Energy requirement for pullulanase secretion by the main terminal branch of the general secretory pathway

Possot, Odile; Letellìer, Lucienne; Pugsley, Anthony P.

doi:10.1046/j.1365-2958.1997.3451726.x

Cited by 63 publications

(51 citation statements)

References 27 publications

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“…The values reported in Fig. 1 for ⌬ and cellular ATP measured in the absence and presence of CCCP or arsenate agree with values reported previously for other Gram-negative respiring bacteria (12,20,21). Of considerable interest, the protease degraded VirB10 to VirB10* from the H 2 0-or EtOH-treated control cells, but failed to degrade VirB10 from the CCCP-or arsenate-treated cells (Fig.…”

Section: Virb10 Protease Susceptibility As a Function Of Cellular Atpsupporting

confidence: 80%

Agrobacterium VirB10, an ATP energy sensor required for type IV secretion

Cascales

Christie

2004

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

136

168

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Bacteria use type IV secretion systems (T4SS) to translocate DNA and protein substrates to target cells of phylogenetically diverse taxa. Recently, by use of an assay termed transfer DNA immunoprecipitation (TrIP), we described the translocation route for a DNA substrate [T-DNA, portion of the Ti (tumor-inducing) plasmid that is transferred to plant cells] of the Agrobacterium tumefaciens VirB͞D4 T4SS in terms of a series of temporally and spatially ordered substrate contacts with subunits of the secretion channel. Here, we report that the bitopic inner membrane protein VirB10 undergoes a structural transition in response to ATP utilization by the VirD4 and VirB11 ATP-binding subunits, as monitored by protease susceptibility. VirB10 interacts with inner membrane VirD4 independently of cellular energetic status, whereas the energy-induced conformational change is required for VirB10 complex formation with an outer membrane-associated heterodimer of VirB7 lipoprotein and VirB9, as shown by coimmunoprecipitation. Under these conditions, the T-DNA substrate is delivered from the inner membrane channel components VirB6 and VirB8 to periplasmic and outer membrane-associated VirB2 pilin and VirB9. We propose that VirD4 and VirB11 coordinate the ATP-dependent formation of a VirB10 ''bridge'' between inner and outer membrane subassemblies of the VirB͞D4 T4SS, and that this morphogenetic event is required for T-DNA translocation across the A. tumefaciens cell envelope. conjugation ͉ energy coupling ͉ pathogenesis ͉ membrane complex ͉ translocation

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Section: Virb10 Protease Susceptibility As a Function Of Cellular Atpsupporting

confidence: 80%

Agrobacterium VirB10, an ATP energy sensor required for type IV secretion

Cascales

Christie

2004

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

136

168

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“…To determine whether the effect of CCCP was reversible, cells treated with CCCP were washed twice with and resuspended in fresh medium. Alternatively, 1 mM bovine serum albumin, to which CCCP adsorbs, was added to the medium (42). In both conditions, the proteinase K-resistant product was no longer detected, showing that the action of the CCCP could be reversed (data not shown).…”

Section: Resultsmentioning

confidence: 99%

Energy-Dependent Conformational Change in the TolA Protein ofEscherichia coliInvolves Its N-Terminal Domain, TolQ, and TolR

et al. 2001

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TolQ, TolR, and TolA inner membrane proteins of Escherichia coli are involved in maintaining the stability of the outer membrane. They share homology with the ExbB, ExbD, and TonB proteins, respectively. The last is involved in energy transduction between the inner and the outer membrane, and its conformation has been shown to depend on the presence of the proton motive force (PMF), ExbB, and ExbD. Using limited proteolysis experiments, we investigated whether the conformation of TolA was also affected by the PMF. We found that dissipation of the PMF by uncouplers led to the formation of a proteinase K digestion fragment of TolA not seen when uncouplers are omitted. This fragment was also detected in ⌬tolQ, ⌬tolR, and tolA(H22P) mutants but, in contrast to the parental strain, was also seen in the absence of uncouplers. We repeated those experiments in outer membrane mutants such as lpp, pal, and ⌬rfa mutants: the behavior of TolA in lpp mutants was similar to that observed with the parental strain. However, the proteinase K-resistant fragment was never detected in the ⌬rfa mutant. Altogether, these results suggest that TolA is able to undergo a PMF-dependent change of conformation. This change requires TolQ, TolR, and a functional TolA N-terminal domain. The potential role of this energy-dependent process in the stability of the outer membrane is discussed.

