Abstract
Type VI secretion system (T6SS) is an apparatus for Gram-negative bacteria to deliver effector proteins directly into target cells. There are three T6SS gene clusters, named H1-, H2-, and H3-T6SS gene cluster, in Pseudomonas aeruginosa PAO1. The aim of this study is to elucidate the role of both sfa2 (PA1663), a putative RpoN transcriptional activator encoding gene located in the H2-T6SS gene cluster and rpoN (PA4462), a transcription factor in regulation of H2-T6SS. In previous studies, the expression of H2-T6SS gene cluster were down-regulated in sfa2 deletion mutant strain. This study constructed an rpoN deletion mutant strain (ΔrpoN) to elucidate the relationship between Sfa2 and RpoN in regulation of H2-T6SS gene expression. Analysis of the upstream region of H2-T6SS with β-glucuronidase (GUS) reporter gene assay confirmed that Sfa2 and RpoN co-regulate the expression of the H2-T6SS gene cluster. Using recombinant Sfa2 and RpoN in an electrophoretic motility shift assay, this study found that the Sfa2 binding site and RpoN binding site are located from nucleotide position -432 to -403 and -672 to -503, respectively in the upstream region of the H2-T6SS gene cluster. This study also showed that the expression of Hcp decreases in both the sfa2 and rpoN deletion mutant strains. By GUS reporter gene assay, PA5267 (hcpB) is the major one of three hcp genes (PA0263, PA1512 and PA5267) regulated by Sfa2 and RpoN. However, only RpoN can bind to the upstream refion of three hcp genes but not Sfa2. Compared to PAO1, twitching motility, swimming motility, iron chelation, the production of pyocyanin and pyoverdin, and biofilm formation reduced in ΔrpoN. Conversely, the hemolysis and quorum sensing ability increased in ΔrpoN. Combined results in this study, we suggest that Sfa2 interacts with RpoN to up-regulate both H2-T6SS gene cluster and hcp genes expression and RpoN plays an important role in regulating phenotypic traits in P. aeruginosa PAO1.