Browsing by Subject "Phagen-Display"

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Biogenese und Virusassembly des filamentösen Coliphagen M13
(2012) Ploß, Martin; Kuhn, Andreas
Taxonomically, the bacteriophage M13 is assigned to the single-stranded DNA phage and belongs to the family of Inoviridae. For propagation the Gram-negative bacteria Escherichia coli with F-pili is required. The host cell is not lysed by the phage. New findings about the M13 phage biogenesis are presented here within four essential areas of the M13 phage cycle concerning the sections infection, assembly, and phage secretion. Phage adsorption experiments in which the host bacterium E. coli K38 was infected by M13 phage showed that the phage adsorption to the cells takes place within the first 5 minutes and because of a limitation of F-pili per cell a maximum of 7 phages per cell were found to be adsorbed. The insertion of the phage coat protein gp9 into the cytoplasmic membrane of the host cell was verified by the periplasmic location of antigenic epitopes introduced into the N-terminal domain of gp9. The membrane insertion of gp9 was found to depend on the host protein YidC. Plasmid-encoded gp9 exhibiting antigenic epitopes at the N-terminal domain did not interfere with the assembly of new progeny phage. Therefore, the development of a phage display system with gp9 by introducing short peptide sequences (17 ? 36 amino acids) is feasible. After overexpression of gp1/11 assembly complexes in E. coli and size exclusion chromatography, respectively, the complex was characterized and a molecular weight of ~ 300 kDa was assigned. Examinations of the purified gp1/11 assembly complexes by transmission electron microscopy (TEM) revealed ring-like structures with ~ 7 ? 8 nm in inner diameter and ~ 11 ? 12 nm in outer diameter. The investigation of M13 wild-type infection showed that the secretion of new progeny phage starts after a short lag period (eclipse). An infected E. coli cell secreted upto 925 progeny in a time period of 115 minutes which corresponds to an average of 7 secreted phages per minute. The generation time of the infected E. coli K38 cells rose from 24 minutes to 48 minutes. Experiments were carried out with genetically manipulated phages which were hindered to synthesize the major coat protein gp8 in the host cell by a nonsense mutation in the phage genome. Therefore, phage replication was only observed in host cells bearing plasmid encoding gene 8. Since the quantity of the protein was limited the lag period (eclipse) was extended to 12 minutes and the efficiency of phage secretion was decreased to about 2 phages per minute. The M13 phage secretion from infected E. coli cells was visualized by atomic force microscopy (AFM). The identity of the phage was verified by labeling with protein-A conjugated gold and transmission electron microscopy (TEM). The secretion of M13 progeny was first observed at the cell poles of E. coli and then spreaded within 4 minutes along the cell surface. After 16 minutes the secretion was observed over the entire cell surface.