Proposed models for nitrate regulation of motility, biofilm formation, and virulence in P. aeruginosa PAO1. (A) Nitrate sensor-response regulator function in motility and biofilm formation. (Top) In wild-type P. aeruginosa PAO1, nitrate activates the autophosphorylation of the nitrate sensor NarX. Transfer of the phosphate to the receiver domain of the response regulator NarL results in either activation or repression of target operon transcription. Pathways leading to motility and biofilm development are operative. (Middle) In the double mutant ΔnarXL, loss of the nitrate sensor NarX results in an inability to activate NarL. Swimming and swarming are both diminished in ΔnarXL, suggesting impairment of flagellar synthesis and/or function. As a consequence, biofilm thickness and biomass are enhanced compared to the wild type, possibly due to a defect in organism dispersal from the biofilm. The membrane nitrate reductase operon narK1K2GHJI is not activated in the nitrate sensor-response regulator double mutant. However, derepression of the periplasmic nitrate reductase operon napEFDABC in ΔnarXL permits growth under microaerobic and anaerobic conditions (unpublished observations), allowing biofilm formation. (Bottom) In the response regulator mutant ΔnarL, the cognate sensor target is absent. Inability of NarX to activate NarL results in a significant increase in rhamnolipid production. As a consequence, ΔnarL displays a hyperswarming phenotype that likely diminishes its ability to form a biofilm. It is unknown whether an alternative sensor activates NarL to stimulate rhamnolipid production or whether phospho-NarL represses rhamnolipid production. Cross talk with other sensors or response regulators that modulate flagellar function may also explain why the individual ΔnarL and ΔnarX mutants swam normally while swimming of the double mutant ΔnarXL was impaired. Despite alterations in biofilm formation and swimming in the regulatory mutants, all were as virulent as wild-type PAO1 in C. elegans. (B) Nitrate dissimilation in motility, biofilm formation, and virulence. (Top) In wild-type PAO1, both membrane nitrate reductase and periplasmic nitrate reductase contribute to nitrite formation. We propose that further reduction of nitrite to nitric oxide (NO) provides a signal to stimulate rhamnolipid production, resulting in swarming and normal biofilm formation. It is unknown whether the regulation of rhamnolipid synthesis by NO is direct or indirect. (Bottom) This model is supported by the phenotypes of the nitrate dissimilation pathway mutants. The membrane nitrate reductase mutant ΔnarGH is defective in rhamnolipid production, swarming, and biofilm formation but not in swimming. Hence, it appears that NO is not involved in regulation of flagellar synthesis and/or function. While the periplasmic nitrate reductase mutant ΔnapA shows no phenotypic differences from wild-type PAO1, swarming is completely ablated in the double nitrate reductase mutant ΔnarGH:ΔnapA. Swarming was also ablated in the nitrite reductase mutant ΔnirS but not in the nitric oxide reductase mutant Tn::norC. For clarity, the latter two mutants are not indicated in the diagram. The absence of an adequate level of NO signaling results in avirulence of both ΔnarGH and ΔnirS in C. elegans.