Book of Abstracts: Albany 2011
June 14-18 2011
©Adenine Press (2010)
Characterization of erp Operator DNA Binding by Borrelia burgdorferi Protein BpaB.
BpaB is an erp Operator DNA binding protein expressed by Borrelia burgdorferi, the causative agent of Lyme disease. BpaB and 2 other transcription regulatory proteins, EbfC and BpuR proteins control erp transcription levels. Erp proteins bind host factor H, a regulator of complement activation. By doing so they help protect the bacterium from the alternative pathway that involves complement-mediated killing. Transcription and expression of Erp proteins occurs only upon association with mammalian host blood or tissue, when multiple environmental signals, including temperature induce expression.
BpaB has recently been identified as an erp transcription repressor and competes for binding to erp Operator DNA with a second B. burgdorferi DNA-binding protein, EbfC (1). Mutagenesis of erp Operator DNA and EMSA binding specificity measurements show that BpaB binds within a 15 bp region just 5’ of the -35 sequence on erp operator 2 DNA. DNA Footprinting experiments also indicate that BpaB binds initially within the 15 bp erp DNA region after which it then initiates nonspecific binding to flanking regions. BpaB binds initially at a stoichiometry of 1, to a region of erp Operator DNA that contains the 15 bp binding region. Further complex formations observed on EMSA gels indicated that 1 protein was being added in a step wise fashion. Ultracentrifugation equilibrium analysis indicated that up to 13 proteins can attach to the same 23 bp erp DNA oligomer thereby supporting footprinting results. Similar analysis also showed that 24 proteins are capable of binding to a 50 bp erp dsDNA oligomer. EMSA binding assays indicate a Kd/dissociation constant of ~0.25 µM for binding to 50 bp erp dsDNA. Experiments are under way to determine if BpaB binding to erp operator DNA is cooperative.
Department of Microbiology, Immunology and Molecular Genetics, University of Kentucky Department of Microbiology, Immunology and Molecular Genetics, University of Kentucky, College of Medicine, Lexington, Kentucky 40536-0298