Tra Proteins Characteristic of F-Like Type IV Secretion Systems Constitute an Interaction Group by Yeast Two-Hybrid Analysis

Harris, Richard C.; Silverman, P.

doi:10.1128/jb.186.16.5480-5485.2004

Cited by 44 publications

(72 citation statements)

References 38 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…There has been speculation that TraG might form a transenvelope bridge and contact TraN in the outer membrane (Firth & Skurray, 1992); however, this interaction has not been detected using cross-linking (Klimke et al, 2005). We are currently exploring the possibility that TraG interacts with TraU, which is a putative Mps protein, and is known to be part of an interaction complex that is involved in pilus synthesis, but is not itself required for this process (Moore et al, 1990;Harris & Silverman, 2004). …”

Section: Discussionmentioning

confidence: 99%

Entry exclusion in F-like plasmids requires intact TraG in the donor that recognizes its cognate TraS in the recipient

et al. 2007

View full text Add to dashboard Cite

Section: Discussionmentioning

confidence: 99%

Entry exclusion in F-like plasmids requires intact TraG in the donor that recognizes its cognate TraS in the recipient

et al. 2007

View full text Add to dashboard Cite

“…TraC ATPase function appears to be essential because a mutation in the TraC Walker A box abolished F-pilus formation (Harris and Silverman, unpublished data). An extension/retraction switch at the base of individual F-pili (30) could regulate TraC ATPase activity in such a way that the protein is functionally equivalent to both PilF and PilT. Alternatively, retraction might not require ATP hydrolysis at all (15).…”

Section: Discussionmentioning

confidence: 99%

F-pili dynamics by live-cell imaging

Clarke

Maddera

Harris

et al. 2008

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

Self Cite

130

139

View full text Add to dashboard Cite

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...

show abstract

“…Therefore, TraF F and TrhF R27 may act as chaperones during pilus assembly. Yeast two-hybrid studies have indicated that TraF F interacts with TraH F (six cysteines) (31), which has a C-terminal coiled-coil domain (42), making it an excellent candidate for chaperone-assisted assembly in the periplasm.…”

Section: Discussionmentioning

confidence: 99%

“…Proteins with no cysteines are emphasized in white. The possible interactions between the proteins are summarized according to Harris and Silverman (31). White arrows indicate inferred interactions from sequence data for TrbC and TraW, which are fused into one protein in R27 (42).…”

Section: Discussionmentioning

confidence: 99%

F-Like Type IV Secretion Systems Encode Proteins with Thioredoxin Folds That Are Putative DsbC Homologues

Elton

Holland²,

Frost³

et al. 2005

J Bacteriol

View full text Add to dashboard Cite

F and R27 are conjugative plasmids of enteric bacteria belonging to the IncF and IncHI1 plasmid incompatibility groups, respectively. Based on sequence analysis, two genes of the F transfer region, traF and trbB, and three genes of the R27 transfer region, trhF, dsbC, and htdT, are predicted to encode periplasmic proteins containing a C-terminal thioredoxin fold. The C-X-X-C active-site motif of thioredoxins is present in all of these proteins except TraF F . Escherichia coli carrying a dsbA mutation, which is deficient in disulfide bond formation, cannot synthesize pili and exhibits hypersensitivity to dithiothreitol (DTT) as monitored by mating ability. Overproduction of the E. coli disulfide bond isomerase DsbC, TrbB F , DsbC R27 , or HtdT R27 , but not TraF F or TrhF R27 , reverses this hypersensitivity to DTT. Site-directed mutagenesis established that the C-X-X-C motif was necessary for this activity. Secretion into the periplasm of the C-terminal regions of TrbB F and DsbC R27 , containing putative thioredoxin folds, but not TrhF R27 , partially complemented the host dsbA mutation. A trbB F deletion mutant showed a 10-fold-lower mating efficiency in an E. coli dsbC null strain but had no phenotype in wild-type E. coli, suggesting redundancy in function between TrbB F and E. coli DsbC. Our results indicate that TrbB F , DsbC R27 , and HtdT R27 are putative disulfide bond isomerases for their respective transfer systems. TraF F is essential for conjugation but appears to have a function other than disulfide bond chemistry.

show abstract

Tra Proteins Characteristic of F-Like Type IV Secretion Systems Constitute an Interaction Group by Yeast Two-Hybrid Analysis

Cited by 44 publications

References 38 publications

Entry exclusion in F-like plasmids requires intact TraG in the donor that recognizes its cognate TraS in the recipient

Entry exclusion in F-like plasmids requires intact TraG in the donor that recognizes its cognate TraS in the recipient

F-pili dynamics by live-cell imaging

F-Like Type IV Secretion Systems Encode Proteins with Thioredoxin Folds That Are Putative DsbC Homologues

Contact Info

Product

Resources

About