Systematic Identification of Hypothetical Bacteriophage Proteins

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Systematic identification of hypothetical bacteriophage proteins targeting key protein complexes of Pseudomonas aeruginosa An Van den Bossche, Pieter-Jan Ceyssens, Jeroen De Smet, Hanne Hendrix, Hannelore Bellon, Nadja Leimer, Jeroen Wagemans, Anne-Sophie Delattre, William Cenens, Abram Aertsen, Bart Landuyt, Leonid Minakhin, Konstantin Severinov, Jean Paul Noben, and Rob Lavigne J. Proteome Res., Just Accepted Manuscript • DOI: 10.1021/pr500796n • Publication Date (Web): 04 Sep 2014 Downloaded from http://pubs.acs.org on September 6, 2014

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Journal of Proteome Research

Systematic identification of hypothetical bacteriophage proteins targeting key protein complexes of Pseudomonas aeruginosa An Van den Bossche1, Pieter-Jan Ceyssens1,6, Jeroen De Smet1, Hanne Hendrix1, Hannelore Bellon1,†, Nadja Leimer1,†, Jeroen Wagemans1, Anne-Sophie Delattre1, William Cenens2, Abram Aertsen2, Bart Landuyt3, Leonid Minakhin4, Konstantin Severinov4, Jean-Paul Noben5 and Rob Lavigne1,*

1

Division of Gene Technology, KU Leuven, B-3001 Leuven, Belgium

2

Laboratory of Food Microbiology, KU Leuven, B-3001 Leuven, Belgium

3

Functional Genomics and Proteomics, Department of Biology, KU Leuven, B-3000 Leuven,

Belgium 4

Waksman Institute for Microbiology, Rutgers University, Piscataway, NJ 08854, US

5

Biomedical Research Institute and Transnational University Limburg, Hasselt University, 3950

Diepenbeek, Belgium 6

Scientific Institute of Public Health (WIV-ISP), 1050 Brussels, Belgium

ACS Paragon Plus Environment

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ABSTRACT Addressing the functionality of predicted genes remains an enormous challenge in the postgenomic era. A prime example of genes lacking functional assignments are the poorly conserved, early expressed genes of lytic bacteriophages, whose products are involved in the subversion of the host metabolism. In this study, we focused on the composition of important macromolecular complexes of Pseudomonas aeruginosa involved in transcription, DNA replication, fatty acid biosynthesis, RNA regulation, energy metabolism and cell division, during infection with members of seven distinct clades of lytic phages. Using affinity purifications of these host protein complexes coupled to mass spectrometric analyses, 37 host complex-associated phage proteins could be identified. Importantly, eight of these show an inhibitory effect on bacterial growth upon episomal expression, suggesting that these phage proteins are potentially involved in hijacking the host complexes. Using complementary protein-protein interaction assays, we further mapped the inhibitory interaction of gp12 of phage 14-1 to the α subunit of the RNA polymerase. Together, our data demonstrate the powerful use of interactomics to unravel the biological role of hypothetical phage proteins, which constitute an enormous untapped source of novel antibacterial proteins. (Data are available via ProteomeXchange with identifier PXD001199.) KEYWORDS: Bacteriophages, P. aeruginosa, Interactomics, Affinity purifications, Functional annotation


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INTRODUCTION In an era in which bacteriophage genomes are published by the dozens

1,2

, the gap between

predicted phage gene sequences and their corresponding functions is continuously expanding. The analysis of lytic phages particularly contributes to this discrepancy because of four basic reasons: (a) the broad availability of affordable high-throughput sequencing, combined with the ease of isolating new phages, (b) the limited availability of straightforward methods to generate phage mutants, restricting functional studies, (c) short infection cycles, which challenge -omics applications technically, and (d) the lack of sequence similarity of gene products, despite continually expanding databases and improving search algorithms

3–6

. As a result, while

structural phage proteins and most phage-encoded enzymes can readily be identified from mass spectrometric and sequence analyses, respectively, ca. 70% of the annotated phage genes are currently deposited as “hypothetical protein” in public databases (NCBI Entrez database). Typically, many of these hypothetical proteins are small polypeptides synthesized soon after the beginning of phage infection. Cases of such proteins that have been studied in detail, show that they serve to inhibit, activate or redirect intracellular processes of the host to obtain efficient production of phage progeny 7. These proteins adapt the phage to a specific host in a particular environmental niche and are prone to rapid evolution

8–10

. Although a number of studies state

that the functional elucidation of these “antibacterial” phage proteins could be a powerful tool in target identification and drug discovery

7,11,12

, only limited progress has been made in the last

decade 13. It has become clear that protein-protein interaction (PPI) networks are key determinants to the understanding of the function of individual proteins, since proteins exert their biological function through these interactions

14

. Typically, PPI networks contain highly connected proteins (hubs)


