A-045. Elucidating the Pseudomonas aeruginosa AmpR Regulon

D. Balasubramanian1, L. Schneper1, O. Caille1, M. Merighi2, A. Brencic2, R. Smith2, E. Zeng1, S. Lory2, G. Narasimhan1, K. Mathee1;
1Florida International Univ., Miami, FL, 2Harvard Med. Sch., Boston, MA.

Background: In Enterobacteriaceae, a member of the LysR-type modulator family, AmpR, regulates the expression of the chromosomal ß-lactamase ampC. In Pseudomonas aeruginosa, an opportunistic pathogen responsible for the morbidity and mortality of patients with cystic fibrosis, very little is known of the regulatory mechanisms that control ß-lactamase expression. In addition to regulating ampC, AmpR also regulates the production of many virulence factors including elastase, staphylolytic protease, pyocyanin, and the quorum sensing systems. This led us to hypothesize that AmpR could be a global regulator. The aim of this study is to further elucidate the AmpR regulon. Methods: The characteristics of a wild-type prototypic P. aeruginosa PAO1 strain with an isogenic ampR mutant, PAOampR were compared. Using Iterative Enhancement of Motifs (IEM), an algorithm that we developed to identify protein-binding sites in promoters, we determined AmpR binding sites upstream of many open reading frames in P. aeruginosa. In order to better understand the ampR regulon, we performed transcriptional profiling using DNA microarrays with an ampR deletion mutant PAOΔampR. Purified histidine-tagged-AmpR was was used in gel mobility shift assays to determine if these genes are directly regulated by AmpR. Results: Our hypothesis that AmpR is part of a regulon was confirmed in our preliminary transcriptome analysis since the deletion of ampR influenced the expression of over 200 genes that are either upregulated or downregulated. Genes that were identified to be AmpR-regulated in previous studies and IEM were also identified in microarray analysis. Similarly, purified AmpR was able to bind to promoters containing putative AmpR binding sites. Conclusions: Our experiments support the hypothesis that AmpR functions as a global regulator in P. aeruginosa. Ongoing research is focused on further elucidating this complex regulatory circuit that has the potential of helping to develop better therapeutic measures against chronic P. aeruginosa infection.