Autonomous Replication of the Conjugative Transposon Tn916

ABSTRACTIntegrative and conjugative elements (ICEs), also known as conjugative transposons, are self-transferable elements that are widely distributed among bacterial phyla and are important drivers of horizontal gene transfer. Many ICEs carry genes that confer antibiotic resistances to their host cells and are involved in the dissemination of these resistance genes. ICEs reside in host chromosomes but under certain conditions can excise to form a plasmid that is typically the substrate for transfer. A few ICEs are known to undergo autonomous replication following activation. However, it is not clear if autonomous replication is a general property of many ICEs. We found that Tn916, the first conjugative transposon identified, replicates autonomously via a rolling-circle mechanism. Replication of Tn916was dependent on the relaxase encoded byorf20of Tn916. The origin of transfer of Tn916,oriT(916), also functioned as an origin of replication. Using immunoprecipitation and mass spectrometry, we found that the relaxase (Orf20) and the two putative helicase processivity factors (Orf22 and Orf23) encoded by Tn916likely interact in a complex and that the Tn916relaxase contains a previously unidentified conserved helix-turn-helix domain in its N-terminal region that is required for relaxase function and replication. Lastly, we identified a functional single-strand origin of replication (sso) in Tn916that we predict primes second-strand synthesis during rolling-circle replication. Together these results add to the emerging data that show that several ICEs replicate via a conserved, rolling-circle mechanism.IMPORTANCEIntegrative and conjugative elements (ICEs) drive horizontal gene transfer and the spread of antibiotic resistances in bacteria. ICEs reside integrated in a host genome but can excise to create a plasmid that is the substrate for transfer to other cells. Here we show that Tn916, an ICE with broad host range, undergoes autonomous rolling-circle replication when in the plasmid form. We found that the origin of transfer functions as a double-stranded origin of replication and identified a single-stranded origin of replication. It was long thought that ICEs do not undergo autonomous replication. Our work adds to the evidence that ICEs replicate autonomously as part of their normal life cycle and indicates that diverse ICEs use the same replicative mechanism.

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Demonstration of Conjugative Transposon (Tn5397)-Mediated Horizontal Gene Transfer between Clostridium difficile and Enterococcus faecalis

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00496-10 ◽

2010 ◽

Vol 54 (11) ◽

pp. 4924-4926 ◽

Cited By ~ 36

Author(s):

Azmiza S. Jasni ◽

Peter Mullany ◽

Haitham Hussain ◽

Adam P. Roberts

Keyword(s):

Gene Transfer ◽

Clostridium Difficile ◽

Horizontal Gene Transfer ◽

Enterococcus Faecalis ◽

Nosocomial Infections ◽

Conjugative Transposon ◽

Antibiotic Resistant ◽

Specific Target ◽

Ribotype 027 ◽

Target Sites

ABSTRACT Antibiotic-resistant Enterococcus faecalis and Clostridium difficile are responsible for nosocomial infections in humans, in which they inhabit the same niche. Here, we demonstrate transfer of the conjugative transposon Tn5397 from C. difficile 630 to E. faecalis JH2-2, the first reported gene transfer between these two bacteria. Furthermore, transfer from the E. faecalis EF20A transconjugant to the epidemic ribotype 027 C. difficile strain R20291 was also demonstrated. Tn5397 was shown to use a single specific target site in E. faecalis; it also has specific target sites in C. difficile. These experiments highlight the importance of continual monitoring for emerging resistances in these bacteria.

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Analysis of transcription of Porcine circovirus type 1

Journal of General Virology ◽

10.1099/0022-1317-83-11-2743 ◽

2002 ◽

Vol 83 (11) ◽

pp. 2743-2751 ◽

Cited By ~ 32

Author(s):

Annette Mankertz ◽

Bernd Hillenbrand

Keyword(s):

