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Journal of Virology, August 2006, p. 8081-8088, Vol. 80, No. 16
0022-538X/06/$08.00+0     doi:10.1128/JVI.00065-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Global Changes in Cellular Gene Expression during Bacteriophage PRD1 Infection{dagger}

Minna M. Poranen,1,2 Janne J. Ravantti,1,2 A. Marika Grahn,1,2,{ddagger} Rashi Gupta,1 Petri Auvinen,1 and Dennis H. Bamford1,2*

Institute of Biotechnology,1 Department of Biological and Environmental Sciences, Viikki Biocenter, P.O. Box 56 (Viikinkaari 5), 00014 University of Helsinki, Helsinki, Finland2

Received 10 January 2006/ Accepted 30 May 2006


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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.


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INTRODUCTION
 
The reproduction of a virus is dependent on the host organism, which provides substrates and synthesis machineries for viral progeny production. Most viruses interfere with the host's normal physiology to promote viral progeny production by downregulating the expression of cellular genes. This phenomenon was discovered in the 1940s after an infection of bacterial cells with T-even bacteriophages (for a review, see reference 35) and has been proven to also apply to animal and plant virus infections (for a review, see reference 4). An extensive shutoff of host gene expression early in the viral infection is not, however, necessarily a universal mechanism (56); other strategies may also exist. In addition to redirecting cellular recourses for viral reproduction, viruses may also change the cellular physiology to prevent subsequent infections of an already infected host cell. This phenomenon, known as superinfection exclusion, operates during infection by a variety of viruses (14, 31, 36, 63, 65) and bacteriophages (29, 34, 38, 43, 60).

Recently, the effect of virus infection on host mRNA synthesis has been broadly studied using microarray technology. While the earlier-applied technologies either provided information at the whole-genome level, without knowledge of the specific genes affected, or covered only a select set of host genes, the array technology can potentially offer information about individual genes in the entire host gene pool. Currently, array technology has been used to analyze animal virus infections or, in a few cases, study changes in infected plant (42, 57, 66) or fungal cells (2, 3). So far, the microarray-based analyses of phage infections have focused on the detection of phage-specific transcripts in infected cells (19, 39), changes in the host genome (21, 41), or transcriptional profiles of hosts parasitized by lysogenic phages (13). In spite of the potential of the array technology, to our knowledge, only a single study (42) of virus-induced changes on host gene expression covers the entire host genome.

To undertake a genome-wide analysis of a bacterial host under phage infection at a resolution from a single gene to the entire genome, we studied changes in Escherichia coli gene expression during bacteriophage PRD1 infection using microarray technology. PRD1 was selected because it represents a structurally complex virus composed of a protein capsid, a lipid membrane, and a DNA genome (1, 15); it shares similarities with viruses infecting animals (adenovirus, family Adenoviridae), algae (PBCV-1, Phycodnaviridae) and insects (Chilo iridescent virus, Iridoviridae) (5, 7, 9, 10); and its life cycle is well characterized (6, 24). Furthermore, PRD1 can infect E. coli, for which gene functions and transcriptional control are characterized in detail.

PRD1 is a lytic bacteriophage of the Tectiviridae family (6). During a PRD1 infection, 24 phage-encoded protein species are synthesized from two early and three late promoters (8, 23). Eighteen of these proteins are incorporated into the virion in a process assisted by both phage- and host-encoded proteins (8, 26, 46). Towards the end of the phage reproduction cycle, virus-encoded holin and endolysin proteins lyse the host cell, releasing the progeny virions (53, 68). Under optimal conditions, the entire PRD1 life cycle takes about an hour.

Here we report changes in E. coli gene expression during bacteriophage PRD1 infection. The aim of the present study was to analyze changes in E. coli cells under the stress from lytic phage infection. We have specifically analyzed PRD1-induced effects by comparing samples from infected cells to that from a noninfected control at 5, 10, 15, 30, and 50 min postinfection (p.i.). Simultaneously, we have analyzed temporal changes in the noninfected E. coli culture to reveal changes that take place during the ageing of the culture. To our knowledge, this report is the first on microarray analysis of bacterial virus-host interaction, focusing on the changes in host gene expression during infection.


