title: Structural and functional characterization of Pseudomonas aeruginosa global regulator AmpR.
abstract: Pseudomonas aeruginosa is a dreaded pathogen in many clinical settings. Its inherent and acquired antibiotic resistance thwarts therapy. In particular, derepression of the AmpC β-lactamase is a common mechanism of β-lactam resistance among clinical isolates. The inducible expression of ampC is controlled by the global LysR-type transcriptional regulator (LTTR) AmpR. In the present study, we investigated the genetic and structural elements that are important for ampC induction. Specifically, the ampC (PampC) and ampR (PampR) promoters and the AmpR protein were characterized. The transcription start sites (TSSs) of the divergent transcripts were mapped using 5' rapid amplification of cDNA ends-PCR (RACE-PCR), and strong σ(54) and σ(70) consensus sequences were identified at PampR and PampC, respectively. Sigma factor RpoN was found to negatively regulate ampR expression, possibly through promoter blocking. Deletion mapping revealed that the minimal PampC extends 98 bp upstream of the TSS. Gel shifts using membrane fractions showed that AmpR binds to PampC in vitro whereas in vivo binding was demonstrated using chromatin immunoprecipitation-quantitative PCR (ChIP-qPCR). Additionally, site-directed mutagenesis of the AmpR helix-turn-helix (HTH) motif identified residues critical for binding and function (Ser38 and Lys42) and critical for function but not binding (His39). Amino acids Gly102 and Asp135, previously implicated in the repression state of AmpR in the enterobacteria, were also shown to play a structural role in P. aeruginosa AmpR. Alkaline phosphatase fusion and shaving experiments suggest that AmpR is likely to be membrane associated. Lastly, an in vivo cross-linking study shows that AmpR dimerizes. In conclusion, a potential membrane-associated AmpR dimer regulates ampC expression by direct binding.
authors.affiliation: Department of Molecular Microbiology and Infectious Diseases, Herbert Wertheim College of Medicine, Florida International University, Miami, Florida, USA.
authors.forename: Olivier
authors.initials: O
authors.lastname: Caille
authors.affiliation: Department of Biological Sciences, College of Arts and Sciences, Florida International University, Miami, Florida, USA.
authors.forename: Diansy
authors.initials: D
authors.lastname: Zincke
authors.affiliation: Department of Microbiology and Molecular Genetics, Harvard Medical School, Boston, Massachusetts, USA.
authors.forename: Massimo
authors.initials: M
authors.lastname: Merighi
authors.affiliation: Department of Biological Sciences, College of Arts and Sciences, Florida International University, Miami, Florida, USA.
authors.forename: Deepak
authors.initials: D
authors.lastname: Balasubramanian
authors.affiliation: Department of Molecular Microbiology and Infectious Diseases, Herbert Wertheim College of Medicine, Florida International University, Miami, Florida, USA.
authors.forename: Hansi
authors.initials: H
authors.lastname: Kumari
authors.affiliation: Department of Biological Sciences, College of Arts and Sciences, Florida International University, Miami, Florida, USA.
authors.forename: Kok-Fai
authors.initials: KF
authors.lastname: Kong
authors.affiliation: Department of Molecular Microbiology and Infectious Diseases, Herbert Wertheim College of Medicine, Florida International University, Miami, Florida, USA.
authors.forename: Eugenia
authors.initials: E
authors.lastname: Silva-Herzog
authors.affiliation: School of Computing and Information Science, College of Engineering and Computing, Florida International University, Miami, Florida, USA.
authors.forename: Giri
authors.initials: G
authors.lastname: Narasimhan
authors.affiliation: Department of Molecular Microbiology and Infectious Diseases, Herbert Wertheim College of Medicine, Florida International University, Miami, Florida, USA.
authors.forename: Lisa
authors.initials: L
authors.lastname: Schneper
authors.affiliation: Department of Microbiology and Molecular Genetics, Harvard Medical School, Boston, Massachusetts, USA.
authors.forename: Stephen
authors.initials: S
authors.lastname: Lory
authors.affiliation: Department of Molecular Microbiology and Infectious Diseases, Herbert Wertheim College of Medicine, Florida International University, Miami, Florida, USA Kalai.Mathee@fiu.edu.
authors.forename: Kalai
authors.initials: K
authors.lastname: Mathee
mesh_terms: D020816:Amino Acid Motifs; D000900:Anti-Bacterial Agents / Q000494:pharmacology; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002874:Chromosome Mapping; D002876:Chromosomes, Bacterial; D016384:Consensus Sequence; D024881:Drug Resistance, Bacterial; D015964:Gene Expression Regulation, Bacterial / Q000502:physiology*; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D011487:Protein Conformation; D011550:Pseudomonas aeruginosa / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D018440:beta-Lactam Resistance; D001618:beta-Lactamases / Q000235:genetics / Q000378:metabolism; D047090:beta-Lactams / Q000494:pharmacology
chemical_list: D000900:Anti-Bacterial Agents; D001426:Bacterial Proteins; D047090:beta-Lactams; C071526:AmpR protein, Bacteria; C048795:AmpC beta-lactamases; D001618:beta-Lactamases