The roles of SaPI1 proteins gp7 (CpmA) and gp6 (CpmB) in capsid size determination and helper phage interference

Abstract

SaPIs are molecular pirates that exploit helper bacteriophages for their own high frequency mobilization. One striking feature of helper exploitation by SaPIs is redirection of the phage capsid assembly pathway to produce smaller phage-like particles with T=4 icosahedral symmetry rather than T=7 bacteriophage capsids. Small capsids can accommodate the SaPI genome but not that of the helper phage, leading to interference with helper propagation. Previous studies identified two proteins encoded by the prototype element SaPI1, gp6 and gp7, in SaPI1 procapsids but not in mature SaPI1 particles. Dimers of gp6 form an internal scaffold, aiding fidelity of small capsid assembly. Here we show that both SaPI1 gp6 (CpmB) and gp7 (CpmA) are necessary and sufficient to direct small capsid formation. Surprisingly, failure to form small capsids did not restore wild-type levels of helper phage growth, suggesting an additional role for these SaPI1 proteins in phage interference.

Figure 1

Effects of deletion of SaPI1 cpmA and cpmB. (A) Cryo-electron micrograph (left) and size distribution plot (right) of SaPI1 wild-type particles (RN10628). The dimensions (nm) of the short (width) and long (length) axes of each particle were plotted on the x and y axes, respectively. (B) Cryo-EM and size distribution plot of SaPI1 ΔcpmA particles (cpmB⁺; ST98). Several particles that appear to be partly filled with DNA are indicated by the arrows. (C) Cryo-EM and size distribution plot of SaPI1 ΔcpmB particles (cpmA⁺; ST100). One particle of each different type is indicated: sf, small full capsid; lf, large full capsid; se, small empty capsid; le, large empty capsid; a, aberrant shell. (D) Cryo-EM and size distribution plot of SaPI1 ΔcpmAB particles (ST126). The scale bars represent 100 nm. The number of particles counted (n) is shown for each plot.

Figure 2

Incorporation of CpmA and CpmB into 80α-sized procapsids. (A) Cryo-EM of SaPI1 procapsids isolated from ST98 (80α, SaPI1 ΔcpmA). The scale bar represents 100 nm. (B) SDS-PAGE of CsCl gradient-separated procapsids from ST116 (80α ΔterS, SaPI1 ΔterS ΔcpmB) and ST117 (80α ΔterS, SaPI1 ΔterS ΔcpmA). M, marker; molecular mass, as indicated (kDa). The positions of the major 80α capsid protein gp47 (CP), scaffold protein gp46 (CP), and contaminating major tail protein gp53 (TP) are indicated, as are positions of SaPI1 proteins CpmA (in ST116) and CpmB (in ST117).

Figure 3

Effects of insertion of SaPI1 cpmA and cpmB into 80α. (A) Cryo-EM (left) and size distribution plot (right) of 80α wild-type particles. (B) Cryo-EM and size distribution plot of 80α::SaPI1 cpmA particles (ST97). (C) Cryo-EM and size distribution plot of 80α::SaPI1 cpmB particles (ST99). (D) Cryo-EM and size distribution plot of 80α::SaPI1 cpmAB particles (ST82). Features in the size distribution plots are the same as those in Figure 1. The scale bars represent 100 nm.