Pseudomonas aeruginosa AlgR Phosphorylation Status Differentially Regulates Pyocyanin and Pyoverdine Production

Abstract

Pseudomonas aeruginosa employs numerous, complex regulatory elements to control expression of its many virulence systems. The P. aeruginosa AlgZR two-component regulatory system controls the expression of several crucial virulence phenotypes. We recently determined, through transcriptomic profiling of a PAO1 ΔalgR mutant strain compared to wild-type PAO1, that algZR and hemCD are cotranscribed and show differential iron-dependent gene expression. Previous expression profiling was performed in strains without algR and revealed that AlgR acts as either an activator or repressor, depending on the gene. Thus, examination of P. aeruginosa gene expression from cells locked into different AlgR phosphorylation states reveals greater physiological relevance. Therefore, gene expression from strains carrying algR 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 algR D54N mutant produced lower levels of pyoverdine but increased expression of the small RNAs prrf1 and prrf2 compared to PAO1. In contrast, the algR D54N mutant produced more pyocyanin than wild-type PAO1. On the other hand, the PAO1 algR D54E mutant produced higher levels of pyoverdine, likely due to increased expression of an iron-regulated gene encoding the sigma factor pvdS, but it had decreased pyocyanin production. AlgR specifically bound to the prrf2 and pvdS promoters in vitro. AlgR-dependent pyoverdine production was additionally influenced by carbon source rather than the extracellular iron concentration per se. AlgR phosphorylation effects were also examined in a Drosophila melanogaster feeding, murine acute pneumonia, and punch wound infection models. Abrogation of AlgR phosphorylation attenuated P. aeruginosa 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 P. aeruginosa to establish and maintain an infection.

Metatranscriptomic Identification of Diverse and Divergent RNA Viruses in Green and Chlorarachniophyte Algae Cultures

“…Unfortunately, this C-terminal region lacks the key functional motifs usually associated with the RdRp, preventing us from definitively establishing the ALG_2_DN594 contig as a true RNA virus. Similarly, the ALG_3_DN34624 alignment with the viral RdRp sequences that comprise the PFAM RdRp_4 profile (PF02123) does not show conservation of the crucial functional residues at the A, B and C-motifs within the RdRp, particularly the canonical motif C that is normally GDD, yet GFD in ALG_3 contig ( Figure S2B ): strikingly, the GFD motif is absent from an alignment of 4627 viral RdRp sequences [ 60 ]. Whether this reflects a newly identified functional motif remains to be determined, but we cannot safely conclude that ALG_3_DN34624 encodes a viral RdRp.…”

“…Considering the high levels of sequence divergence between our viral sequences and those associated with fungi and within the clade formed by Ostreobium sp.-associated viruses, it seems likely that any such horizontal gene transfer events are not recent and may have occurred in Ulvophyceae or even Chlorophyta ancestors. It will be of considerable interest to examine this new group of mitoviruses across a larger set of green microalgae species, particularly whether their putative mitochondrial subcellular location is the result of an escape from cytoplasmic dsRNA silencing (as suggested for newly characterized plant mitoviruses [ 85 ]) or if they are relics of the eukaryotic endosymbiosis event, particularly as mitoviruses have bacterial counterparts—the Leviviridae [ 60 ]. More broadly, these newly-reported mitovirus-like sequences further illustrate the enormous diversity of hosts infected by the Narnaviridae , including such eukaryotic microorganisms as Apicomplexa, Excavates and Oomycetes hosts [ 86 , 87 , 88 , 89 ].…”

Pseudomonas aeruginosa type IV minor pilins and PilY1 regulate virulence by modulating FimS-AlgR activity

“…The observation that the loss of algR had no impact on virulence towards amoebae [ 38 ] or nematodes ( Fig 5A and 5B ) suggests that the AlgR-activated genes may not contribute to virulence, although the mechanisms of killing could differ. In mouse models, fimS and algR deletion mutants are attenuated, though overexpression of AlgR also markedly reduces virulence [ 55 , 65 , 87 ]. Further, Little et al demonstrated that PAO1 algR D54E had WT virulence in Drosophila melanogaster and mouse infection models, while an algR D54A mutant was highly attenuated [ 87 ].…”

The Pseudomonas aeruginosa PrrF1 and PrrF2 Small Regulatory RNAs Promote 2-Alkyl-4-Quinolone Production through Redundant Regulation of the antR mRNA

“…Alternatively, differential expression of the PrrF1 and PrrF2 sRNAs may allow diverse environmental signals, in addition to iron depletion, to affect PrrF regulation of metabolism and virulence. Indeed, recent work by Little and colleagues demonstrated that the P. aeruginosa AlgZR two-component regulatory system activates PrrF expression through specific interaction with the prrF2, but not the prrF1, promoter (65). The tandem duplication of the prrF genes also results in the production of a longer heme response sRNA named PrrH (66).…”