Pseudomonas aeruginosa mediates PqsA-dependent iron regulation of the RsmY and RsmZ sRNAs in static conditions

Pseudomonas aeruginosa is an opportunistic Gram-negative pathogen that causes acute and chronic lung infection in compromised hosts. During infection, the host innate immune system restricts iron to limit microbial growth. In response, P. aeruginosa induces expression of numerous virulence genes. Recently, our lab showed that some virulence factors are responsive to iron limitation in static but not shaking growth conditions, the former of which is likely to mimic growth in the chronically-infected lung. One of these novel iron-responsive factors is the HSI-2-type six secretion system (T6SS), which is also induced during chronic infection. Iron regulation of T6SS was partially impacted by deletion of the iron-responsive PrrF sRNA and completely dependent upon the Pseudomonas quinolone signal (PQS) biosynthetic gene pqsA. Here, we analyzed the impact of iron on the expression of two small regulatory RNAs (sRNAs), RsmY and RsmZ, that activate expression of T6SS by sequestering the RsmA translation inhibitor. Our results demonstrate that iron starvation induces expression of RsmY and RsmZ in static but not shaking cultures. We further show that this induction occurs through the rsmY and rsmZ promoters and is dependent upon PqsA. We identified interrupted palindromes in the rsmY and rsmZ promoters as putative PqsR binding sites, and disruption of these sites eliminated iron-dependent regulation of rsmY and rsmZ promoter activity. To determine if iron-dependent regulation of the Rsm sRNAs is likely responsible for iron regulation of HSI-2 T6SS, we constructed translational and transcriptional reporters of the hsiA2 T6SS gene. Analysis of these reporters revealed robust PqsA-mediated iron regulation of the transcriptional reporter, as well as modest PrrF-dependent iron regulation of the translational reporter. Taken together, our results show novel iron regulatory pathways that are promoted by static growth, highlighting the importance of studying regulatory mechanisms in static communities that are likely more representative of chronic P. aeruginosa infections. IMPORTANCEIron is a central component of various bacterial metabolic pathways making it an important host acquired nutrient for pathogens to establish infection. Previous iron regulatory studies primaried relied on shaking bacterial cultures; while these ensure cultural homogeneity they do not reflect growth conditions during infection. We recently showed that static growth of Pseudomonas aeruginosa promotes iron-dependent regulation of a type six secretion system (T6SS), a virulence factor that is induced during chronic infections. In the current study, we found that static growth also promotes iron-dependent regulation of the RsmY and RsmZ sRNAs, which are global regulators that affect T6SS during chronic P. aeruginosa lung infection. Hence, our work demonstrates the Rsm sRNAs as potential effectors of iron regulation during static growth that may also be relevant in chronic infection.