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MATERIALS AND METHODS
 
Sample collection. E. coli K-12 strain JE2571 (leu thr thi lacY thy pil) (11), carrying conjugative plasmid RP4 (17), was used as a host for bacteriophage PRD1 (49). Cells were grown at 37°C in MOPS (morpholinepropanesulfonic acid) (pH 7.4)-buffered synthetic rich medium (47) supplemented with 4 mM leucine and 2 mM threonine.

An overnight culture was diluted into fresh medium (1.5 ml to 50 ml) and grown with aeration at 37°C to an optical density at 600 nm (OD600) of 1.6. The dilution was repeated, now 12-fold (to an OD600 of 0.14 to 0.16), and the culture was split in half. The aeration was continued at 37°C until the OD600 reached 1.6. One portion of the cell culture was infected with PRD1 with a multiplicity of infection of 30; the other portion was a noninfected control culture. Cultures were continuously aerated at 37°C.

Samples for RNA isolation were taken from infected and noninfected cultures at time points of 5, 10, 15, 30, and 50 min p.i. A sample at the zero time point was collected from the noninfected culture. In addition, a sample was collected from the infected culture 2 h p.i. to verify the productivity of the infection and to calculate the number of viral progenies. All of the samples were immediately further processed. The experiment was repeated four times (three times for microarray analysis and once for quantitative real-time PCR [qPCR]).

Isolation of total RNA. Total RNA was isolated from ~1 x 109 cells (10 ml of cell culture) using two phenol and three ether extractions. In the first extraction, the phenol (Sigma P-4682, pH 4.3) was preheated to 95 to 100°C and the sample was supplemented with 1 ml of lysis buffer (0.5 M Tris-HCl, pH 7.5, 0.2 M EDTA, 10% sodium dodecyl sulfate [SDS]) for rapid cell lysis. The nucleic acids were precipitated from the aqueous phase by NaOAc and ethanol. Further purification was performed using an RNeasy midikit (QIAGEN; catalog no. 75144) and on-column DNase (QIAGEN; catalog no. 79254) digestion. The purified RNA samples were stored at –80°C. Concentrations and qualities of the RNA samples were determined by measuring absorbance values at 260 and 280 nm, and the integrity of the total RNA was conformed by gel electrophoresis and ethidium bromide staining. The A260-to-A280 ratio was ≥2.1 in all RNA samples.

mRNA enrichment and synthesis of fluorescent cDNA probes. The 16S and 23S rRNAs were removed from the total RNA samples by a capture hybridization approach using the MicroExpress bacterial mRNA purification procedure (Ambion; catalog no. 1905). The synthesis of Cy3- and Cy5-labeled cDNA for microarray hybridization was carried out according to the Amersham Biosciences protocol for CyScribe (catalog no. RPN5660) cDNA postlabeling. mRNA from 12 µg of total RNA was used as a template for cDNA synthesis primed by random nonamers (Amersham Biosciences). After cDNA synthesis, the template RNA was degraded by RNase H (USB) at 37°C for 15 min. The amino-allyl-labeled cDNA was purified with a QIAquick PCR purification kit (QIAGEN; catalog no. 28104) using phosphate buffers (28). The cDNA was precipitated with NaAc and ethanol and then labeled with a freshly made suspension of Cy3 or Cy5 dye (in NaHCO3, pH 9) according to the protocol for CyScribe postlabeling (Amersham Biosciences; catalog no. RPN5660). The labeled cDNA was purified using the QIAGEN PCR purification procedure. Samples were stored at –20°C in the dark. Test samples were labeled with Cy3, and reference samples were labeled with Cy5. Reverse labeling of the RNA was performed with one of the three samples to verify transcriptional induction rations.

Hybridization. Differently labeled test and reference samples were combined together with nonspecific unlabeled E. coli rRNA (Boehringer Mannheim; catalog no. 206938) and single-stranded DNA (ssDNA) from herring sperm (Sigma; catalog no. D7290). The mixed samples were denatured and hybridized on glass DNA microarrays obtained from the Institute of Food Research (Norwich, United Kingdom; http://www.ifr.bbsrc.ac.uk/Safety/Microarrays/default.html). The hybridization was performed in 50% formamide, 6x SSC (1x SSC is 0.15 M NaCl plus 0.015 M sodium citrate), 0.5% SDS, and 5x Denhardt's solution (0.1% Ficoll 400, 0.1% polyvinylpyrrolidone, 0.1% bovine serum albumin) at 45°C for ~16 h. After the hybridization, the slides were washed once with 0.1x SSC-0.1% SDS (for 10 min), twice with 0.1x SSC (for 5 min), and three times with H2O (for 2 min).

