Deletion mutant, Δ tssB showed lower growth rate and lower antibacterial activity in Flavobacterium columnare

Type VI secretion system (T6SS) in Gram-negative bacteria is involved in interbacterial competition. Although partial T6SS has been reported in Flavobacterium columnare , its possible function has never been characterized. In this study, one of the contractile sheath protein genes, tssB was cloned in F. columnare G ₄ with 450 bp open reading frame (ORF) encoding 149 amino acids (aa) predicted to be 16.59 kDa. The tssB in F. columnare G ₄ was phylogenetically clustered with the molecule in F. columnare ATCC 49512, and then with F. johnsoniae , implying that the T6SS in F. columnare may also belong to T6SS ⁱⁱⁱ as reported in F. johnsoniae . With the available genetic manipulation technique, the knockout mutant, Δ tssB was constructed with the establishment of complementation strains, tssB ⁺ strain and tssB ⁺ tssC ⁺ strain. The Δ tssB showed similar colony morphology with rhizoids as observed in the wildtype F. columnare G ₄ , but exhibited significantly lower growth rate than the wildtype, and also lower antibacterial effect against Aeromonas hydrophila F4, as shown with the significantly lower concentration of A. hydrophila F4 in comparison with the wildtype. The differentially expressed genes (DEGs) of the Δ tssB strain comprised 13 upregulated and 102 downregulated genes, which were enriched in the Cellular Component term membrane and the nitrogen metabolism pathway, as revealed by GO and KEGG enrichment analyses. It is thus indicated that a potentially functional T6SS may exist in F. columnare , and further investigation should be conducted to reveal the exact composition of T6SS and its function in F. columnare .