Infectious Clone ◽

Porcine Circovirus Type ◽

Open Reading Frames ◽

Porcine Circovirus ◽

Origin Of Replication ◽

Rolling Circle Replication ◽

Structural Protein ◽

Rolling Circle ◽

Rep Protein

Porcine circovirus type 1 (PCV1) contains two major open reading frames encoding the replication initiator proteins, Rep and Rep′, and the structural protein, Cap. The promoters of these two genes (P cap and P rep ) have been mapped. P cap is located within the rep open reading frame (nt 1328–1252). P rep has been mapped to the intergenic region immediately upstream of the rep gene (nt 640–796) and overlaps the origin of replication of PCV1. Although binding of both rep gene products to a fragment containing P rep and the overlapping origin of replication has been reported, only the full-length Rep protein repressed P rep , while the spliced isoform Rep′ did not. P rep repression is mediated by binding of the Rep protein to the two inner hexamers, H1 and H2, located in the origin of PCV1, whereas binding of Rep to hexamers H3 and H4 was not necessary. Use of Rep mutants indicated that the conserved rolling-circle replication domain II as well as the P loop are essential for repression of P rep . In contrast to P rep , transcription of P cap was not influenced by viral proteins. Additionally, the ratio of the rep and rep′ transcripts was analysed. Twelve hours after transfection of PK15 cells with an infectious clone of PCV1, similar amounts of both transcripts were detected, but later the amount of the two transcripts varied, indicating a balanced expression of the two rep transcripts.

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ISCR Elements: Novel Gene-Capturing Systems of the 21st Century?

Microbiology and Molecular Biology Reviews ◽

10.1128/mmbr.00048-05 ◽

2006 ◽

Vol 70 (2) ◽

pp. 296-316 ◽

Cited By ~ 377

Author(s):

Mark A. Toleman ◽

Peter M. Bennett ◽

Timothy R. Walsh

Keyword(s):

Antibiotic Resistance ◽

Resistance Genes ◽

Antibiotic Resistance Genes ◽

Origin Of Replication ◽

Rolling Circle Replication ◽

Antibiotic Resistant ◽

Rolling Circle ◽

Genetic Mechanisms ◽

Genetic Context ◽

Accurate Reflection

SUMMARY “Common regions” (CRs), such as Orf513, are being increasingly linked to mega-antibiotic-resistant regions. While their overall nucleotide sequences show little identity to other mobile elements, amino acid alignments indicate that they possess the key motifs of IS91-like elements, which have been linked to the mobility ent plasmids in pathogenic Escherichia coli. Further inspection reveals that they possess an IS91-like origin of replication and termination sites (terIS), and therefore CRs probably transpose via a rolling-circle replication mechanism. Accordingly, in this review we have renamed CRs as ISCRs to give a more accurate reflection of their functional properties. The genetic context surrounding ISCRs indicates that they can procure 5′ sequences via misreading of the cognate terIS, i.e., “unchecked transposition.” Clinically, the most worrying aspect of ISCRs is that they are increasingly being linked with more potent examples of resistance, i.e., metallo-β-lactamases in Pseudomonas aeruginosa and co-trimoxazole resistance in Stenotrophomonas maltophilia. Furthermore, if ISCR elements do move via “unchecked RC transposition,” as has been speculated for ISCR1, then this mechanism provides antibiotic resistance genes with a highly mobile genetic vehicle that could greatly exceed the effects of previously reported mobile genetic mechanisms. It has been hypothesized that bacteria will surprise us by extending their “genetic construction kit” to procure and evince additional DNA and, therefore, antibiotic resistance genes. It appears that ISCR elements have now firmly established themselves within that regimen.

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Plasmid Characteristics Modulate the Propensity of Gene Exchange in Bacterial Vesicles

Journal of Bacteriology ◽

10.1128/jb.00430-18 ◽

2019 ◽

Vol 201 (7) ◽

Cited By ~ 4

Author(s):

Frances Tran ◽

James Q. Boedicker

Keyword(s):