Data collection. Each microarray contained 7,680 spots arranged in 16 blocks, with each block containing 20 by 24 spots. A total of 4,266 of the probes were unique and specific for E. coli K-12 (according to E. coli genome annotation U00096). Every glass carried a duplicate printed microarray.

The microarray glasses were scanned with ScanArray 5000 (Perkin Elmer) using 5-µm resolution. Spot segmentation and intensity calculations were performed using GenePix image analysis software (Axon Instruments, Inc., 1999 version). The local background was subtracted from the raw spot intensity to produce background-subtracted intensities, and the median intensity value for each spot was taken as a representative intensity value. Negative median values were set to zero prior to further processing.

Data processing. The data contained three types of values: E. coli zero-minute expression intensities (E0); E. coli expression intensities for 5, 10, 15, 30, and 50 min p.i. (Ex, where x = 5, 10, 15, 30, or 50); and PRD1-infected E. coli expression intensities for 5, 10, 15, 30, and 50 min p.i. (Px, where x = 5, 10, 15, 30, or 50). There were 34 repeats of E0 (including self-hybridization), 12 repeats of Ex, and 6 repeats of Px at each time point. The total hybridization intensities summed over all of the E. coli K-12-specific spots within each array were set to a constant value, and a median value for an open reading frame (ORF) was taken as the representative intensity value at each time point and condition. Finally, different experiments were brought back to register by their mean total ratios, which were calculated from the original spot intensities by first scaling the Ex/E0 pair of expressions and then, with the scaled Ex, the pair of expressions Px/Ex. When the mean values were used (instead of the median values) throughout the procedure described above, the differences in global scale were negligible, as was the effect of a dye swap.

After the spot intensities were normalized, the data were made to reflect the current annotation of the E. coli K-12 genome (GenBank accession no. U00096.2; version 24 June 2004) according to the information at http://biocyc.org/ecocyc/release-notes.shtml. As a result, the number of E. coli K-12-specific ORFs in our analysis was 4,207.

Genes with signal values of less than 15% (0.15) of the mean value from all of the ORFs (mean, 1.0) were considered to be not expressed at a given time point. This cutoff value is based on median standard deviation values calculated from the E0 data set for each ORF. Over 98% of the calculated median minus standard deviation values were >0, if genes with signal values less that 0.15 were omitted. Based on this criterion, 1,672 ORFs, corresponding to ~39% of all E. coli ORFs, were not expressed in the noninfected E. coli cells at the zero time point. This is in good accordance with data previously reported for an E. coli culture grown in rich medium (1,776 silent genes) (64). Of the 1,672 nonexpressed ORFs in the E0 sample, only 552 have established functions.

Differences in gene expression were considered significant if the expression values differed more than threefold between two conditions. Using this criterion, 99% of the calculated [(SD + median)/median] ratios for each ORF within the E0 data set were <3 (calculated using signal values of >0.15). The same criterion was used to detect the induction of genes which were considered nonexpressed (signal values of >[3 x 0.15]). In order to detect more subtle changes in expression levels than a threefold cutoff would reveal, a t test was performed using the R program (http://www.r-project.org) for all Ex/E0 and Px/Ex conditions. A P value of <0.01 was used to indicate a statistically significant change between conditions. Operon organization and gene functions are based on information on the EcoCyc (Encyclopedia of Escherichia coli K12 Genes and Metabolism; http://ecocyc.org/) website (32).

Validation of transcript levels using qPCR. Total RNA samples for qPCR were isolated from a cell culture independent of those used for microarray sample preparation. The isolated RNA was subjected to additional DNase treatment (RQ1 DNase; Promega) and purification with the RNeasy MinElute cleanup kit (QIAGEN, catalog no. 74204). The purified RNA was used as a template for cDNA production using random hexamer primers and MultiScribe reverse transcriptase (TaqMan reverse transcription reagents; Applied Biosystems; catalog no. N8080234). The resulting cDNA samples were used as templates for qPCR. Primer pairs specific to several genes of interest were designated using PrimerExpress software (Applied Biosystems) (data not shown), and the PCR was carried out using SYBR green PCR master mix (Applied Biosystems). Thermal cycling and real-time monitoring of SYBR green fluorescence were performed with an ABI Prism 7000 sequence detector system (Applied Biosystems). Changes in expression levels were analyzed using ABI Prism 7000 SDS software (Applied Biosystems) and ftsZ (a cell division gene) as a reference.

