Agrobacterium tumefaciens is a plant-pathogenic bacterium capable of secreting several virulence factors into extracellular space or the host cell. In this study, we used shotgun proteomics analysis to investigate the secretome of A. tumefaciens, which resulted in identification of 12 proteins, including 1 known secretory protein (VirB1*) and 11 potential secretory proteins. Interestingly, one unknown protein, which we designated hemolysin-coregulated protein (Hcp), is a predicted soluble protein without a recognizable N-terminal signal peptide. Western blot analysis revealed that A. tumefaciens Hcp is expressed and secreted when cells are grown in both minimal and rich media. Further biochemical and immunoelectron microscopy analysis demonstrated that intracellular Hcp is localized mainly in the cytosol, with a small portion in the membrane system. To investigate the mechanism of secretion of Hcp in A. tumefaciens, we generated mutants with deletions of a conserved gene, icmF, or the entire putative operon encoding a recently identified type VI secretion system (T6SS). Western blot analysis indicated that Hcp was expressed but not secreted into the culture medium in mutants with deletions of icmF or the t6ss operon. The secretion deficiency of Hcp in the icmF mutant was complemented by heterologous trans expression of icmF, suggesting that icmF is required for Hcp secretion. In tumor assays with potato tuber disks, deletion of hcp resulted in approximately 20 to 30% reductions in tumorigenesis efficiency, while no consistent difference was observed when icmF or the t6ss operon was deleted. These results increase our understanding of the conserved T6SS used by both plant-and animal-pathogenic bacteria.

Agrobacterium tumefaciens causes crown gall disease in a wide range of plants by transforming plants through the transfer and integration of its transferred DNA (T-DNA) into the host genome. In the present study, we used two-dimensional gel electrophoresis to examine the protein expression profiles of A. tumefaciens in response to the phenolic compound acetosyringone (AS), a known plant-released virulence (vir) gene inducer. Using mass spectrometry, we identified 11 proteins consisting of 9 known AS-induced Vir proteins and 2 newly discovered AS-induced proteins, an unknown protein Y4mC (Atu6162) and a small heat shock protein HspL (Atu3887). Further expression analysis revealed that the AS-induced expression of Y4mC and HspL is regulated by the VirA/VirG two-component system. This report presents the first proteomics study successfully identifying both known and new AS-induced proteins that are implicated in Agrobacterium virulence.