Phage G Structure at 6.1 Å Resolution, Condensed DNA, and Host Identity Revision to a Lysinibacillus - PubMed (original) (raw)

Phage G Structure at 6.1 Å Resolution, Condensed DNA, and Host Identity Revision to a Lysinibacillus

Brenda González et al. J Mol Biol. 2020.

Abstract

Phage G has the largest capsid and genome of any known propagated phage. Many aspects of its structure, assembly, and replication have not been elucidated. Herein, we present the dsDNA-packed and empty phage G capsid at 6.1 and 9 Å resolution, respectively, using cryo-EM for structure determination and mass spectrometry for protein identification. The major capsid protein, gp27, is identified and found to share the HK97-fold universally conserved in all previously solved dsDNA phages. Trimers of the decoration protein, gp26, sit on the 3-fold axes and are thought to enhance the interactions of the hexameric capsomeres of gp27, for other phages encoding decoration proteins. Phage G's decoration protein is longer than what has been reported in other phages, and we suspect the extra interaction surface area helps stabilize the capsid. We identified several additional capsid proteins, including a candidate for the prohead protease responsible for processing gp27. Furthermore, cryo-EM reveals a range of partially full, condensed DNA densities that appear to have no contact with capsid shell. Three analyses confirm that the phage G host is a Lysinibacillus, and not Bacillus megaterium: identity of host proteins in our mass spectrometry analyses, genome sequence of the phage G host, and host range of phage G.

Keywords: DNA condensates; DNA packaging; Lysinibacillus; decoration proteins; phage G.

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Figures

Figure 1:

Figure 1:. Phage G capsid cryo-EM structure.

A) A representative micrograph shows particles with various capsid states. B) Phage G dsDNA-full capsid reconstruction at 6.1 Å resolution with 2,564 particles. The structure displayed is surface colored radially using the color bar on the right.

Figure 2:

Figure 2:. Phage G major capsid protein hexamer and decoration protein trimer arrangement.

A) Phage G’s major capsid protein, gp27, and its decoration protein, gp26 arrangement. The structure shows gp27 hexamers and gp26 trimers positioned at the 3-fold axes around the hexamers. B) An overview of phage G’s gp27 homology model. All 3 domains of the structure are consistent with the domains described in HK97’s major capsid protein including the A domain, E loop, P domain. C) The phage G decoration, gp26, protein oligomerizes into trimers. The first 15 amino acids were omitted in modeling because of their flexibility. Arrows indicate the direction the N-terminus extends in the capsid density contacting neighboring gp27 subunits.

Figure 3:

Figure 3:. Lack of concentric dsDNA rings in the phage G DNA-full structure.

A) Middle volume slice of phage G’s DNA-full capsid. B) Middle volume slice of phage G’s empty capsid. C) Radial distance plots were generated for the dsDNA-full (blue) and empty (orange) phage G structures.

Figure 4:

Figure 4:. Multiple DNA states in phage G capsids seen in cryo-EM micrographs.

Multiple states of DNA packaged inside phage G capsid in our cryo-EM micrographs were categorized into 4 classes: full, empty, partial, and ruptured. Representative particles for each category are shown.

Figure 5:

Figure 5:. Histogram of partial DNA diameter.

We further analyzed the group of 84 particles categorized as having partially full, compacted DNA density, by plotting a histogram of their DNA density width measured in the shortest dimension. The measured DNA density diameter values within the group of heterogeneous DNA density phage G capsids ranged from 495 Å to 1606 Å. Some particles appear to have toroid-like organizations from the side and top views as shown in the left two insets in this figure.

Figure 6:

Figure 6:. Identification of major Phage G head proteins by mass spectrometry.

(A) Phage G genome region encoding the major head morphogenesis genes, including the terminase, portal, candidate prohead protease, major capsid protein (MCP) and head decoration protein (Dec). Genes whose products were identified by mass spectrometry are shaded blue. (B) SDS-PAGE gel of sucrose gradient purified Phage G virions. Individual gel slices that were subjected to mass spectrometric analyses are indicated. The mass spectral protein sequence coverage of the major capsid protein gp27 from different gel slices are shown in (C) gel slice 4, (D) gel slice 3, and (E) gel slice 2. Red arrow indicates cleavage by the prohead protease, gp19. The mass spectral protein sequence coverage of the head decoration protein gp26 from gel slice 2 is shown in (F). Amino-acids matched to a mass spectrum are shaded in yellow. Amino-acids marked in green potentially have a post-translational modification (e.g., phosphorylation).

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