Multiple Mechanisms Are Involved in Repression of Filamentous Phage SW1 Transcription by the DNA-Binding Protein FpsR |
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| Affiliation: | 1. State Key Laboratory of Microbial Metabolism, School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai 200240, PR China;2. State Key Laboratory of Ocean Engineering, School of Naval Architecture, Ocean and Civil Engineering, Shanghai Jiao Tong University, Shanghai 200240, PR China;3. Laboratory for Marine Biology and Biotechnology, Qingdao National Laboratory for Marine Science and Technology, Qingdao 266237, PR China;4. Department of Pharmacology and Chemical Biology, Institute of Medical Sciences, School of Medicine, Shanghai Jiao Tong University, Shanghai 200025, PR China;1. Departamento de Ciencia y Tecnología, CONICET, Universidad Nacional de Quilmes, Roque Sáenz Peña 352, B1876BXD, Bernal, Provincia de Buenos Aires, Argentina;2. Department of Biomedical Sciences, University of Padua, Padua, Italy;1. Department of Biochemistry and Biophysics, Stockholm University, SE-106 91 Stockholm, Sweden;2. Science for Life Laboratory, Stockholm University, Box 1031, SE-171 21 Solna, Sweden;1. Great Lakes Bioenergy Research Center, Madison, WI 53706 USA;2. Department of Biochemistry, University of Wisconsin, Madison, WI 53706 USA;3. Research Faculty of Agriculture, Hokkaido University, Sapporo, 060-8589 Japan;4. Department of Chemistry, University of Wisconsin, Oshkosh, 54901 USA;5. DOE Joint Genome Institute, Walnut Creek, CA 94598 USA |
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| Abstract: | SW1 is the first filamentous phage isolated from a deep-sea environment. Nevertheless, the mechanism by which the SW1 genetic switch is controlled is largely unknown. In this study, the function of the phage-encoded FpsR protein was characterized by molecular biological and biochemical analyses. The deletion of fpsR increased the copy number of SW1 ssDNA and mRNA, indicating that FpsR functions as a repressor. In addition, transcription from the fpsR promoter was shown to be increased in an fpsR deletion mutant, suggesting self-repression by FpsR. Purified FpsR bound to four adjacent operator sites (O1–O4) embedded within the fpsA promoter and the fpsA–fpsR intergenic region. A surface plasmon resonance experiment showed that FpsR can bind to the O1–O4 operators separately and with different binding affinity, and the dissociation constants of FpsR with O2 and O3 were found to be lower at 4 °C than at 20 °C. A gel permeation chromatography assay revealed that FpsR oligomerized to form tetramers. Point mutation analysis indicated that the C-terminal domain influenced the binding affinity and regulatory function of FpsR. Collectively, these data support a model in which FpsR actively regulates phage production by interacting with the corresponding operators, thus playing a crucial role in the SW1 genetic switch. |
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