The Dynamics of Synthesis and Localization of Jumbo Phage RNA Polymerases inside Infected Cells

Abstract

A nucleus-like structure composed of phage-encoded proteins and containing replicating viral DNA is formed in Pseudomonas aeruginosa cells infected by jumbo bacteriophage phiKZ. The PhiKZ genes are transcribed independently from host RNA polymerase (RNAP) by two RNAPs encoded by the phage. The virion RNAP (vRNAP) transcribes early viral genes and must be injected into the cell with phage DNA. The non-virion RNAP (nvRNAP) is composed of early gene products and transcribes late viral genes. In this work, the dynamics of phage RNAPs localization during phage phiKZ infection were studied. We provide direct evidence of PhiKZ vRNAP injection in infected cells and show that it is excluded from the phage nucleus. The nvRNAP is synthesized shortly after the onset of infection and localizes in the nucleus. We propose that spatial separation of two phage RNAPs allows coordinated expression of phage genes belonging to different temporal classes.

Keywords: giant phages; jumbo phages; phage nucleus; phiKZ; pseudo-nucleus; transcription regulation; virion RNA-polymerase.

Figure 1

Accumulation of PhiKZ Gp180 and Gp55 in the course of the infection. Changes in RNAP subunit amounts (determined by the intensity of bands on Western blots such as the ones shown below) are presented. For each time point, means from three independent measurements are plotted; error bars represent standard deviations. Black arrows indicate the positions of Gp180 (a) and Gp55 (b) bands; Gp55 migrates as two bands.

Figure 2

Localization of mCherry fluorescent protein, and Gp180 and Gp55 mCherry fusions during the PhiKZ infection. White arrows point to the phage nucleus (see also Figure S3). Times post-infection are indicated. All scale bars are 2 μm.

Figure 3

Localization of PhiKZ vRNAP in infected cells. (a). Co-localization of DAPI-stained DNA and the mCherry signals in PhiKZ phage particles in the lysate of mCherry-gp180-containing cells revealed by fluorescent microscopy. The scale bars are 1 μm. Phage particles from the lysate of mCherry-containing and native PAO1 strain cells are shown as controls. (b). Images of PAO1 cells infected with mCherry-Gp180-containing phiKZ phage. Yellow arrows point to mCherry-Gp180, red arrows point to the area of concentrated DNA, and white arrows to phage nucleus. Times indicate minutes post infection. In the top row, cell contour is delineated in gray. The scale bars are 2 μm.