Hcp of the Type VI Secretion System (T6SS) in Acidovorax citrulli Group II Strain Aac5 Has a Dual Role as a Core Structural Protein and an Effector Protein in Colonization, Growth Ability, Competition, Biofilm Formation, and Ferric Iron Absorption

A type VI secretion system (T6SS) gene cluster has been reported in Acidovorax citrulli. Research on the activation conditions, functions, and the interactions between key elements in A. citrulli T6SS is lacking. Hcp (Hemolysin co-regulated protein) is both a structural protein and a secretion protein of T6SS, which makes it a special element. The aims of this study were to determine the role of Hcp and its activated conditions to reveal the functions of T6SS. In virulence and colonization assays of hcp deletion mutant strain Delta hcp, tssm (type VI secretion system membrane subunit) deletion mutant strain Delta tssm and double mutant Delta hcp Delta tssm, population growth was affected but not virulence after injection of cotyledons and seed-to-seedling transmission on watermelon. The population growth of Delta hcp and Delta tssm were lower than A. citrulli wild type strain Aac5 of A. citrulli group II at early stage but higher at a later stage. Deletion of hcp also affected growth ability in different culture media, and the decline stage of Delta hcp was delayed in KB medium. Biofilm formation ability of Delta hcp, Delta tssm and Delta hcp Delta tssm was lower than Aac5 with competition by prey bacteria but higher in KB and M9-Fe3+ medium. Deletion of hcp reduced the competition and survival ability of Aac5. Based on the results of Western blotting and qRT-PCR analyses, Hcp is activated by cell density, competition, ferric irons, and the host plant. The expression levels of genes related to bacterial secretion systems, protein export, and several other pathways, were significantly changed in the Delta hcp mutant compared to Aac5 when T6SS was activated at high cell density. Based on transcriptome data, we found that a few candidate effectors need further identification. The phenotypes, activated conditions and transcriptome data all supported the conclusion that although there is only one T6SS gene cluster present in the A. citrulli group II strain Aac5, it related to multiple biological processes, including colonization, growth ability, competition and biofilm formation.