The Transmembrane Morphogenesis Protein gp1 of Filamentous Phages Contains Walker A and Walker B Motifs Essential for Phage Assembly

Abstract

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.

Keywords: ATPase; M13; Walker motifs; assembly complex; filamentous phage; gp1; membrane protein; molecular hinge; phage assembly; secretion; zonula occludens toxin (Zot).

Figure 1

(A) Representative “spot assay” in which serial dilutions of phages were spotted on a bacterial lawn in order to analyse the amount of complementation compared to the wild-type. As controls, amberI phages were always spotted on the non-suppressor strain Escherichia coli K38 (a) and the amber suppressor strain E. coli K37 (b) to determine the phage titer of the solution. For complementation in trans, the E. coli strain M15 was used (c, d). Plasmid-encoded wild-type gp1 showed plaques to about the same dilution level as E. coli K37 (c), demonstrating that the induction of the protein in trans is able to fully complement the amber gene. A mutation of the internal start codon to leucine (M241L) had no effect, since gp11 was still made in the amber phage (d). (B) Quantification of phage titer from in vivo complementations of geneI. The amount of phages produced using plasmid-encoded wild-type gp1 is near-identical to the phage titer determined by using the amber-suppressor strain E. coli K37. Plasmid-encoded gp1 was normalised to 1, plotted on a logarithmic scale, and compared with the gp1 mutant M241L. The M241L mutation has no effect in this assay, although only gp1 is produced from plasmid-derived mRNA. The amberI phage still allows the expression of gp11, thus allowing full complementation of the gene, as both gene products are essential for the functionality of the protein [13]. (C) Schematic representation of the protein gp1 with its internal open reading frame (ORF), gp11, at methionine at position 241. The transmembrane (TM) segment is depicted in green. Numbers below indicate amino acid residue positions.

Figure 2

(A) Quantified phage titer in in vivo complementation assays of geneI mutants in the Walker A motif. The amount of phages produced using plasmid-encoded wild-type gp1 was normalised to 1 and compared with gp1 mutants, plotted on a logarithmic scale. The codon for lysine residue in position 14 (K14) in the putative Walker A motif was mutated to alanine (K14A), arginine (K14R), proline (K14P), glutamic acid (K14E), and tryptophan (K14W). None of the mutations can substitute for the lysine, indicating a crucial catalytic role of the residue contained within the motif. In addition, the effect of the mutation of lysine in position 9 to alanine (K9A) is shown on the right. The mutation in this position does not influence phage production. (B) In vivo complementation of geneI mutants in the putative Walker B motif. A mutation of the codon in position 88 from aspartate to asparagine (D88N) abolishes phage production. Similarly, the exchange of the residue glutamate to glutamine in position 89 (E89Q) results in the loss of phage production. (C) Schematic representation of the gp1 protein with its internal ORF g11p. The two Walker motifs, Walker A and Walker B, are shown in purple with the respective sequences below. Numbers indicate amino acid residues. Key residues crucial for phage production are in bold. The transmembrane (TM) segment is depicted in green.

Figure 3

(A) Quantified phage titer in in vivo complementation assays of geneI cysteine mutants. The amount of phages produced using plasmid-encoded wild-type gp1 was normalized to 1 and compared with gp1 mutants, plotted on a logarithmic scale. While the substitution of cysteine with the chemically similar residue serine had no effect on protein function in position 30 (C30S), 146 (C146S), and 256 (C256S), mutations in positions 90 (C90S), 332 (C332S), and 347 (C347S) severely affected the production of phages. (B) Western blot analysis showing the migration pattern of His-tagged gp1 quadruple mutant (C30S, C90S, C146S, C256S) under reducing and non-reducing conditions. Samples were either not boiled (✗) or boiled (✓). (C) Schematic representation of the protein gp1 with its internal ORF gp11. The two Walker motifs, Walker A and Walker B, are shown in purple, the TM in green, and the cysteine residues are depicted in yellow. Numbers refer to the residue of the cysteines within gp1/gp11.

Figure 4

(A) Quantified phage titer in in vivo complementation assays of geneI glycine mutants. The amount of phages produced using plasmid-encoded wild-type gp1 was normalised to 1 and compared with gp1 mutants, plotted on a logarithmic scale. While the drastic structural substitution from glycine to proline had no effect on protein function in position 229 (G229P), 260 (G260P), 47 (G47P), and 197 (G197P), mutations in positions 29 (G29P) and 118 (G118P) severely affected the production of phages. Statistical analysis shown is a result of three independent experiments. (B) Model of gp1 illustrating the possible function of G29 and G118 in mediating conformational change. (C) Schematic representation of the gp1 protein with its internal ORF g11p. The two Walker motifs, Walker A and Walker B, are shown in purple, the TM in green, and the conserved glycine residues are depicted in light blue. The numbers refer to the glycine residues within gp1/gp11.