Functional role of conserved residues in the characteristic secretion NTPase motifs of the Pseudomonas aeruginosa type IV pilus motor proteins PilB, PilT and PilU

Chiang, Poney; Sampaleanu, L.M.; Ayers, M.; Pahuta, Markian; Howell, P. Lynne; Burrows, Lori L.

doi:10.1099/mic.0.2007/011320-0

Cited by 114 publications

(111 citation statements)

References 46 publications

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

“…Second, type IV pili extend and retract at rates approaching 1 m/sec (25,26), much faster than F-pili. Finally, the energy sources for type IV pilus dynamics are hexameric ring ATPases, one for extension (PilF) and a different one for retraction (PilT) (29). In contrast, F encodes only a single candidate ATPase, TraC, that is required for F-pilus assembly (8).…”

Section: Discussionmentioning

confidence: 99%

F-pili dynamics by live-cell imaging

Clarke

Maddera

Harris

et al. 2008

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

131

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

Section: Discussionmentioning

confidence: 99%

F-pili dynamics by live-cell imaging

Clarke

Maddera

Harris

et al. 2008

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

131

139

View full text Add to dashboard Cite

show abstract

“…Therefore, the coordination of EpsEArg 336 by both Asp 293 and Glu 334 is likely crucial for correct positioning of the latter for catalysis. A mutation in either Arg 336 or Asp 293 would have negative consequences on function as seen both in this study and in previous ones looking at the effects of mutating the EpsE-Asp 293 equivalent in PaPilT (34) and PulE (17,34). Interestingly, the equivalent salt bridges in the PaPilT structure are observed only in active subunit B.…”

mentioning

confidence: 57%

“…Asp 293 and with the catalytic Glu 334 , a residue essential for ATPase activity (34). Therefore, the coordination of EpsEArg 336 by both Asp 293 and Glu 334 is likely crucial for correct positioning of the latter for catalysis.…”

mentioning

confidence: 99%

Oligomerization of EpsE Coordinates Residues from Multiple Subunits to Facilitate ATPase Activity

Patrick

Korotkov

Hól

et al. 2011

Journal of Biological Chemistry

View full text Add to dashboard Cite

EpsE is an ATPase that powers transport of cholera toxin and hydrolytic enzymes through the Type II secretion (T2S) apparatus in the Gram-negative bacterium, Vibrio cholerae. Our data suggest that these arginines are essential for ATP hydrolysis, although their roles in shaping the active site of EpsE are varied. Specifically, we have shown that replacements of these arginine residues abrogate the T2S process due to a reduction of ATPase activity yet do not have any measurable effect on nucleotide binding or oligomerization of EpsE. We have further demonstrated that point mutations in the EpsE intersubunit interface also reduce ATPase activity without disrupting oligomerization, strengthening the idea that residues from multiple subunits must precisely interact in order for EpsE to be sufficiently active to support T2S. Our findings suggest that the action of EpsE is similar to that of other Type II/IV secretion ATPase family members, and thus these results may be widely applicable to the family as a whole.

show abstract

“…Three predicted TFP ATPases-PilB, PilT, and PilUare supposed to be necessary for the concerted action of twitching motility in P. aeruginosa. PilB is involved in polymerization of pilin subunits to form the pilus, whereas PilT and PilU are involved in depolymerization, suggesting that PilB and PilT play antagonistic roles-with PilB promoting pilin association and fiber formation and PilT contributing to pilus retraction via pilin disassembly-whereas the role of PilU is less clear (10,14,15,21). The phage protein Tip directly interacts with PilB, but not with PilT, at the internal 40-aa regions (187-226) that are not functionally conserved in other proteins, leading to the complete loss of both structural and functional TFPs.…”

Section: Discussionmentioning

confidence: 99%

“…Because any defect in reciprocal ATPases results in the loss of twitching motility (14), it was important to further study whether the Tip-PilB interaction agonizes or antagonizes normal PilB function, based on phage adsorption and surface piliation assays. Because TFP directly binds to various TFP-specific phages, a phage adsorption assay was performed by using Tip-expressing cells as described (4).…”

Section: Significancementioning

confidence: 99%

A phage protein that inhibits the bacterial ATPase required for type IV pilus assembly

Chung

Jang

Bae

et al. 2014

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

View full text Add to dashboard Cite

Type IV pili (TFPs) are required for bacterial twitching motility and for phage infection in the opportunistic human pathogen Pseudomonas aeruginosa. Here we describe a phage-encoded protein, D3112 protein gp05 (hereafter referred to as Tip, representing twitching inhibitory protein), whose expression is necessary and sufficient to mediate the inhibition of twitching motility. Tip interacts with and blocks the activity of bacterial-encoded PilB, the TFP assembly/ extension ATPase, at an internal 40-aa region unique to PilB. Tip expression results in the loss of surface piliation. Based on these observations and the fact that many P. aeruginosa phages require TFPs for infection, Tip-mediated twitching inhibition may represent a generalized strategy for superinfection exclusion. Moreover, because TFPs are required for full virulence, PilB may be an attractive target for the development of novel antiinfectives.phage-host interaction | protein-protein interaction | lysogenic conversion | antipathogenics T emperate phages generally lysogenize rather than kill host bacteria, leading to alteration of host traits by the expression of phage-encoded genes (1). Superinfection exclusion is one example of a phage-mediated physiological change, in which a successful lysogenized phage blocks potential competition by another phage. Superinfection exclusion of phages of similar lineage generally involves phage repressors (i.e., homoimmunity) (1, 2). Some temperate phages, however, have developed repressor-independent superinfection exclusion/immunity systems that block phage adsorption or DNA uptake, enabling infected hosts to become resistant to superinfection by phages sharing the same uptake mechanism (1, 3). We previously reported that Pseudomonas aeruginosa temperate phage D3112 blocks infection by a second phage, MP22, but not vice versa. Both D3112 and MP22 require type IV pilus (TFP)-mediated twitching motility for infection (4). This finding is most likely accounted for by the twitching-inhibitory activity unique to D3112 (5). We reasoned that the elucidation of the superinfection exclusion system of phage D3112 may provide insights into the mechanisms of TFP assembly and function. Moreover, because twitching motility is one of the group behaviors critical to biofilm formation and pathogenicity of P. aeruginosa, elucidation of D3112-mediated superinfection exclusion may also be important in the development of new antibacterial therapies. In the present study, we have identified a phage protein for twitching inhibition and its bacterial target. ResultsExpression of D3112 gp05 Is Necessary and Sufficient for Twitching Inhibition. Zegans et al. previously reported that P. aeruginosa swarming motility is inhibited by phage DMS3 and that the inhibition involves the bacterial Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)/Cas system present in some P. aeruginosa strains such as PA14 (6). To investigate the potential involvement of the CRISPR/Cas system in twitching inhibition by phage D3112, we first determ...

show abstract

Functional role of conserved residues in the characteristic secretion NTPase motifs of the Pseudomonas aeruginosa type IV pilus motor proteins PilB, PilT and PilU

Cited by 114 publications

References 46 publications

F-pili dynamics by live-cell imaging

F-pili dynamics by live-cell imaging

Oligomerization of EpsE Coordinates Residues from Multiple Subunits to Facilitate ATPase Activity

A phage protein that inhibits the bacterial ATPase required for type IV pilus assembly

Contact Info

Product

Resources

About