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An Agrobacterium VirB10 Mutation Conferring a Type IV Secretion System Gating Defect

Banta, L.M. and Kerr, J.E. and Cascales, E. and Giuliano, M.E. and Bailey, M.E. and McKay, C. and Chandran, Vidya and Waksman, Gabriel and Christie, P.J. (2011) An Agrobacterium VirB10 Mutation Conferring a Type IV Secretion System Gating Defect. Journal of Bacteriology 193 (10), pp. 2566-2574. ISSN 0021-9193.

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Official URL: http://dx.doi.org/10.1128/JB.00038-11

Abstract

Agrobacterium VirB7, VirB9, and VirB10 form a "core complex" during biogenesis of the VirB/VirD4 type IV secretion system (T4SS). VirB10 spans the cell envelope and, in response to sensing of ATP energy consumption by the VirB/D4 ATPases, undergoes a conformational change required for DNA transfer across the outer membrane (OM). Here, we tested a model in which VirB10 regulates substrate passage by screening for mutations that allow for unregulated release of the VirE2 secretion substrate to the cell surface independently of target cell contact. One mutation, G272R, conferred VirE2 release and also rendered VirB10 conformationally insensitive to cellular ATP depletion. Strikingly, G272R did not affect substrate transfer to target cells (Tra(+)) but did block pilus production (Pil(+)). The G272R mutant strain displayed enhanced sensitivity to vancomycin and SDS but did not nonspecifically release periplasmic proteins or VirE2 truncated of its secretion signal. G272 is highly conserved among VirB10 homologs, including pKM101 TraF, and in the TraF X-ray structure the corresponding Gly residue is positioned near an alpha-helical domain termed the antenna projection (AP), which is implicated in formation of the OM pore. A partial AP deletion mutation (Delta AP) also confers a Tra(+) Pil(-) phenotype; however, this mutation did not allow VirE2 surface exposure but instead allowed the release of pilin monomers or short oligomers to the milieu. We propose that (i) G272R disrupts a gating mechanism in the core chamber that regulates substrate passage across the OM and (ii) the G272R and Delta AP mutations block pilus production at distinct steps of the pilus biogenesis pathway.

Item Type: Article
School or Research Centre: Birkbeck Schools and Research Centres > School of Science > Biological Sciences
Depositing User: Administrator
Date Deposited: 27 Jun 2011 13:27
Last Modified: 17 Apr 2013 12:20
URI: http://eprints.bbk.ac.uk/id/eprint/3563

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