The F pilus of Escherichia coli appears to support stable DNA transfer in the absence of wall-to-wall contact between cells

Harrington, L.; Rogerson, Allen C.

doi:10.1128/jb.172.12.7263-7264.1990

Cited by 60 publications

(40 citation statements)

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“…1 whereas conjugative systems lacking retractable pili function well only on solid surfaces (7,27). DNA transfer from F ϩ cells also occurs on solid and even in liquid media without stabilization, albeit at a very much reduced frequency (21,22,28).…”

Section: Discussionmentioning

confidence: 99%

“…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 suggest a hypothesis for F-pilus function that emphasizes the mechanical properties of the filament.…”

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confidence: 99%

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F-pili dynamics by live-cell imaging

Clarke

Maddera

Harris

et al. 2008

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

131

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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Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

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F-pili dynamics by live-cell imaging

Clarke

Maddera

Harris

et al. 2008

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

131

139

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“…However, it has been suggested that F-plasmid transfer can occur through the pilus between donor and recipient cells held at a distance from one another (6,12). In addition, the demonstration that mating could occur between organisms with very different cell envelopes suggested that the establishment of a productive mating pair need not require tight junction formation.…”

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The Tra2 core of the IncP(alpha) plasmid RP4 is required for intergeneric mating between Escherichia coli and Streptomyces lividans

et al. 1996

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Escherichia coli cells and Streptomyces mycelia are able to form close contacts in the absence of a conjugative system which might facilitate intergeneric plasmid transfer without the genes required for mating pair formation (Tra2) of the RP4 plasmid. The same Tra2 genes found to be essential for RP4 plasmid transfer, RSF1010 mobilization, and donor-specific phage propagation in E. coli were also required for intergeneric transfer between E. coli and Streptomyces lividans.Bacterial conjugation, the horizontal transmission of genetic material, usually requires close contact between donor and recipient cells, as demonstrated for F-plasmid-mediated transfer (4). However, it has been suggested that F-plasmid transfer can occur through the pilus between donor and recipient cells held at a distance from one another (6, 12). In addition, the demonstration that mating could occur between organisms with very different cell envelopes suggested that the establishment of a productive mating pair need not require tight junction formation.Following reports that RP4-mediated intergeneric plasmid transfer was possible between Escherichia coli and Streptomyces lividans (11), we wished to investigate whether the genes for pilus assembly were required for this event. The RP4 pilus, unlike its F counterpart, is rarely found attached to the cell surface, suggesting that it has no role in initiating contact between cells (5). However, E. coli and S. lividans can form close contacts in the absence of a known conjugation system in either cell (see below). Thus, plasmid transfer might require only the genes for DNA metabolism (Tra1) during conjugation. The requirement of genes for pilus formation (Tra2) would be evidence supporting an important role for the pilus in DNA transfer.Adhesion between E. coli and S. lividans. Examination of mixtures of E. coli and S. lividans with a scanning electron microscope revealed that E. coli was closely associated with the mycelial surface, usually via the pole of the E. coli cell. This phenomenon was observed with E. coli S17-1 (14) and S. lividans 1326 (7) (Fig. 1A) as well as other strains of E. coli and S. lividans (data not shown). The presence of a conjugative system in either strain did not have an effect on the ability of the cells to adhere to one another. Further examination by transmission electron microscopy of samples prepared by cryofixation (4) revealed a defined structure between the two cells (Fig. 1B) which resembled the F-plasmid-mediated conjugative junctions visualized in thin sections of mating E. coli cells (4). We were intrigued by these close contacts and wondered if they could substitute for the functions that encode mating pair formation in RP4 during intergeneric plasmid transfer from E. coli to S. lividans.Mobilization of pSET152 from E. coli to S. lividans. Intergeneric transfer of pSET152 (contains the multiple cloning site and replicon of pUC plasmids, the origin of transfer [oriT] of RK2, int C31 of the Streptomyces phage C31, and the apramycin resistance marker which ...

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“…The process of retraction is controversial and is based on electron microscopic examination of cells during mating pair formation and filamentous phage (f1, M13, or fd) infection. In addition, a second role for the F pilus involving the actual transfer of DNA has been proposed on the basis of evidence of DNA transfer between cells separated physically from one another but connected by a pilus (11). This latter function for the F pilus is in agreement with genetic analysis of the F tra region, since mutations in genes that affect pilus synthesis block DNA transfer while mutations in the recipient cell which decrease mating pair formation have only a modest effect (2 logs) on transfer efficiency.…”

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Analysis of the traLEKBP sequence and the TraP protein from three F-like plasmids: F, R100-1 and ColB2

et al. 1996

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The sequence of a region of the F plasmid containing the traLEKBP genes involved in plasmid transfer was compared to the equivalent regions of two IncFII plasmids, R100-1 and ColB2. The traLEK gene products of all three plasmids were virtually identical, with the most changes occurring in TraE. The traB genes were also nearly identical except for an 11-codon extension at the 3 end of the R100-1 traB gene. The TraP protein of R100-1 differed from those of F and ColB2 at its N terminus, while the ColB2 TraP protein contained a change of sequence in a predicted loop which was shown to be exposed in the periplasmic space by TnphoA mutagenesis. The effect of the altered TraP sequences was determined by complementing a traP mutant with clones expressing the traKBP genes of F, R100-1, and ColB2. The traP mutation in pOX38 (pOX38-traP474), a derivative of F, was found to have little effect on pilus production, pilus retraction, and filamentous phage growth and only a moderate effect on transfer. The transfer ability of pOX38-traP474 was shown to be affected by mutations in the rfa (lipopolysaccharide) locus and in ompA in the recipient cell in a manner similar to that for the wild-type pOX38-Km plasmid itself and could be complemented with the traP analogs from R100-1 and ColB2 to give an F-like phenotype. Thus, the TraP protein appears to play a minor role in conjugation and may interact with TraB, which varies in sequence along with TraP, in order to stabilize the proposed transmembrane complex formed by the tra operon products.

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The F pilus of Escherichia coli appears to support stable DNA transfer in the absence of wall-to-wall contact between cells

Abstract: Separation of HfrC-F- mating pairs of Escherichia coli by a filter 6 microns thick with straight-through pores 0.01 to 0.1 micron in diameter did not prevent DNA transfer. We conclude that the F pilus alone is capable of acting as a stable conduit for cell-to-cell DNA transfer.

Cited by 60 publications

References 5 publications

F-pili dynamics by live-cell imaging

F-pili dynamics by live-cell imaging

The Tra2 core of the IncP(alpha) plasmid RP4 is required for intergeneric mating between Escherichia coli and Streptomyces lividans

Analysis of the traLEKBP sequence and the TraP protein from three F-like plasmids: F, R100-1 and ColB2

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