Loh Belinda, Haase Maximilian, Mueller Lukas, Kuhn Andreas, Leptihn Sebastian
Sebastian Leptihn, Institute of Microbiology and Molecular Biology, University of Hohenheim, Garbenstrasse 30, 70599 Stuttgart, Germany.
Viruses. 2017 Apr 9;9(4):73. doi: 10.3390/v9040073.
In contrast to lytic phages, filamentous phages are assembled in the inner membrane and secreted across the bacterial envelope without killing the host. For assembly and extrusion of the phage across the host cell wall, filamentous phages code for membrane-embedded morphogenesis proteins. In the outer membrane of Escherichia coli, the protein gp4 forms a pore-like structure, while gp1 and gp11 form a complex in the inner membrane of the host. By comparing sequences with other filamentous phages, we identified putative Walker A and B motifs in gp1 with a conserved lysine in the Walker A motif (K14), and a glutamic and aspartic acid in the Walker B motif (D88, E89). In this work we demonstrate that both, Walker A and Walker B, are essential for phage production. The crucial role of these key residues suggests that gp1 might be a molecular motor driving phage assembly. We further identified essential residues for the function of the assembly complex. Mutations in three out of six cysteine residues abolish phage production. Similarly, two out of six conserved glycine residues are crucial for gp1 function. We hypothesise that the residues represent molecular hinges allowing domain movement for nucleotide binding and phage assembly.
与裂解性噬菌体不同，丝状噬菌体在内膜中组装，并穿过细菌包膜分泌，而不会杀死宿主。为了使噬菌体在宿主细胞壁上组装和挤出，丝状噬菌体编码膜嵌入的形态发生蛋白。在大肠杆菌的外膜中，gp4蛋白形成一种孔状结构，而gp1和gp11在宿主的内膜中形成复合物。通过将序列与其他丝状噬菌体进行比较，我们在gp1中鉴定出推测的沃克A和沃克B基序，沃克A基序中有一个保守的赖氨酸（K14），沃克B基序中有一个谷氨酸和一个天冬氨酸（D88、E89）。在这项研究中，我们证明沃克A和沃克B对噬菌体产生都是必不可少的。这些关键残基的关键作用表明，gp1可能是驱动噬菌体组装的分子马达。我们进一步鉴定了组装复合物功能的必需残基。六个半胱氨酸残基中的三个发生突变会导致噬菌体产生消失。同样，六个保守甘氨酸残基中的两个对gp1功能至关重要。我们推测这些残基代表分子铰链，允许结构域移动以进行核苷酸结合和噬菌体组装。
