Pseudomonas aeruginosa is a versatile opportunistic bacterial pathogen. Its ability to successfully colonize various environments and hosts is partly due to the capability of P. aeruginosa to outcompete other bacteria. The Type Six Secretion System (T6SS) is one key weapon giving a competitive advantage. It is a supramolecular contractile machine capable of delivering a plethora of potent antibacterial toxins. P. aeruginosa possesses three T6SSs (H1- to H3-T6SS) and several T6SS-related islands scattered throughout the genome. Some of these islands encode the T6SS puncturing device, VgrG, and additional T6SS-related function such as Toxin-Immunity pairs. The vgrG1b operon is present downstream of the H1-T6SS cluster and is composed of seven genes. This operon is highly conserved in P. aeruginosa genomes except for the toxin-immunity gene pair, tse7-tsi7. This study demonstrates the Cterminal DNase activity of Tse7. Moreover, the study shows that delivery of Tse7 requires the H1-T6SS and the N-terminus of Tse7 PAAR domain tops the VgrG1b spike. Tsi7, protects producing cells from the DNase activity through direct interactions with Tse7. Additional putative nucleases were identified (TpnA and TpnB). These nucleases are encoded in genetic clusters also encoding PAAR proteins. The tpnB cluster encodes a novel T6SS-chaperone type, TapN, which harbours a DUF4123 domain. TapN is required for the association of the toxin TpnB to the PAAR (PA3904). Another protein, PA3906, encoded within the tpnB cluster is also proposed to have a putative chaperone function by interacting with the PAAR tip. 4 Finally, this study investigates the post-translational regulation of the H1-T6SS activity via the repressor TagF. Two mechanisms of TagF action have been proposed. Firstly, TagF directly counteracts Fha, a T6SS activator. Secondly, TagF directly interacts with T6SS components making the so-called baseplate subcomplex, suggesting a new mode of inhibition by blocking T6SS conformational changes and subsequent sheath contraction.