leading to diversification of phenotypes in the entire community and allowing rapid changes in the population structure to meet the demands of a changing environment.

scholarly journals Chromosomal Toxin-Antitoxin Systems May Act as Antiaddiction Modules

2008 ◽  
Vol 190 (13) ◽  
pp. 4603-4609 ◽  
Author(s):  
Manuel Saavedra De Bast ◽  
Natacha Mine ◽  
Laurence Van Melderen
Keyword(s):  
Physiological Role ◽  
Mobile Genetic Elements ◽  
Selective Advantage ◽  
Vertical Inheritance ◽  
Genetic Elements ◽  
Daughter Cells ◽  
Fitness Advantage ◽  
Bacterial Chromosomes ◽  
Postsegregational Killing

ABSTRACT Toxin-antitoxin (TA) systems are widespread among bacterial chromosomes and mobile genetic elements. Although in plasmids TA systems have a clear role in their vertical inheritance by selectively killing plasmid-free daughter cells (postsegregational killing or addiction phenomenon), the physiological role of chromosomally encoded ones remains under debate. The assumption that chromosomally encoded TA systems are part of stress response networks and/or programmed cell death machinery has been called into question recently by the observation that none of the five canonical chromosomally encoded TA systems in the Escherichia coli chromosome seem to confer any selective advantage under stressful conditions (V. Tsilibaris, G. Maenhaut-Michel, N. Mine, and L. Van Melderen, J. Bacteriol. 189:6101-6108, 2007). Their prevalence in bacterial chromosomes indicates that they might have been acquired through horizontal gene transfer. Once integrated in chromosomes, they might in turn interfere with their homologues encoded by mobile genetic elements. In this work, we show that the chromosomally encoded Erwinia chrysanthemi ccd (control of cell death) (ccdEch ) system indeed protects the cell against postsegregational killing mediated by its F-plasmid ccd (ccd F) homologue. Moreover, competition experiments have shown that this system confers a fitness advantage under postsegregational conditions mediated by the ccd F system. We propose that ccdEch acts as an antiaddiction module and, more generally, that the integration of TA systems in bacterial chromosomes could drive the evolution of plasmid-encoded ones and select toxins that are no longer recognized by the antiaddiction module.

scholarly journals Potential for Isolation of Immortalized Hepatocyte Cell Lines by Liver-DirectedIn VivoGene Delivery of Transposons in Mice

2019 ◽  
Vol 2019 ◽  
pp. 1-12 ◽  
Author(s):  
Masahiro Sato ◽  
Issei Saitoh ◽  
Emi Inada ◽  
Shingo Nakamura ◽  
Satoshi Watanabe
Keyword(s):  
Nucleic Acids ◽  
Cell Lines ◽  
Mobile Genetic Elements ◽  
Isolated Hepatocytes ◽  
Proliferative Potential ◽  
Genetic Elements ◽  
Cellular Immortalization

Isolation of hepatocytes and their culturein vitrorepresent important avenues to explore the function of such cells. However, these studies are often difficult to perform because of the inability of hepatocytes to proliferatein vitro. Immortalization of isolated hepatocytes is thus an important step toward continuousin vitroculture. For cellular immortalization, integration of relevant genes into the host chromosomes is a prerequisite. Transposons, which are mobile genetic elements, are known to facilitate integration of genes of interest (GOI) into chromosomesin vitroandin vivo. Here, we proposed that a combination of transposon- and liver-directed introduction of nucleic acids may confer acquisition of unlimited cellular proliferative potential on hepatocytes, enabling the possible isolation of immortalized hepatocyte cell lines, which has often failed using more traditional immortalization methods.

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