Gene Transfer ◽

Horizontal Gene Transfer ◽

Extracellular Vesicles ◽

Copy Number ◽

Genetic Material ◽

Plasmid Copy Number ◽

Gene Exchange ◽

Origin Of Replication ◽

Plasmid Copy ◽

The Impact

ABSTRACTHorizontal gene transfer is responsible for the exchange of many types of genetic elements, including plasmids. Properties of the exchanged genetic element are known to influence the efficiency of transfer via the mechanisms of conjugation, transduction, and transformation. Recently, an alternative general pathway of horizontal gene transfer has been identified, namely, gene exchange by extracellular vesicles. Although extracellular vesicles have been shown to facilitate the exchange of several types of plasmids, the influence of plasmid characteristics on genetic exchange within vesicles is unclear. Here, a set of different plasmids was constructed to systematically test the impact of plasmid properties, specifically, plasmid copy number, size, and origin of replication, on gene transfer in vesicles. The influence of each property on the production, packaging, and uptake of vesicles containing bacterial plasmids was quantified, revealing how plasmid properties modulate vesicle-mediated horizontal gene transfer. The loading of plasmids into vesicles correlates with the plasmid copy number and is influenced by characteristics that help set the number of plasmids within a cell, including size and origin of replication. Plasmid origin also has a separate impact on both vesicle loading and uptake, demonstrating that the origin of replication is a major determinant of the propensity of specific plasmids to transfer within extracellular vesicles.IMPORTANCEExtracellular vesicle formation and exchange are common within bacterial populations. Vesicles package multiple types of biomolecules, including genetic material. The exchange of extracellular vesicles containing genetic material facilitates interspecies DNA transfer and may be a promiscuous mechanism of horizontal gene transfer. Unlike other mechanisms of horizontal gene transfer, it is unclear whether characteristics of the exchanged DNA impact the likelihood of transfer in vesicles. Here, we systematically examine the influence of plasmid copy number, size, and origin of replication on the loading of DNA into vesicles and the uptake of DNA containing vesicles by recipient cells. These results reveal how each plasmid characteristic impacts gene transfer in vesicles and contribute to a greater understanding of the importance of vesicle-mediated gene exchange in the landscape of horizontal gene transfer.

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Massive Gene Flux Drives Genome Diversity between Sympatric Streptomyces Conspecifics

mBio ◽

10.1128/mbio.01533-19 ◽

2019 ◽

Vol 10 (5) ◽

Cited By ~ 10

Author(s):

Abdoul-Razak Tidjani ◽

Jean-Noël Lorenzi ◽

Maxime Toussaint ◽

Erwin van Dijk ◽

Delphine Naquin ◽

...

Keyword(s):

Gene Transfer ◽

Horizontal Gene Transfer ◽

Public Goods ◽

Significant Proportion ◽

Gene Clusters ◽

Recent Common Ancestor ◽

Natural Soil ◽

Gene Pools ◽

Bacterial Gene ◽

Integrative And Conjugative Elements

ABSTRACT In this work, by comparing genomes of closely related individuals of Streptomyces isolated at a spatial microscale (millimeters or centimeters), we investigated the extent and impact of horizontal gene transfer in the diversification of a natural Streptomyces population. We show that despite these conspecific strains sharing a recent common ancestor, all harbored significantly different gene contents, implying massive and rapid gene flux. The accessory genome of the strains was distributed across insertion/deletion events (indels) ranging from one to several hundreds of genes. Indels were preferentially located in the arms of the linear chromosomes (ca. 12 Mb) and appeared to form recombination hot spots. Some of them harbored biosynthetic gene clusters (BGCs) whose products confer an inhibitory capacity and may constitute public goods that can favor the cohesiveness of the bacterial population. Moreover, a significant proportion of these variable genes were either plasmid borne or harbored signatures of actinomycete integrative and conjugative elements (AICEs). We propose that conjugation is the main driver for the indel flux and diversity in Streptomyces populations. IMPORTANCE Horizontal gene transfer is a rapid and efficient way to diversify bacterial gene pools. Currently, little is known about this gene flux within natural soil populations. Using comparative genomics of Streptomyces strains belonging to the same species and isolated at microscale, we reveal frequent transfer of a significant fraction of the pangenome. We show that it occurs at a time scale enabling the population to diversify and to cope with its changing environment, notably, through the production of public goods.

