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. 2004 Dec 7;101(49):17228-33.
doi: 10.1073/pnas.0405843101. Epub 2004 Nov 29.

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

Eric Cascales  1 , Peter J Christie
Affiliations

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

Eric Cascales et al. Proc Natl Acad Sci U S A. .

Abstract

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 VirB/D4 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 VirB/D4 T4SS, and that this morphogenetic event is required for T-DNA translocation across the A. tumefaciens cell envelope.

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Figures

Fig. 1.
Fig. 1.
Identification of an energy-dependent VirB10 conformational switch. A348 (WT) or a virB10-null mutant (ΔB10) were treated with the energy poisons arsenate (Ars) or CCCP, or H2O or EtOH as controls. Spheroplasts from these cells were incubated in the presence (+) or absence (–) of the S. griseus protease. Immunoblots were probed with the antibodies to VirB10 (Top) or, as controls for spheroplast integrity, the VirB4 integral membrane subunit (Middle), or cytosolic VirE2 (Bottom). The positions of full-length VirB10 (B10) and the degradation product (B10*) are indicated at right. Transmembrane potential (Δψ) in millivolts (mV) and ATP levels in percent relative to the H2O-treated control were quantitated as described in the text. nd, Not determined.
Fig. 2.
Fig. 2.
Dependence of the VirB10 conformation on energetic subunits. Protease susceptibility of VirB10 in the WT or virB4, virB11 or virD4 mutant strains (A); the virB11 strain (ΔB11) producing native VirB11 (B11) or Walker A mutant proteins (K175Q and ΔGKT) (B); or the virD4 strain producing native VirD4 (D4) or a Walker A mutant protein (K152Q) (C). Spheroplasts of strains indicated were incubated in the presence (+) or absence (–) of protease. Immunoblots were developed with anti-VirB10 antibodies. Positions of the intact VirB10 protein and the degradation product (B10*) are indicated at the left. In A and C, immunoblots show the presence of nonspecific VirB10 degradation products of ≤40-kDa detected previously (42).
Fig. 3.
Fig. 3.
Effects of cellular energy on formation of VirB10 complexes. WT or mutant strains were treated with CCCP, arsenate (Ars), or the corresponding EtOH or H2O controls, then detergent-solubilized as described in Experimental Procedures. (A) Material precipitated with anti-VirB10 antibodies from extracts of energized (EtOH- or H2O-treated) or energy-depleted (Ars- or CCCP-treated) WT cells, or the ΔvirB9 (ΔB9) or ΔvirB10 (ΔB10) control strains. (B) Material precipitated with anti-VirB10 antibodies from extracts of energized (H2O) or energy-depleted (Ars) WT cells in the absence (–) or presence of reducing agent (DTT). (C) Material precipitated with anti-VirD4 antibodies from extracts of energized and energy-depleted WT cells or the ΔvirB10 or virD4 control strains. Immunoblots were developed with antibodies to the T4SS subunits listed at the right. Crossreactive material was immunoreactive heavy (filled circle) and light (open circle) chain IgG. T, total cell extract; S, material in supernatant after the immunoprecipitation; IP, immunoprecipitated material.
Fig. 4.
Fig. 4.
Effects of virD4- and virB11-null and Walker A mutations on VirB10 complex formation. (A) Material precipitated with anti-VirB10 antibodies from extracts of the ΔvirB11 strain producing native VirB11 or the VirB11 Walker A mutants (K175Q and ΔGKT). (B) Material precipitated with anti-VirB10 antibodies from extracts of the virD4 mutant producing native VirD4 or the VirD4 Walker A mutant (K152Q). (C) Material precipitated with anti-VirD4 antibodies from extracts of the ΔvirB10 mutant or the ΔvirB11 mutant producing VirB11 or VirB11K175Q. (D) Material precipitated with anti-VirD4 antibodies from extracts of the virD4 mutant producing VirD4 or VirD4K152Q. Immunoblots were developed with antibodies to the T4SS subunits listed at the left. Crossreactive material was immunoreactive heavy (filled circle) and light (open circle) chain IgG. S, material in supernatant after the immunoprecipitation; IP, immunoprecipitated material.
Fig. 5.
Fig. 5.
TrIP studies with the virB10 strain. (A) TrIP and QTrIP measurements of the T-strand interaction with VirB proteins from the WT or the ΔvirB10 strain. S, supernatant after immunoprecipitation with the anti-VirB antibodies listed on bottom; IP, immunoprecipitated material. For TrIP, the T-strand substrate (T-DNA) and the Ti control fragment (ophDC) were detected by PCR amplification and gel electrophoresis. For QTrIP, quantitative data (Upper) are presented as cpm of incorporated radionucleotide during one cycle in the logarithmic phase of PCR amplication and reported as a ratio of cpm recovered with a given antibody from the virB10 mutant vs. the WT strain. QTrIP data are reported for a single experiment; several repetitions of these experiments showed <5% deviation of the values shown for a given T4SS subunit. (B) Schematic showing the proposed contribution of VirB10 energy coupling to assembly and function of the VirB/D4 T4SS.
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References

    1. Cascales, E. & Christie, P. J. (2003) Nat. Rev. Microbiol. 1, 137–150. - PMC - PubMed
    1. Llosa, M. & O'Callaghan, D. (2004) Mol. Microbiol. 53, 1–8. - PubMed
    1. Cascales, E. & Christie, P. J. (2004) Science 304, 1170–1173. - PMC - PubMed
    1. Holland, B. (2004) Biochim. Biophys. Acta, in press.
    1. Christie, P. J. (2004) Biochim. Biophys. Acta, in press.

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