Abstract 2369 Biophysical and Mechanistic Characterization of the Pseudomonas aeruginosa BqsR/BqsS Two-Component System

Pseudomonas aeruginosa (P. aeruginosa) is a ubiquitous Gram-negative bacterium that is one of the major causes of chronic nosocomial infections. P. aeruginosa can grow either as planktonic or within a biofilm, a complex network that allows bacteria to be significantly more resistant to antibiotics. Studies have shown that the two-component signal transduction system BqsR/S regulates biofilm formation/decay in P. aeruginosa through the detection of extracellular Fe2+. PaBqsS has been identified as a transmembrane sensor histidine kinase (HK) while PaBqsR has been identified as a cytosolic response regulator (RR) that binds to DNA and is capable of altering the transcription of genes involved in biofilm formation, but the mechanism of this regulatory sensing process is unknown. In this work, we have expressed, purified, and initially characterized PaBqsR using NMR structural techniques. These results reveal the presence of a well-folded, monomeric protein both in the absence of phosphorylation and in a pseudo-phosphorylated state. Using X-ray crystallography, we have solved the structure of the N-terminal phosphorylation domain of PaBqsR to 1.3 Å resolution, revealing a canonical (βα)5 response regulator assembly that consists of a central five-stranded parallel β-sheet surrounded by five helices. Interestingly, we have found that the reported PaBqsR DNA-binding consensus sequence is located upstream of important iron transport genes within the P. aeruginosa genome. Electrophoretic mobility shift assays (EMSAs) show that PaBqsR is indeed capable of binding upstream of these genes, but only in the pseudo-phosphorylated state. We have also explored the metal-binding capabilities of PaBqsS. We first tested the ability of purified PaBqsS to bind Fe2+ and we show that the WT protein binds 1 metal ion per HK dimer. We have used X-ray absorption spectroscopy to show that Fe2+ is bound in an octahedral coordination with hard Lewis acid (N/O) donors surrounding the metal ion. Using site-directed mutagenesis experiments, we altered a key periplasmic RExxE motif to show that this site is the location of Fe2+ binding at a dimeric interface. When taken together, these results provide the first biophysical characterization of the Bqs system and demonstrate an important connection between the BqsR/S system and a conserved iron acquisition system that functions as an important P. aeruginosa virulence factor. This work was sponsored by HHMI Gilliam Fellowship GT15765 and NIH grant R35GM133497.