norCBD disruption affects the H2-type six secretion system and multiple virulence factors in Pseudomonas aeruginosa

The type six secretion system (T6SS) is a macromolecular weapon used by many Gram-negative bacteria. The T6SS functions as a needle injection system that delivers effector proteins directly into neighboring bacterial cells, thereby affecting their gene expression and physiological processes. <i>Pseudomonas aeruginosa</i> possesses at least three distinct T6SSs, designated as H1-, H2-, and H3-T6SS. Although extensive studies have been carried out on these T6SS systems in recent years, the regulatory mechanisms of T6SS remain incomplete. Here, we report the identification of <i>norCBD</i> as an operon that modulates the transcriptional activity of H2-T6SS. Both transposon insertion at <i>norCBD</i> and the deletion of the <i>norCBD</i> genes significantly reduced the CTX-<i>H2</i>-T6SS reporter activity. The <i>norCBD</i> operon encodes nitric oxide reductase (NorCBD), which reduces nitric oxide (NO) to nitrous oxide (N₂O), a crucial step in reducing the toxic level of intracellular NO and facilitating anaerobic respiration. As the transcriptional regulator Dnr activates H2-type VI secretion system (H2-T6SS) in response to NO, experiments were carried out to examine whether <i>norCBD</i> deletion caused intracellular NO accumulation, which in turn disrupted Dnr-dependent regulation of H2-T6SS and virulence factors. The NO levels and Dnr-regulated gene expression were measured, and several virulence-related phenotypes were examined. The effects of NO donor sodium nitroprusside (SNP) and NO scavenger carboxy-phenyl-tetramethylimidazolineoxyl (CPTIO) were also tested. The data obtained indicate that deletion of <i>norCBD</i> led to intracellular NO accumulation, reduced H2-T6SS expression, and affected motility, pyocyanin production, and biofilm formation. Complementation of <i>norCBD</i> on a plasmid in the deletion mutant was able to restore H2-T6SS expression and the examined phenotypes to the wild-type levels. Treatment with CPTIO also restored H2-T6SS expression in the PAO1(Δ<i>norCBD</i>). These results indicate that NorCBD plays a critical role in maintaining NO homeostasis that is necessary for effective Dnr-mediated gene regulation and multiple virulence-related traits, highlighting the importance of redox balance in coordinating respiration and pathogenesis in <i>P. aeruginosa</i>.