Complete Title of Thesis:
"Characterization of the AlgZR two-component signal transduction system and the regulation of hydrogen cyanide production in Pseudomonas aeruginosa"
Pseudomonas aeruginosa is an opportunistic pathogen that causes chronic infection in
individuals suffering from the genetic disorder cystic fibrosis. In P. aeruginosa, the transcriptional
regulator AlgR controls a variety of virulence factors, including alginate production, twitching
motility, biofilm formation, quorum sensing, and hydrogen cyanide (HCN) production. This study characterizes the AlgZR two-component signal transduction system and the regulation of cyanogenesis.
HCN production was measured by cyanide ion-selective electrode (ISE) assay, which revealed that cyanogenesis was elevated in bacteria grown on an agar surface compared to bacteria grown in planktonic culture. Additionally, HCN production was optimal in a 5% 02 environment as the bacteria entered stationary phase and was RpoN and RpoS dependent. Nuclease protection mapping of the hcnA promoter identified a previously unidentified transcriptional start site. A subset of laboratory and clinical isolates lacking a portion of the hcnA promoter, including the newly identified transcriptional start site and the AlgR binding site, produced small amounts of cyanide.
Strains lacking the response regulator, AlgR or the kinase sensor AlgZ, produced significantly less HCN than did a non-mucoid isogenic parent. In contrast, algR and algZ mutants showed increased HCN production in an alginate-producing (mucoid) background. This regulation only occurred when strains were grown on solid media, as opposed to broth or static biofilm and is independent of the AlgR dependent fimU-pilVWXY1Y2E operon. While autophosphorylation of purified AlgZ, or a C-terminal truncation, has yet to be demonstrated, both the conserved AlgR phosphorylation site, which is required for twitching motility but not alginate production, and the conserved histidine within the AlgZ "H" box (amino acid position 175) were found to be critical for cyanide production. Contrary to predictions by topology algorithms, Western blot analysis of both a N-terminal S-protein tagged AlgZ and a C-terminal Strepll tag AlgZ indicates that the protein is located in the cytoplasm.
This study shows HCN is maximally produced under microaerobic conditions when the organism is surface attached and that production is regulated by the interaction of AlgZ with AlgR, indicating that the pair represent a cytoplasmic sensor kinase and response regulator in a two component signal transduction system.