Multiple Mechanisms Are Involved in Repression of Filamentous Phage SW1 Transcription by the DNA-Binding Protein FpsR

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 fpsRpromoterwas shown to be increased in an fpsRdeletion 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 FpsRwith O2andO3were 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.

Schematic model of FpsR involvement in the genetic switch of phage SW1. FpsR binds to four operators that are located in the fpsA promoter and in the intergenic region between fpsA and fpsR. The transcriptions of PA and PR by RNA polymerase were inhibited due to the steric hindrance and “Roadblock” mechanisms. The genes and regulatory elements are not drawn to scale.

Reference:

Huahua Jian, Guanpeng Xu, Shunzhang Liu, Yali Hao, Canxing Meng, Jianrong Xu, Yue Zhang, Xipeng Liu, Xiang Xiao. (2019). Multiple Mechanisms Are Involved in Repression of Filamentous Phage SW1 Transcription by the DNA-Binding Protein FpsR. Journal of Molecular Biology 431, 1113-1126.

Link: www.sciencedirect.com/science/article/abs/pii/S0022283618309215