ABSTRACTPseudomonas aeruginosaemploys numerous, complex regulatory elements to control expression of its many virulence systems. TheP. aeruginosaAlgZR two-component regulatory system controls the expression of several crucial virulence phenotypes. We recently determined, through transcriptomic profiling of a PAO1 ΔalgRmutant strain compared to wild-type PAO1, thatalgZRandhemCDare cotranscribed and show differential iron-dependent gene expression. Previous expression profiling was performed in strains withoutalgRand revealed that AlgR acts as either an activator or repressor, depending on the gene. Thus, examination ofP. aeruginosagene expression from cells locked into different AlgR phosphorylation states reveals greater physiological relevance. Therefore, gene expression from strains carryingalgRalleles encoding a phosphomimetic (AlgR D54E) or a phosphoablative (AlgR D54N) form were compared by microarray to PAO1. Transcriptome analyses of these strains revealed 25 differentially expressed genes associated with iron siderophore biosynthesis or heme acquisition or production. The PAO1algR D54Nmutant produced lower levels of pyoverdine but increased expression of the small RNAsprrf1andprrf2compared to PAO1. In contrast, thealgR D54Nmutant produced more pyocyanin than wild-type PAO1. On the other hand, the PAO1algR D54Emutant produced higher levels of pyoverdine, likely due to increased expression of an iron-regulated gene encoding the sigma factorpvdS, but it had decreased pyocyanin production. AlgR specifically bound to theprrf2andpvdSpromotersin vitro. AlgR-dependent pyoverdine production was additionally influenced by carbon source rather than the extracellular iron concentrationper se. AlgR phosphorylation effects were also examined in aDrosophila melanogasterfeeding, murine acute pneumonia, and punch wound infection models. Abrogation of AlgR phosphorylation attenuatedP. aeruginosavirulence in these infection models. These results show that the AlgR phosphorylation state can directly, as well as indirectly, modulate the expression of iron acquisition genes that may ultimately impact the ability ofP. aeruginosato establish and maintain an infection.IMPORTANCEPyoverdine and pyocyanin production are well-knownP. aeruginosavirulence factors that obtain extracellular iron from the environment and from host proteins in different manners. Here, we show that the AlgR phosphorylation state inversely controls pyoverdine and pyocyanin production and that this control is carbon source dependent.P. aeruginosaexpressing AlgR D54N, mimicking the constitutively unphosphorylated state, produced more pyocyanin than cells expressing wild-type AlgR. In contrast, a strain expressing an AlgR phosphomimetic (AlgR D54E) produced higher levels of pyoverdine. Pyoverdine production was directly controlled through theprrf2small regulatory RNA and the pyoverdine sigma factor, PvdS. Abrogating pyoverdine or pyocyanin gene expression has been shown to attenuate virulence in a variety of models. Moreover, the inability to phosphorylate AlgR attenuates virulence in three different models, aDrosophila melanogasterfeeding model, a murine acute pneumonia model, and a wound infection model. Interestingly, AlgR-dependent pyoverdine production was responsive to carbon source, indicating that this regulation has additional complexities that merit further study.