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Journal of Bacteriology, July 2009, p. 4316-4329, Vol. 191, No. 13
0021-9193/09/$08.00+0     doi:10.1128/JB.00029-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

An IcmF Family Protein, ImpL_M, Is an Integral Inner Membrane Protein Interacting with ImpK_L, and Its Walker A Motif Is Required for Type VI Secretion System-Mediated Hcp Secretion in Agrobacterium tumefaciens ^,

Lay-Sun Ma,^1,2,3 Jer-Sheng Lin,¹ and Erh-Min Lai^1,2,3*

Institute of Plant and Microbial Biology, Academia Sinica, Taipei, Taiwan,¹ Molecular and Biological Agricultural Sciences Program, Taiwan International Graduate Program, National Chung-Hsing University and Academia Sinica, Taipei, Taiwan,² Graduate Institute of Biotechnology, National Chung-Hsing University, Taichung, Taiwan³

Received 12 January 2009/ Accepted 17 April 2009

An intracellular multiplication F (IcmF) family protein is a conserved component of a newly identified type VI secretion system (T6SS) encoded in many animal and plant-associated Proteobacteria. We have previously identified ImpL_M, an IcmF family protein that is required for the secretion of the T6SS substrate hemolysin-coregulated protein (Hcp) from the plant-pathogenic bacterium Agrobacterium tumefaciens. In this study, we characterized the topology of ImpL_M and the importance of its nucleotide-binding Walker A motif involved in Hcp secretion from A. tumefaciens. A combination of β-lactamase-green fluorescent protein fusion and biochemical fractionation analyses revealed that ImpL_M is an integral polytopic inner membrane protein comprising three transmembrane domains bordered by an N-terminal domain facing the cytoplasm and a C-terminal domain exposed to the periplasm. impL_M mutants with substitutions or deletions in the Walker A motif failed to complement the impL_M deletion mutant for Hcp secretion, which provided evidence that ImpL_M may bind and/or hydrolyze nucleoside triphosphates to mediate T6SS machine assembly and/or substrate secretion. Protein-protein interaction and protein stability analyses indicated that there is a physical interaction between ImpL_M and another essential T6SS component, ImpK_L. Topology and biochemical fractionation analyses suggested that ImpK_L is an integral bitopic inner membrane protein with an N-terminal domain facing the cytoplasm and a C-terminal OmpA-like domain exposed to the periplasm. Further comprehensive yeast two-hybrid assays dissecting ImpL_M-ImpK_L interaction domains suggested that ImpL_M interacts with ImpK_L via the N-terminal cytoplasmic domains of the proteins. In conclusion, ImpL_M interacts with ImpK_L, and its Walker A motif is required for its function in mediation of Hcp secretion from A. tumefaciens.

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* Corresponding author. Mailing address: 128, Sec. 2, Academia Road, Nankang, Taipei, Taiwan 11529. Phone: 886-2-27871158. Fax: 886-2-27827954. E-mail: emlai{at}gate.sinica.edu.tw

Published ahead of print on 24 April 2009.

Supplemental material for this article may be found at http://jb.asm.org/.

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Journal of Bacteriology, July 2009, p. 4316-4329, Vol. 191, No. 13
0021-9193/09/$08.00+0     doi:10.1128/JB.00029-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.