A Pseudomonas aeruginosa type VI secretion system regulated by CueR facilitates copper acquisition.

The type VI secretion system (T6SS) is widely distributed in Gram-negative bacteria, whose function is known to translocate substrates to eukaryotic and prokaryotic target cells to cause host damage or as a weapon for interbacterial competition. Pseudomonas aeruginosa encodes three distinct T6SS clusters (H1-, H2-, and H3-T6SS). The H1-T6SS-dependent substrates have been identified and well characterized; however, only limited information is available for the H2- and H3-T6SSs since relatively fewer substrates for them have yet been established. Here, we obtained P. aeruginosa H2-T6SS-dependent secretomes and further characterized the H2-T6SS-dependent copper (Cu2+)-binding effector azurin (Azu). Our data showed that both azu and H2-T6SS were repressed by CueR and were induced by low concentrations of Cu2+. We also identified the Azu-interacting partner OprC, a Cu2+-specific TonB-dependent outer membrane transporter. Similar to H2-T6SS genes and azu, expression of oprC was directly regulated by CueR and was induced by low Cu2+. In addition, the Azu-OprC-mediated Cu2+ transport system is critical for P. aeruginosa cells in bacterial competition and virulence. Our findings provide insights for understanding the diverse functions of T6SSs and the role of metal ions for P. aeruginosa in bacteria-bacteria competition. Author summary The type VI secretion system (T6SS) is a specific macromolecular protein export apparatus, and widely distributed in Gram-negative bacteria. T6SS plays an important role in anti-bacterial competition or delivers effector proteins to both eukaryotic and prokaryotic cells. In the present study, we performed secretomes analysis and identified 21 substrates of P. aeruginosa H2-T6SS-dependent. Specifically, we report a Cu2+-scavenging pathway consisting of a copper transporter, OprC, and a type VI secretion system (H2-T6SS)-secreted Cu2+-binding protein, Azu. Both of them are under control of the transcriptional regulator CueR. Indeed, the Azu-OprC-mediated Cu2+ transport system is critical for P. aeruginosa cells in bacterial competition and virulence. These findings exemplify how P. aeruginosa deploys this metal system to adapt to the complex environment during evolution.