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A Bacillus anthracis-Based In Vitro System Supports Replication of Plasmid pXO2 as Well as Rolling-Circle-Replicating Plasmids

Applied and Environmental Microbiology ◽

10.1128/aem.00240-07 ◽

2007 ◽

Vol 73 (15) ◽

pp. 5005-5010 ◽

Cited By ~ 4

Author(s):

Eowyn Tinsley ◽

Saleem A. Khan

Keyword(s):

Bacillus Anthracis ◽

Virulence Plasmid ◽

Origin Of Replication ◽

Free System ◽

In Vitro System ◽

Rolling Circle ◽

A Cell ◽

Rolling Circle Mechanism ◽

Plasmid Pt181

ABSTRACT Capsule-encoding virulence plasmid pXO2 of Bacillus anthracis is predicted to replicate by a unidirectional theta-type mechanism. To gain a better understanding of the mechanism of replication of pXO2 and other plasmids in B. anthracis and related organisms, we have developed a cell-free system based on B. anthracis that can faithfully replicate plasmid DNA in vitro. The newly developed system was shown to support the in vitro replication of plasmid pT181, which replicates by the rolling-circle mechanism. We also demonstrate that this system supports the replication of plasmid pXO2 of B. anthracis. Replication of pXO2 required directional transcription through the plasmid origin of replication, and increased transcription through the origin resulted in an increase in plasmid replication.

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Why are rhizobial symbiosis genes mobile?

Philosophical Transactions of the Royal Society B Biological Sciences ◽

10.1098/rstb.2020.0471 ◽

2021 ◽

Vol 377 (1842) ◽

Cited By ~ 1

Author(s):

Grace E. Wardell ◽

Michael F. Hynes ◽

Peter J. Young ◽

Ellie Harrison

Keyword(s):

Gene Transfer ◽

Horizontal Gene Transfer ◽

Complex Trait ◽

Mobile Genetic Elements ◽

Host Bacterium ◽

Genetic Elements ◽

Integrative And Conjugative Elements ◽

Legume Symbiosis ◽

Bacterial Genes ◽

Intracellular Infections

Rhizobia are one of the most important and best studied groups of bacterial symbionts. They are defined by their ability to establish nitrogen-fixing intracellular infections within plant hosts. One surprising feature of this symbiosis is that the bacterial genes required for this complex trait are not fixed within the chromosome, but are encoded on mobile genetic elements (MGEs), namely plasmids or integrative and conjugative elements. Evidence suggests that many of these elements are actively mobilizing within rhizobial populations, suggesting that regular symbiosis gene transfer is part of the ecology of rhizobial symbionts. At first glance, this is counterintuitive. The symbiosis trait is highly complex, multipartite and tightly coevolved with the legume hosts, while transfer of genes can be costly and disrupt coadaptation between the chromosome and the symbiosis genes. However, horizontal gene transfer is a process driven not only by the interests of the host bacterium, but also, and perhaps predominantly, by the interests of the MGEs that facilitate it. Thus understanding the role of horizontal gene transfer in the rhizobium–legume symbiosis requires a ‘mobile genetic element's-eye view' on the ecology and evolution of this important symbiosis. This article is part of the theme issue ‘The secret lives of microbial mobile genetic elements’.

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The Arcanobacterium(Actinomyces) pyogenes Plasmid pAP1 Is a Member of the pIJ101/pJV1 Family of Rolling Circle Replication Plasmids

Journal of Bacteriology ◽

10.1128/jb.180.12.3233-3236.1998 ◽

1998 ◽

Vol 180 (12) ◽

pp. 3233-3236 ◽

Cited By ~ 19

Author(s):

Stephen J. Billington ◽

B. Helen Jost ◽

J. Glenn Songer

Keyword(s):

Escherichia Coli ◽

Resistance Gene ◽

Sequence Similarity ◽

Kanamycin Resistance ◽

Corynebacterium Pseudotuberculosis ◽

Rolling Circle Replication ◽

Kanamycin Resistance Gene ◽

Rolling Circle ◽

Rolling Circle Mechanism ◽

Actinomyces Pyogenes

ABSTRACT The 2.4-kb plasmid pAP1 from Arcanobacterium(Actinomyces) pyogenes had sequence similarity within the putative replication protein and double-stranded origin with the pIJ101/pJV1 family of plasmids. pJGS84, a derivative of pAP1 containing a kanamycin resistance gene, was able to replicate inEscherichia coli and Corynebacterium pseudotuberculosis, as well as in A. pyogenes. Detection of single-stranded DNA intermediates of pJGS84 replication suggested that this plasmid replicates by the rolling circle mechanism.

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