Pseudomonas aeruginosa is a Gram-negative, opportunistic pathogens that causes life-threatening, antimicrobial resistant infections in vulnerable patient populations, including patients with cystic fibrosis, cancer, and chronic wounds. During infection, P. aeruginosa requires iron to maintain critical aspects of its metabolism, and possesses numerous virulence factors and iron uptake mechanisms that allow it to compete for this essential metallo-nutrient in the iron-limiting host environment. These systems are tightly regulated by iron-responsive regulatory mechanisms, which ensure adequate uptake while preventing iron toxicity. Because of the essential role of these regulatory mechanisms in maintaining iron homeostasis, they are considered a promising approach for treating P. aeruginosa infections. One prominent regulator of P. aeruginosa iron homeostasis is the PrrF small RNA (sRNA) regulator, which is essential for virulence in acute murine lung infection. Unfortunately, the exact contribution of PrrF to P. aeruginosa pathogenesis has not yet been elucidated. Moreover, our current understanding of PrrF and other iron regulatory mechanisms is largely based on studies using shaking and highly aerated cultures, which are not likely representative of microbial communities in vivo. To address these gaps, the work in this thesis utilizes proteomic, metabolic, and genetic approaches to determine the impact of static growth on iron-responsive regulatory mechanisms in P. aeruginosa, including PrrF sRNAs. We demonstrate that iron regulation paradigms in P. aeruginosa are dramatically altered in static conditions, due in part to changes in PrrF activity. Notably, we identify type 6 secretion systems (T6SS) as a target of enhanced iron regulation in P. aeruginosa in static conditions, and demonstrate that this altered regulation is caused by changes in the production and activity of the PrrF-regulated quorum signaling molecules, 2-alkyl-4(1H)-quinolones (AQs). Furthermore, we demonstrate that altered AQ activity may modulate clinically-significant interactions with other opportunistic pathogens, such as S. aureus, In turn, the work described herein has broad implications for the study of P. aeruginosa infections, and highlights the need to further probe essential P. aeruginosa iron homeostasis mechanisms in static conditions.