Western blot analysis. Western blotting was carried out as described previously (54) using primary antibody to Hsp60 (groL gene product). Polyclonal anti-Hsp60 serum was raised by immunizing a rabbit with purified Hsp60 (GroEL) protein (Boehringer) as described previously (27).


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RESULTS AND DISCUSSION
 
Dissection of virus-induced changes. To distinguish the virus-induced changes from the E. coli growth-dependent changes, we constructed two experimental setups: one to analyze the noninfected E. coli culture under the experimental conditions and time scale of the infection (noninfected x-min sample versus noninfected 0-min sample; Ex/E0) and another to evaluate the difference between the infected and noninfected cultures within each experimental time point (infected x-min sample versus noninfected x min sample; Px/Ex). The results of these two measurements establish the mRNA content of the infected cell in comparison to that before the infection (infected x-min sample versus noninfected 0-min sample; Ex/E0 x Px/Ex = Px/E0). We made six replicates of each condition and time point, three biological replicates (Fig. 1A) (RNA isolated from three independent cultures) and two technical replicates from each of them (altogether 124 measurements). Our analysis covers over 99% (4,207 ORFs) of the currently known E. coli ORFs (4,242 ORFs, GenBank version U00096.2).


Figure 1
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  		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.

Initially, the selection of differentially expressed genes was based on differences (n-fold) calculated from the normalized expression values of two different conditions (as described in Materials and Methods). A threefold cutoff value was applied throughout the analysis, and this discussion is focused on genes with known physiological functions. The similar expression patterns for genes within the same operons and regulons strengthen the significance of the results obtained. The microarray expression values were verified for selected genes using qPCR and Western blot analyses. In addition, statistical analysis was applied to determine the significance of the results and to allow the detection of genes with lower changes (n-fold) but with high confidence levels in terms of P value.

Gene expression changes in the noninfected E. coli culture. The physiological state and the transcriptional activity of the noninfected E. coli cells were not constant during the measuring period that is optimal for virus production. The growth rate of the culture started to decrease at the time point equivalent to 15 min p.i. (Fig. 1A), indicating that the bacteria were facing nutrient exhaustion and preparing for the stationary phase. Previous studies have identified the stimulation of 215 E. coli genes during the transition into the stationary phase (62). We detected the induction and repression of 263 and 99, respectively, ORFs in the noninfected E. coli culture during the 50-min experimental period (Fig. 2A). Most notably, the entire cysteine regulon (cysB, cysDNC, cysJIH, cysPUWAM, cysK, sbp, cbl, and tauABCD) was upregulated as well as many genes responding to phosphate starvation (phoH, phoBR, phoA, pstSCAB, and phoU) or involved in the uptake and biosynthesis of methionine (metA, metBL, metC, metF, metNIQ, metJ, metK, metE, and metR) and arginine (argC, argE, argF, argI, and carAB), indicating that some of the essential components of the growth medium became limited. The aging of the culture resulted also in the downregulation of several genes functioning in energy metabolism (atpBFAGC, cyoCDE, and nuoEFGHIJLMN) and transport of small molecules (e.g., dppBCDF, potABCD, and rbsDACB).


Figure 2
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  		FIG. 2. Global changes in E. coli gene expression during the PRD1 infection. (A) The number of up- and downregulated ORFs in noninfected (Ex/E0) and PRD1-infected (Px/Ex) 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.

