AlgR Phosphorylation Status Differentially Regulates Pyocyanin and Pyoverdine Production.

employs numerous, complex regulatory elements to control expression of its many virulence systems. The AlgZR two-component regulatory system controls the expression of several crucial virulence phenotypes. We recently determined, through transcriptomic profiling of a PAO1 Δ mutant strain compared to wild-type PAO1, that and are cotranscribed and show differential iron-dependent gene expression. Previous expression profiling was performed in strains without and revealed that AlgR acts as either an activator or repressor, depending on the gene. Thus, examination of gene expression from cells locked into different AlgR phosphorylation states reveals greater physiological relevance. Therefore, gene expression from strains carrying alleles 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 PAO1 mutant produced lower levels of pyoverdine but increased expression of the small RNAs and compared to PAO1. In contrast, the mutant produced more pyocyanin than wild-type PAO1. On the other hand, the PAO1 mutant produced higher levels of pyoverdine, likely due to increased expression of an iron-regulated gene encoding the sigma factor , but it had decreased pyocyanin production. AlgR specifically bound to the and promoters AlgR-dependent pyoverdine production was additionally influenced by carbon source rather than the extracellular iron concentration AlgR phosphorylation effects were also examined in a feeding, murine acute pneumonia, and punch wound infection models. Abrogation of AlgR phosphorylation attenuated virulence 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 of to establish and maintain an infection. Pyoverdine and pyocyanin production are well-known virulence 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. expressing 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 the small 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, a feeding 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.