LTQ-XL mass spectrometry proteome analysis expands the Pseudomonas aeruginosa AmpR regulon to include cyclic di-GMP phosphodiesterases and phosphoproteins, and identifies novel open reading frames

Pseudomonas aeruginosa is well known for its antibiotic resistance and intricate regulatory network, contributing to its success as an opportunistic pathogen. This study is an extension of our transcriptomic analyses (microarray and RNA-Seq) to understand the global changes in PAO1 upon deleting a gene encoding a transcriptional regulator AmpR, in the presence and absence of β-lactam antibiotic. This study was performed under identical conditions to explore the proteome profile of the ampR deletion mutant (PAOδ. ampR) using LTQ-XL mass spectrometry. The proteomic data identified ~. 53% of total PAO1 proteins and expanded the master regulatory role of AmpR in determining antibiotic resistance and multiple virulence phenotypes in P. aeruginosa. AmpR proteome analysis identified 853 AmpR-dependent proteins, which include 102 transcriptional regulators and 21 two-component system proteins. AmpR also regulates cyclic di-GMP phosphodiesterases (PA4367, PA4969, PA4781) possibly affecting major virulence systems. Phosphoproteome analysis also suggests a significant role for AmpR in Ser, Thr and Tyr phosphorylation. These novel mechanisms of gene regulation were previously not associated with AmpR. The proteome analysis also identified many unannotated and misannotated ORFs in the P. aeruginosa genome. Thus, our data sheds light on important virulence regulatory pathways that can potentially be exploited to deal with P. aeruginosa infections. Biological significance: The AmpR proteome data not only confirmed the role of AmpR in virulence and resistance to multiple antibiotics, but also expanded the perimeter of AmpR regulon. The data presented here points to the role of AmpR in regulating cyclic di-GMP levels and phosphorylation of Ser, Thr and Tyr, adding another dimension to the regulatory functions of AmpR. We also identify some previously unannotated/misannotated ORFs in the P. aeruginosa genome, indicating the limitations of existing ORF analyses software. This study will contribute towards understanding complex genetic organization of P. aeruginosa. Whole genome proteomic picture of regulators at higher nodal positions in the regulatory network will not only help us link various virulence phenotypes but also design novel therapeutic strategies.