Virus-induced changes on host gene expression. The growth rate of the E. coli culture was not affected by the PRD1 infection; the turbidity (OD600) of PRD1-infected and noninfected cultures rises with equal rates until the phage induces lysis of the infected cells at ~50 min p.i (Fig. 1A). Also, the cell division rate of the infected and noninfected cultures was the same (Fig. 1B). Surprisingly, the PRD1-induced changes in host gene expression were relatively moderate and the total number of ORFs affected due to the infection (327) (Fig. 2A) was less than the number of differentially expressed ORFs (462) in the noninfected cells during the experimental period (Fig. 2A). Genes that are involved in cell growth and division were not affected during infection, which is in accordance with the data shown in Fig. 1. The majority of the virus-induced changes occurred after the production of virion components, during the assembly of progeny virions and the expression of the lysis components (30 to 50 min p.i.) (Fig. 2) (46, 68). Thus, there was no major reprogramming of the cell physiology early in the infection cycle to promote the production of viral components. Unlike what was expected, pronounced transcriptional activation of genes involved in the biosynthesis of amino acids, phospholipids, or nucleotides (the building blocks for the virion synthesis) was not observed. Instead, the upregulation of several genes involved in the biosynthesis of different amino acids was delayed or suppressed in infected cells (see below) (see Fig. 4N and P). Owing to this unexpected result, we calculated the quantity of substrate molecules utilized for the production of progeny virions and compared it to the total supply in the replicating E. coli cell (Table 1). We observed that the PRD1 progeny production utilizes only a fraction of the host capacity to synthesize DNA, proteins, and phospholipids (5 to 15%). This observation rationalizes the invariability in the expression of biosynthetic enzymes during infection.


Figure 4
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  		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 (Ex/E0); dashed line, infected x-min sample versus noninfected x-min sample (Px/Ex); dotted line, sum of the previous ones (infected x-min sample versus noninfected 0-min sample; Px/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).


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  		TABLE 1. Quantity of substrate molecules utilized for the production of PRD1 virions and the total supply in a replicating E. coli cell

Early phase of PRD1 infection—induction of uptake pathways. The changes detected in the host transcriptosome early in the infection (5 to 15 min p.i.) included genes involved in osmotic adaptation (osmB, osmY, and treR), anaerobic respiration (fdoHI, fdnG, hypC, and nuoK), uptake of organic compounds (dctA, galP, glpTQ, gntT, manY, nupG, and treB), and export of cations (zntA). PRD1 genome delivery into the cell (Fig. 2B) induces transient permeability changes in the host's outer and plasma membranes and the release of cytoplasmic compounds into the external milieu (18). We propose that the activation of uptake pathways and anaerobic respiration systems early in the PRD1 infection is aimed toward compensating these effects and restoring normal cytoplasmic conditions.

Cellular stress responses. Many of the most highly induced genes during the assembly of progeny virions (30 to 50 min p.i.) (Fig. 2) were chaperonins, proteases, and other stress-inducible genes of the {sigma}32-dependent heat shock regulon (as shown in Table 2 for dnaKJ, groSL, grpE, hslVU, htpG, clpB, ibpAB, rfaFCL and Fig. 3A for groL and ibpB). Also, the hslO gene for the molecular chaperone Hsp33 (30) was induced in infected cells (Table 2). A general signal for the {sigma}32-dependent transcription is the stress-induced unfolding of proteins in the cytoplasm (67). Apparently, the amount of unfolded polypeptides within the cell increases due to infection, inducing the {sigma}32-regulon. Previous studies have revealed that PRD1 reproduction is actually dependent on several cellular chaperonins; the correct folding of the PRD1 major capsid proteins P3 and P5 is dependent on the Hsp60 (groL) and Hsp10 (groS) chaperonins (26). The morphogenesis of other bacteriophages, such as lambda and T4, also relies on host chaperonins (22, 61).


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  		TABLE 2. Regulation of selected stress-inducible genes during PRD1 infection


Figure 3
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  		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 (Ex/E0); dashed line (black), infected x-min sample versus noninfected 0-min sample; (Px/E0); dashed line (gray), infected x-min sample versus noninfected x-min sample (Px/Ex). 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.

One of the best-characterized bacterial stress responses is the SOS response, which is activated under different stress conditions, the main trigger being ssDNA (44). In the beginning of PRD1 infection, the viral linear double-stranded DNA (dsDNA) genome is replicated (Fig. 2B), potentially exposing ssDNA. However, none of the approximately 15 genes of the SOS response regulon were activated during the PRD1 infection. This suggests that the viral ssDNA binding proteins P12 (which is the most highly expressed viral protein during PRD1 infection) and P19 (50, 51) can efficiently shield any exposed ssDNA. Neither of the viral ssDNA binding proteins is needed for viral genome replication in vitro (55), suggesting that the evolutional driving force on P12- and P19-encoding genes may have been to prevent the SOS induction.