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“…How, then, could retraction occur? We propose that elongation and retraction of PulG pseudopili are both stimulated by PulE or that the electrochemical potential across the plasma membrane that is required for secretion (30) is directly or indirectly responsible for PulG pilus disassembly. We further propose that elongation may be arrested and disassembly (retraction) may be triggered by incorporation into the base of the filament of a minor pseudopilin, possible PulK (Fig.…”

Section: Pseudopili or Real Pili?mentioning

confidence: 99%

Type IV-Like Pili Formed by the Type II Secreton: Specificity, Composition, Bundling, Polar Localization, and Surface Presentation of Peptides

Vignon

Köhler

Larquet³

et al. 2003

J Bacteriol

Self Cite

118

148

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The secreton or type II secretion machinery of gram-negative bacteria includes several type IV pilin-like proteins (the pseudopilins) that are absolutely required for secretion. We previously reported the presence of a bundled pilus composed of the pseudopilin PulG on the surface of agar-grown Escherichia coli K-12 cells expressing the Klebsiella oxytoca pullulanase (Pul) secreton genes at high levels (N. Sauvonnet, G. Vignon, A. P. Pugsley, and P. Gounon, EMBO J. 19:2221-2228, 2000). We show here that PulG is the only pseudopilin in purified pili and that the phenomenon is not restricted to the Pul secreton reconstituted in E. coli or to PulG. For example, high-level expression of the endogenous E. coli gsp secreton genes caused production of bundled pili composed of the pseudopilin GspG, and the Pul secreton was able to form pili composed of PulG-like proteins from secreton systems of other bacteria. PulG derivatives in which the C terminus was extended by the addition of eight different peptides were also assembled into pili and functioned in secretion. Three of the C-terminal peptides were shown to be exposed along the entire length of the assembled pili. Hence, the C terminus of PulG may represent a permissive site for the insertion of immunogenic epitopes or other peptide sequences. One of these PulG variants, with a six-histidine tag at its C terminus, formed nonpolar, nonbundled pili, suggesting that bundle formation and polar localization are not correlated with the ability of PulG to function in secretion. We propose that the PulG pilus is an artifactual manifestation of a periplasmic "pseudopilus" and that cycles of pseudopilus extension and retraction within the periplasm propel pullulanase through secretin channels in the outer membrane. Abnormally long pili that extend beyond the outer membrane are produced only when pilus length control and retraction are deregulated by overproduction of the major pseudopilus subunit (PulG).The secreton or type II secretion (T2S) system permits the energy-dependent secretion of a limited number of specific proteins from the periplasm in gram-negative bacteria (33). Many of the 12 or more secreton components share extensive sequence similarity with components of the type IV piliation (T4P) system in gram-negative bacteria (24, 33). In the T2S system, these "shared" components include the pilins themselves (called pseudopilins in the secreton system) and prepilin peptidase, the enzyme that removes a short N-terminal peptide and then N-methylates type IV pilin precursors (25,29,35,37) and type IV pilin-like proteins. The N-terminal prepeptide and approximately 20-residue hydrophobic domains of the type IV pilins and pseudopilins are highly conserved (24, 33). In the pullulanase (PulA)-specific secreton from Klebsiella oxytoca, there are five type IV pseudopilins (PulG, PulH, Pull, PulJ, and PulK) (31,36,40).In the absence of any direct evidence for the assembly of pseudopilins into a pilus (34, 38), they were proposed to form a small, intraperiplasmic structure (the ...

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Energy requirement for pullulanase secretion by the main terminal branch of the general secretory pathway

Cited by 63 publications

References 27 publications

Agrobacterium VirB10, an ATP energy sensor required for type IV secretion

Agrobacterium VirB10, an ATP energy sensor required for type IV secretion

Energy-Dependent Conformational Change in the TolA Protein ofEscherichia coliInvolves Its N-Terminal Domain, TolQ, and TolR

Type IV-Like Pili Formed by the Type II Secreton: Specificity, Composition, Bundling, Polar Localization, and Surface Presentation of Peptides

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