Highly variable individual donor cell fates characterize robust horizontal gene transfer of an integrative and conjugative element

Delavat, François; Mitri, Sara; Pelet, Serge; Meer, Jan Roelof van der

doi:10.1073/pnas.1604479113

Cited by 44 publications

(112 citation statements)

References 47 publications

(93 reference statements)

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“…For example, when activated, ICEclc, an ICE active in Pseudomonas species, can inhibit host cell growth (ϳ50% of activated cells stop dividing) and cause cell lysis (77,82). Despite the damage incurred by host cells, 75% of donors with excised ICEclc that contact a recipient cell successfully transfer the element (82). Single-cell microscopy studies, such as those conducted on ICEclc, are required to assess the effect of Tn916 induction on host cell fate.…”

Section: Discussionmentioning

confidence: 99%

Autonomous Replication of the Conjugative Transposon Tn916

2016

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Integrative 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 Tn916 was dependent on the relaxase encoded by orf20 of 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 Tn916 likely interact in a complex and that the Tn916 relaxase 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 Tn916 that 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. IMPORTANCE Integrative 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. Integrative and conjugative elements (ICEs), also called conjugative transposons, are mobile genetic elements that encode proteins that mediate transfer of the element from the host cell (donor) to a recipient by conjugation. ICEs often contain additional (cargo) genes that can provide a selective advantage to the host cells (reviewed in references 1 and 2). Most ICEs have been identified based on their cargo genes and the phenotypes conferred. For example, many ICEs carry genes encoding antibiotic resistances. The horizontal dissemination of ICEs and their associated cargo genes is a major driver of bacterial genome plasticity and evolution and the s...

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Section: Discussionmentioning

confidence: 99%

Autonomous Replication of the Conjugative Transposon Tn916

2016

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“…Previous experimental data suggested that the tc cells indeed do not return to a silent ICE clc state, but become irreparably damaged, arrest their division and wither [13]. However, because their number is proportionally low, there is no fitness cost on the population carrying the ICE [48,49]. The advantage of prolonged feedback loop output is that constant levels of the BisCD regulator can be maintained, allowing coordinated and organized production of the components necessary for the ICE clc transfer itself.…”

Section: Discussionmentioning

confidence: 99%

“…Up to ten images at different positions were acquired using Visiview software (Visitron systems GMbH), with exposures set to 40 ms, 500 ms and 500 ms for phase contrast (PhC), eGFP and eCherry, respectively. Cells were automatically segmented on image sets using pipelines described previously [12,48], from which their fluorescence (either eGFP or eCherry, or both) was quantified. Subpopulations of transfer competent cells were quantified using quantile-quantile-plotting as described previously [12,65].…”

Section: Methodsmentioning

confidence: 99%

A four-step regulatory cascade controls bistable transfer competence development of the integrative and conjugative element ICEclcinPseudomonas

Carraro

Richard

Sulser

et al. 2019

Preprint

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Genetic bistability controls different phenotypic programs in defined subpopulations of genetically identical bacteria. Conjugative transfer of the integrative and conjugative element ICEclc in Pseudomonas requires development of a transfer competence state in stationary phase, but this state arises only in 3-5% of individual cells. The mechanisms controlling and underlying the bistable switch between non-active and transfer competence cells have long remained enigmatic. Using a variety of genetic tools combined with stochastic modeling, we characterize here the factors and overall network architecture controlling bistable ICEclc activation of transfer competence. Two new key regulators (BisR and BisDC) were uncovered, that link the hierarchical cascade of ICEclc transfer competence activation to in total four regulatory nodes. The final activator complex named BisDC drives a positive feedback on its own transcription, and directly controls the “late” ICE promoters for excision and transfer. Stochastic mathematical modeling conceptually explained the arisal and maintenance of bistability by the feedback loop, and demonstrated its importance to guarantee consistent prolonged downstream output in activated cells. A minimized gene set allowing controllable bistable output in a Pseudomonas putida in absence of the ICEclc largely confirmed model predictions. Phylogenetic analyses further showed that the two new ICEclc regulatory factors are widespread among putative ICEs found in Gamma- and Beta- proteobacteria, highlighting the conceptual importance of our findings for the behaviour of this wide family of conjugative elements.Author summaryIntegrative and conjugative elements (ICEs) are mobile genetic elements present in virtually every bacterial species, which can confer adaptive functions to their host, such as antibiotic resistance or xenometabolic pathways. Integrated ICEs maintain by replication along with the genome of their bacterial host, but in order to transfer, the ICE excises and conjugates into a new recipient cell. Single-cell studies on a unique but widely representative ICE model from Pseudomonas (ICEclc) showed that transfer only occurs from a small dedicated subpopulation of cells that arises during stationary phase conditions. This bistable subpopulation differentiation is highly significant for ICE behaviour and fitness, but how it is regulated has remained largely unknown. The present work unveiled the architecture of the ICEclc transfer competence regulation, and showed its widespread occurrence among ICEs of the same family. Stochastic mathematical modeling explained how bistability is generated and maintained, prolonging the capacity of stationary phase cells to complete all stages of ICE activation.

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“…[19] In this case they used clever tricks to indirectly visualize the excision, transfer and re-integration of a mobile genetic element from the chromosome of a donor cell into the chromosome of a recipient cell. By tracing each cell's history by imaging the transfer of the genetic element, they define the donor cell by reduced cell division rates or growth arrest, persistence, or lysis after the transfer has started.…”

Section: Virologymentioning

confidence: 99%