Bacterial conjugative transfer: visualization of successful mating pairs and plasmid establishment in live <i>Escherichia coli</i>

Lawley, Trevor D.; Gordon, G. Scott; Wright, Andrew; Taylor, Diane E.

doi:10.1046/j.1365-2958.2002.02938.x

Cited by 43 publications

(44 citation statements)

References 30 publications

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“…It is too early to conclude whether this behavior is general for horizontal gene transfer or specific for ICEs from the ICEclc family, as few other conjugative systems have been studied at the single-cell level. Studies on F and R751 plasmid transfer from Escherichia coli (33,34) and ICEBs1 transfer in B. subtilis (32) did not particularly describe phenotypic variability among donor cells.…”

Section: Discussionmentioning

confidence: 99%

“…Our starting hypothesis based on previous results (25) was that tc donor cells, when they have been initiated, would efficiently deliver the ICEclc at a high rate of successful transfer. Single-cell studies have been previously used to follow plasmid conjugation in which case donor cell fates have not been particularly addressed (33)(34)(35), but because ICEs behave very differently, those results are not immediately adaptable. Hence, to better study and understand donor cell fates, we develop a fluorescence reporter that distinguishes the four key steps of ICE transmission: activation, excision, actual transfer, and integration into new recipients.…”

mentioning

confidence: 99%

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

Delavat

Mitri

Pelet

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

106

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Horizontal gene transfer is an important evolutionary mechanism for bacterial adaptation. However, given the typical low transfer frequencies in a bacterial population, little is known about the fate and interplay of donor cells and the mobilized DNA during transfer. Here we study transfer of an integrative and conjugative element (ICE) among individual live bacterial cells. ICEs are widely distributed mobile DNA elements that are different than plasmids because they reside silent in the host chromosome and are maintained through vertical descent. Occasionally, ICEs become active, excise, and transmit their DNA to a new recipient, where it is reintegrated. We develop a fluorescent tool to differentiate excision, transfer, and reintegration of a model ICE named ICEclc (for carrying the clc genes for chlorocatechol metabolism) among single Pseudomonas cells by using timelapse microscopy. We find that ICEclc activation is initiated in stationary phase cells, but excision and transfer predominantly occur only when such cells have been presented with new nutrients. Donors with activated ICE develop a number of different states, characterized by reduced cell division rates or growth arrest, persistence, or lysis, concomitant with ICE excision, and likely, ICE loss or replication. The donor cell state transitions can be described by using a stochastic model, which predicts that ICE fitness is optimal at low initiation rates in stationary phase. Despite highly variable donor cell fates, ICE transfer is remarkably robust overall, with 75% success after excision. Our results help to better understand ICE behavior and shed a new light on bacterial cellular differentiation during horizontal gene transfer.

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Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

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

Delavat

Mitri

Pelet

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

106

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“…donor-recipient interactions: whereas bacterial mating pairs have been observed to form in all possible relative positions (side-to-side, pole-to-pole, side-to-pole, and pole-to-side of donor and recipient, respectively) (Lawley et al, 2002), the attachment of A. tumefaciens to plant cells seems to occur mainly in a polar manner (Matthysse, 1987). Sex pili are composed of a small subunit, the pilin (VirB2), which assembles into a Wlamentous pilus structure at the cell surface (Eisenbrandt et al, 1999;Lai and Kado, 1998).…”

Section: The Foreplay Of Bacterial Sex: Mating Pair Formationmentioning

confidence: 99%

The mating pair formation system of conjugative plasmids—A versatile secretion machinery for transfer of proteins and DNA

Schröder

Lanka

2005

Plasmid

188

146

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The mating pair formation (Mpf) system functions as a secretion machinery for intercellular DNA transfer during bacterial conjugation. The components of the Mpf system, comprising a minimal set of 10 conserved proteins, form a membrane-spanning protein complex and a surface-exposed sex pilus, which both serve to establish intimate physical contacts with a recipient bacterium. To function as a DNA secretion apparatus the Mpf complex additionally requires the coupling protein (CP). The CP interacts with the DNA substrate and couples it to the secretion pore formed by the Mpf system. Mpf/CP conjugation systems belong to the family of type IV secretion systems (T4SS), which also includes DNA-uptake and -release systems, as well as eVector protein translocation systems of bacterial pathogens such as Agrobacterium tumefaciens (VirB/VirD4) and Helicobacter pylori (Cag). The increased eVorts to unravel the molecular mechanisms of type IV secretion have largely advanced our current understanding of the Mpf/CP system of bacterial conjugation systems. It has become apparent that proteins coupled to DNA rather than DNA itself are the actively transported substrates during bacterial conjugation. We here present a uniWed and updated view of the functioning and the molecular architecture of the Mpf/CP machinery. 

