JB Accepts, published online ahead of print on 8 February 2008

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J. Bacteriol. doi:10.1128/JB.01775-07
Copyright (c) 2008, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.

Secretome analysis uncovers an Hcp-family protein secreted via a type VI secretion system in Agrobacterium tumefaciens

Hung-Yi Wu, Pei-Che Chung, Hsiao-Wei Shih, Sy-Ray Wen, and Erh-Min Lai^*

Institute of Plant and Microbial Biology, Academia Sinica, Taipei, Taiwan

^* To whom correspondence should be addressed. Email: emlai{at}gate.sinica.edu.tw.

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, resulting in the identification of 12 proteins including one known (VirB1*) and 11 potential secretory proteins. Interestingly, one unknown protein, which we have named 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 mainly localized in the cytosol, with a small portion in the membrane system. To investigate the secretion mechanism 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 carrying deletions of icmF or t6ss. 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 on potato tuber discs, deletion of hcp results in about 20-30% reduction of tumorigenesis efficiency, while no consistent difference can be observed in deletions of icmF or t6ss. These results further our understanding of the conserved T6SS protein secretion system used by both plant and animal pathogenic bacteria in virulence.

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