Components of the phage shock regulon (pspABCDE and yjbO) (37) were among the most highly induced genes during late PRD1 infection (30 and 50 min p.i.) (Table 2). The phage shock protein (Psp) system responds to stress that reduces the energy status of the cell, including extreme temperatures and osmolarity, mislocalization of envelope proteins, and the presence of ionophores or ethanol (16). Initially, phage shock protein A (PspA) was detected at high concentrations in filamentous phage-infected E. coli cells (12). However, an induction of the Psp response during an infection of other coliphages has not been described so far. Apparently PspA has a role in maintaining the proton motive force under different conditions of cellular stress (33). The induction of the pspA gene during PRD1 infection could explain the increase in membrane voltage detected during infection (18). The upregulation of the pspA gene and the whole psp operon also coincides with the production of PRD1 holin protein (about 30 min p.i.). The activation of the lysis functions by holin can be triggered prematurely by ATP depletion (68). The induction of PspA production may be a host defense mechanism for maintaining the cellular energy status in order to postpone the viral lysis functions. Alternatively, the phage has evolved a mechanism to prevent premature lysis by activating the expression of the psp operon.

Induction of exopolysaccharide synthesis. E. coli cells have the potential to produce a number of exopolysaccharides on the cell surface. These exopolysaccharides often protect bacteria from harsh environments, chemicals, or bacteriophages. We detected a significant induction of several genes involved in the synthesis of the colonic acid capsule (wza, wzc, wcaABDE, gmd, wcaGHI, cpsBG, wcaJ, wzxC, wcaKL, and rcsA) (32, 58), starting with the stimulation of the rcsA gene (a positive regulator of capsule polysaccharide synthesis) at 10 min p.i. Exopolysaccharide induction can be viewed as a host defense mechanism for protecting the bacterial population from further phage infections. Alternatively, the synthesis of the host colonic acid capsule is a mechanism for superinfection immunity.

Host protein synthesis shutoff. The expression of the guanosine-3',5'-bis pyrophosphate, (p)ppGpp, synthetase II (spoT) was transiently increased at 30 min after PRD1 infection. (Fig. 3A). The increase in (p)ppGpp concentration is known to be connected to nutrient limitation and leads to a reduction in rRNA synthesis and changes in cellular protein synthesis (40). Accordingly, a reduction in host protein synthesis has been detected in PRD1-infected, pulse-labeled cells beginning at 40 min p.i. (45). Host protein synthesis shutoff is considered to be a common phenomenon associated with animal and plant virus infections (for examples, see references 4 and 20). Host macromolecular synthesis is also drastically reduced during the first few minutes of bacteriophage T2, T4, and T6 infections (35). It has been suggested that the downregulation of protein synthesis is specifically induced by the virus and is aimed to channel host recourses for the virus reproduction. The timing of the spoT upregulation and the host protein synthesis reduction in PRD1-infected cells suggests that the host protein synthesis downregulation in PRD1-infected cells is rather a secondary effect of nutrient limitation not aimed at boosting viral protein synthesis. Similar to PRD1, bacteriophage {Phi}29 has little impact on host transcription until late in infection (56).

Verification of microarray data by qPCR and Western blot analysis. qPCR and Western blot analyses were performed for selected genes to confirm the microarray results (Fig. 3). Five genes (cysB, groL, ibpB, metB, and spoT) that showed different expression patterns in microarray analysis (Fig. 3A, left panel) were selected for qPCRs. The qPCR results shown in Fig. 3A (right panel) were in good agreement with the microarray data. Western blot analysis was performed to detect cellular levels of Hsp60 protein (encoded by the groL gene) in PRD1-infected E. coli cultures. Both microarray and qPCR analyses suggested that, in noninfected cells, the expression of the groL gene is reduced, but in PRD1-infected cells, this normally occurring downregulation is suppressed (Fig. 3A). Accordingly, the Hsp60 levels in infected cells were constant throughout the infection (Fig. 3B).