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“…F-pilus retraction as an essential stage of DNA transfer was first proposed by Marvin and Hohn (12) and by Curtiss (13). Various lines of indirect evidence since then have supported the retraction hypothesis (14)(15)(16)(17)(18), although fundamental uncertainties remain. For example, retraction was initially regarded as a triggered event, occurring only when a recipient cell or bacteriophage bound to the filament tip (12).…”

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confidence: 99%

F-pili dynamics by live-cell imaging

Clarke

Maddera

Harris

et al. 2008

Proc. Natl. Acad. Sci. U.S.A.

130

139

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Bacteria have evolved numerous mechanisms for cell-cell communication, many of which have important consequences for human health. Among these is conjugation, the direct transfer of DNA from one cell to another. For Gram-negative bacteria, conjugation requires thin, flexible filaments (conjugative pili) that are elaborated by DNA donor cells. The structure, function, and especially the dynamics of conjugative pili are poorly understood. Here, we have applied live-cell imaging to characterize the dynamics of F-pili (conjugative pili encoded by the F plasmid of Escherichia coli). We establish that F-pili normally undergo cycles of extension and retraction in the absence of any obvious triggering event, such as contact with a recipient cell. When made, such contacts are able to survive the shear forces felt by bacteria in liquid media. Our data emphasize the role of F-pilus flexibility both in efficiently sampling a large volume surrounding donor cells in liquid culture and in establishing and maintaining cell-cell contact. Additionally and unexpectedly, we infer that extension and retraction are accompanied by rotation about the long axis of the filament.conjugation ͉ conjugative pilus ͉ extension ͉ retraction A mong Gram-negative bacteria, conjugative DNA transfer is mediated by multicomponent type IV secretion systems commonly encoded by large plasmids (1, 2). By virtue of such activity, these plasmids rapidly spread within bacterial populations, contributing to the dissemination of antibiotic resistance (3) among other traits pertinent to human health (2, 4-6).A hallmark of Gram-negative bacterial cells capable of acting as DNA donors is the presence of surface filaments collectively designated conjugative pili (7). F-pili, characteristic of Escherichia coli carrying the conjugative plasmid F, are helical polymers of one subunit, F-pilin (8). Conjugation commences when the tips of F-pili make contact with recipient cells (9, 10). F-pili tips also bind filamentous DNA bacteriophages, such as M13, whereas icosohedral RNA bacteriophages such as R17 bind along the filament sides (11).Indirect evidence has suggested that F-pili are dynamic structures, capable of both extension and retraction. F-pilus retraction as an essential stage of DNA transfer was first proposed by Marvin and Hohn (12) and by Curtiss (13). Various lines of indirect evidence since then have supported the retraction hypothesis (14-18), although fundamental uncertainties remain. For example, retraction was initially regarded as a triggered event, occurring only when a recipient cell or bacteriophage bound to the filament tip (12). Later it was proposed that F-pili normally undergo cycles of extension and retraction (14,17,19). The idea that DNA can be conducted from donor to recipient through extended F-pili (10, 20) has also received experimental support (21,22).Here, we have applied laser-scanning confocal microscopy to study F-pilus dynamics with living cells. Our results clarify some of the uncertainties surrounding F-pilus function and sugge...

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Bacterial conjugative transfer: visualization of successful mating pairs and plasmid establishment in live Escherichia coli

Cited by 43 publications

References 30 publications

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

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

The mating pair formation system of conjugative plasmids—A versatile secretion machinery for transfer of proteins and DNA

F-pili dynamics by live-cell imaging

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