Global changes in cellular gene expression during bacteriophage PRD1 infection

Abstract

Virus-induced changes in cellular gene expression and host physiology have been studied extensively. Still, there are only a few analyses covering the entire viral replication cycle and whole-host gene pool expression at the resolution of a single gene. Here we report changes in Escherichia coli gene expression during bacteriophage PRD1 infection using microarray technology. Relative mRNA levels were systematically measured for over 99% of the host open reading frames throughout the infection cycle. Although drastic modifications could be detected in the expression of individual genes, global changes at the whole-genome level were moderate. Notably, the majority of virus-induced changes took place only after the synthesis of virion components, indicating that there is no major reprogramming of the host during early infection. The most highly induced genes encoded chaparones and other stress-inducible proteins.

FIG. 1.

Experimental setup. E. coli K-12 JE2571 (RP4) cells, cultured in synthetic rich medium with aeration to the exponential growth phase, were infected with PRD1. A high multiplicity of infection (30 infective viruses/cell) was used to ensure that practically all of the cells were infected. (A) Turbidity values (OD₆₀₀) from three independent experiments plotted as a function of time (dashed lines, infected cells; solid lines, noninfected cells). The addition of the virus is indicated by an arrow. Samples (∼10⁹ cells) were collected for total RNA isolation at five time points (circles, 5, 10, 15, 30, and 50 min p.i.) from both infected and noninfected cultures. In addition, a sample from the zero time point was collected from the noninfected control culture. (B and C) Phase-contrast images (Olympus; model no. BX50) of PRD1-infected (B) and noninfected (C) cells around 40 min p.i. The frequency of cells within different stages of cell division was equal in both samples. Bar, 10 μm.

FIG. 2.

Global changes in E. coli gene expression during the PRD1 infection. (A) The number of up- and downregulated ORFs in noninfected (E_x/E₀) and PRD1-infected (P_x/E_x) E. coli cells. Changes in expression levels were considered significant when the relative expression value between two conditions was greater than 3.0 (upregulated) or less than 0.33 (downregulated). Percentages of up- or downregulated ORFs from all of the measured ORFs are shown in parentheses. “Changed” indicates the total number of genes which have changed expression during the experiment. (B) Time scale of viral life cycle. PRD1 recognizes a specific receptor on the host cell outer membrane and delivers its linear dsDNA genome into the host cytoplasm using its internal membrane as a genome delivery device (25). This process induces changes in the permeability and energetic state of the host plasma membrane (18). Early viral proteins involved in viral genome replication are synthesized immediately after infection (∼5 min p.i.) (45), leading to phage DNA replication (10 to 20 min p.i.) (52). Protein components of the virions are produced at about 15 min p.i (45). Viral membrane proteins associated with the host plasma membrane interact with soluble viral capsid proteins to form empty procapsids at about 30 min p.i. (6, 46). The viral genome is packaged into empty particles, and at about 40 min p.i., DNA containing viral particles can be detected inside infected cells (46, 59). Already at about 35 min p.i., viral lysis components are present within infected cells and become active around 55 min p.i. due to decreases in the cellular ATP levels (68). Approximately 200 progeny virions are released from each infected cell.

FIG. 3.

Verification of microarray results by qPCR and Western blot analysis. (A) Comparison of mRNA levels in noninfected and PRD1-infected cells by microarray and qPCR for selected E. coli genes. Genes are indicated within the upper left corner of each panel. Solid line, noninfected x-min sample versus noninfected 0-min sample (E_x/E₀); dashed line (black), infected x-min sample versus noninfected 0-min sample; (P_x/E₀); dashed line (gray), infected x-min sample versus noninfected x-min sample (P_x/E_x). Notice the different scale for ibpB and metB. (B) Detection of the groL gene product Hsp60 from PRD1-infected cells by Western blot analysis. Samples taken from PRD1-infected cells at different time points of infection were analyzed by Western blotting using Hsp60-specific antibodies.

FIG. 4.

Representatives of gene expression pattern clusters. E. coli ORFs were divided into three expression categories within each time point and experiment: downregulated (−1), upregulated (1), and not changed (0). The simplified expression profiles were clustered so that the ORFs having the same temporal expression pattern in both conditions (infected and noninfected) were placed in the same cluster. The number of possible clusters was 59,049 (three classes in five time points in two conditions). However, only 116 different clusters were obtained. All clusters containing more than 10 members are shown (17 clusters, panels A to Q). Solid line, noninfected x-min sample versus noninfected 0-min sample (E_x/E₀); dashed line, infected x-min sample versus noninfected x-min sample (P_x/E_x); dotted line, sum of the previous ones (infected x-min sample versus noninfected 0-min sample; P_x/E₀). The number of ORFs in each cluster is indicated within each panel. Clusters can be divided into seven classes based on the virus effect: (i) no changes in gene expression in comparison to the noninfected culture (A to G), (ii) upregulation of gene-expression in the infected cell (H to L), (iii) downregulation of gene expression in the infected cell (M), (iv) delayed upregulation of gene expression in infected cell in comparison to the noninfected culture (N), (v) delayed downregulation in the infected cell in comparison to the noninfected culture (O), (vi) no change in expression level during the infection due to the virus-induced suppression of upregulation (P), and (vii) no change in expression level during infection due to virus-induced suppression of downregulation (Q).