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

Abstract

ABSTRACT 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 .