Biochemical Characterization of the Enhancer-Binding Protein DdaR of Pseudomonas aeruginosa
Date of Award
Christopher T. Nomura
bacteria, cystic fibrosis
Pseudomonas aeruginosa is a bacterium that forms biofilms in the lungs of individuals with cystic fibrosis. Biofilm formation is regulated by nitric oxide synthase, which is known to produce nitric oxide from the substrate L-arginine. Pathogenic bacteria are released from biofilms in the presence of nitric oxide. A product of methylarginine metabolism in P. aeruginosa is NG,NG-dimethyl-L-arginine (ADMA), which inhibits nitric oxide synthase and prevents the formation of nitric oxide. The PA1195 (ddaH) gene of P. aeruginosa encodes a dimethylarginine dimethylaminohydrolase (DdaH) enzyme for methylarginine degradation, such as ADMA to L-citrulline. The protein DdaR may act as an enhancer-binding protein (EBP) and regulate the activation of σ54-dependent transcription for this gene. In this study we heterologously expressed the DdaR protein as an N-terminal Maltose Binding Protein (MBP) fusion-protein in Escherichia coli BL21 cells, and subsequently purified the MBP-DdaRdbd fusion-protein using amylose affinity chromatography. Polymerase chain reaction (PCR) was used to replicate the DNA probes ZS458 and ZS406. The ZS458 probe contains the ddaH promoter sequence and is the expected target promoter of DdaR. The ZS406 probe contains the gcvH2 promoter. DdaR is not expected to bind to this probe and hence it will serve as a negative control probe. The ZS406 and ZS458 probes were purified at a concentration of 9.1 ng/μL and 8.2 ng/μL, respectively. Future research will focus on utilizing the purified DdaR protein and ZS458 and ZS406 probes in electrophoretic mobility shift assays (EMSAs) to determine if DdaR binds to the promoter region of the ddaH gene in P. aeruginosa.
Allison, Christina M., "Biochemical Characterization of the Enhancer-Binding Protein DdaR of Pseudomonas aeruginosa" (2018). Honors Theses. 131.