Temporal changes in E. coli gene expression. To further analyze the dynamic behavior of the cellular transcriptosome during the viral infection, all of the E. coli ORFs included in our experiment were clustered based on their expression patterns in infected and noninfected cells. The specific clusters formed are presented in Fig. 4, and the genes within each cluster are listed in Table S1 in the supplemental material. The majority of the ORFs (3,803; corresponding to 90% of the genome) follow the same expression profile in both noninfected and infected cells throughout the infection cycle (Fig. 4A to G). Certain processes were delayed in infected cells; for example, the induction of genes involved in methionine uptake and biosynthesis (metA, metBL, metC, metF, metNIQ, metJ, metE, metR, and mmuP) occurred later in infected cells compared to that in noninfected cells (Fig. 4N). Also, the expression of several biosynthetic genes for lipopolysaccharide (rfaFC), membrane-associated oligosaccharide (mdoGH), and enterobacterial common antigen (rffH, wecB, and wecC) were repressed earlier in noninfected cells than in infected cells (Fig. 4O). Genes associated with taurine and sulfate transport and transcriptional regulation of the cysteine regulon (cysB, cysM, cbl, sbp, tauABCD, and fliY) were upregulated in noninfected cells, but this induction was suppressed in infected cells (Fig. 4P). In addition, the downregulation of several genes was suppressed in infected cells (Fig. 4Q).

Interestingly, most of the induced (Fig. 4H to L) or repressed (Fig. 4M) ORFs during PRD1 infection were expressed at constant levels in the noninfected cells through the experimental period (Fig. 4H to J and L to M), whereas the majority of the ORFs that changed expression in the noninfected cells were not affected by infection (Fig. 4B to G). This indicates that the pathways connected to starvation and phage infection do not significantly overlap.

Detection of additional genes with significantly changed expression. The use of a high cutoff (n-fold) may exclude genes with lower changes (n-fold) but with high confidence levels. To investigate this possibility, we applied statistical methods as described in Materials and Methods. The results are summarized in Table S2 in the supplemental material. The overall results of this analysis support our initial gene selection based on differences (n-fold) (see Table S2 in the supplemental material). By applying criteria of changes of >2-fold and a P value of <0.01, it was possible to identify one more heat shock-regulated gene, hslJ, which was induced late during PRD1 infection. The hslJ protein is a membrane-associated heat shock protein.

Conclusions. There seems to be several reproduction tactics among viruses. Viruses may either harness the host cell for their own reproduction by efficient takeover and reprogramming of the host physiology or, as presented here, replicate within the cell without affecting major biosynthetic pathways during the period of progeny production. This latter strategy may ensure virus propagation without the induction of host defense mechanisms, while the previous strategy likely activates host responses. The reproduction of PRD1 phage within an actively dividing E. coli cell consumes only a fraction of the host's biosynthetic capacity. The induction of host defense systems would probably have an adverse effect on phage progeny production and decrease phage fitness. Such overtaking activity would also consume the coding capacity of the viral genome and extend the replication time.

The reprogramming of host cell physiology by viruses should not solely be considered as a way to redirect biosynthetic materials for viral reproduction. This assumption may be correct for the large phages for which such phenomenon was initially described (for a review, see reference 35). The modification of host cell physiology should also be considered as a potential mechanism of superinfection exclusion or host defense. This could be the case, especially among viruses that do not significantly utilize the host's replication capacity. It would be intriguing to obtain results of the consumption of the host's replication capacity in other viral systems to evaluate the role of host reprogramming.


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ACKNOWLEDGMENTS
 
We thank Marja-Leena Perälä for assistance with sample collection.

This investigation was supported by the Finnish Centre of Excellence Program 2006-2011 (1213467 and 1213992) from the Academy of Finland to D.H.B.


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FOOTNOTES
 
* Corresponding author. Mailing address: Viikki Biocenter, P.O. Box 56 (Viikinkaari 5), 00014 University of Helsinki, Helsinki, Finland. Phone: (358) 9 191 59100. Fax: (358) 9 191 59098. E-mail: dennis.bamford{at}helsinki.fi. Back

{dagger} Supplemental material for this article may be found at http://jvi.asm.org/. Back

{ddagger} Present address: Roal Oy, P.O. Box 57, 05201 Rajamäki, Finland. Back


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Journal of Virology, August 2006, p. 8081-8088, Vol. 80, No. 16
0022-538X/06/$08.00+0     doi:10.1128/JVI.00065-06
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