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that form the metabolic core of a cell. Due to improved affinity tags and technical advances in mass spectrometry, affinity purifications combined with mass spectrometry (AP-MS) have become the method of choice to study these networks in vivo and under physiologically relevant conditions 15–18. In this study, we focus on Pseudomonas aeruginosa, one of the most common opportunistic pathogens causing respiratory infections in hospitalized patients as well as other nosocomial infections, and increasingly capable of resisting multiple antibiotics by intrinsic and adapted mechanisms 19–21. Surprisingly, apart from a machine learning-based approach, no PPI study has been published for this bacterium to date, leaving the P. aeruginosa PPI network largely unknown 22. Lytic phages infecting P. aeruginosa have been studied for a long time 2 and current ICTV (International Committee on Taxonomy of Viruses) taxonomy distinguishes six unrelated genera of lytic phages 2. An overview of the genomes (41-280 kb) and the known gene functions of representatives of the genera used in this study is presented in Table 1 and Figure S1. The diversity of these phage genomes implicates that each phage uses substantially different strategies to take over the host and relies on different bacterial functions to complete the respective infection cycle. For example, while all of these phages encode DNA polymerases, only three genera (represented by phages φKZ, LUZ19 and PEV2) supply one or more viral RNA polymerases during infection, whereby only the φKZ-apparatus functions completely independently from the host RNA polymerase 23. Some of the phages are broadly similar to the well-studied coliphages like T7 (phage LUZ19 and LKA1) or N4 (phage PEV2)

24,25

. However,

almost none of their early expressed genes are conserved or have a known function. Consequently, a total of 75% of the 808 genes of the seven phages used, were annotated as “hypothetical protein of unknown function” at the start of this study. Based on analogy with the


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model phages, one can predict that many uncharacterized proteins are involved in phage-host PPIs to reorient specific pathways in P. aeruginosa 26,27. In this work, we equipped seven P. aeruginosa protein complexes involved in transcription, DNA replication, fatty acid biosynthesis, RNA regulation, energy metabolism and cell division with affinity tags (Table 2), enabling AP-MS analyses of host and phage interactors during infection with the different lytic phages (Figure S1). By systematic mass spectrometric analyses of the complexes prepared from cells infected by each phage, we identified 37 non-structural phage proteins. Of these, eight proteins inhibited the growth of P. aeruginosa upon in vivo expression. The P. aeruginosa RNA polymerase binding protein gp12 of phage 14-1 was further characterized by complementary PPI methods, validating the AP-MS strategy as a method of choice to provide functional clues towards understanding phage biology and demonstrating its ability as a target-based method for antibiotic drug discovery.

EXPERIMENTAL SECTION Bacterial strains, phages and media All manipulations and phage infections in P. aeruginosa were performed on the P. aeruginosa PAO1 strain 28. P. aeruginosa strains with a Strep-tag® II or a Protein A-tag (PrA) fused to the Cterminus of the target proteins were made by targeted homologous recombination of the genome 29

. Three E. coli strains were used: E. coli TOP10 (Life Technologies) was used during cloning

procedures, E. coli BL21 (DE3) (Life Technologies) was used for heterologous expression of proteins and E. coli BTH101 (Euromedex) was used for bacterial two-hybrid assays. Unless stated elsewhere, bacteria were grown in Lysogeny Broth (LB) (with appropriate antibiotics) at 37°C.


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The phages used in this research are listed in Table 1. The phages were propagated on P. aeruginosa PAO1 or Li010 (in case of phage LUZ24) cells using standard soft agar overlay followed by PEG8000 precipitation, as previously described

30

and stored in phage buffer (10

mM Tris-HCl pH 7.5, 10 mM MgSO4, 150 mM NaCl). The ‘efficiency of plating’ was determined by using standard soft agar overlay and comparing the titer of phages on the mutant strains to the wild type P. aeruginosa PAO1 strain. Affinity purification For affinity purification, a 600 ml culture of the constructed P. aeruginosa PAO1 strain was infected by one of the phages at a multiplicity of infection of 10 or 5 (in case of phage φKZ) to ensure complete and synchronous infection of the cell culture, at an optical density of 0.3 at 600 nm. The infection was stopped in the early stage of infection (Table 1) by rapidly chilling the culture in an icy water bath. Cells were collected and a PrA-tag pull-down was performed as previously described

31

. For pull-downs using the Strep®-tag II, 1 ml pre-washed Strep-Tactin®

Sepharose beads (IBA) were used according to the manufacturer’s protocol. The eluted fractions were concentrated by ultrafiltration (Amicon Ultra 3K, Millipore). Protein expression and purification For overexpression, phage protein gp12 of phage 14-1 was C-terminally fused to a selfcleavable intein tag by cloning into the expression vector pTYB1 (New England Biolabs) and transformation to E. coli BL21 (DE3) cells. The protein was expressed and purified using manufacturer’s procedures. The protein was dialyzed to a storage buffer (20 mM Tris pH 7.0, 200 mM NaCl, 0.5 mM EDTA and 50% glycerol).


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Recombinant E. coli RNAP as well as the σ70-factor of P. aeruginosa were prepared and purified as described previously

32

. The P. aeruginosa RNAP was purified similar to the

procedure to prepare native E. coli RNAP 33. Briefly, the cell lysate of 15 g P. aeruginosa PAO1 RpoC::PrA cells resuspended in buffer A (40 mM Tris pH 8.0, 5% glycerol, 10 mM EDTA, 5 mM 2-mercapthoethanol) supplemented with 200 mM NaCl and 0.2 mM PMSF, was precipitated with 0.8% polymin-P. The polymin-P pellet was washed with buffer A with 400 mM NaCl, and the RNAP fraction was eluted with buffer A + 1 M NaCl. After polymin-P and salt removal by precipitation with 40% ammonium sulphate, the eluted fraction was dissolved in buffer B (10 mM Tris pH 8.0, 5% glycerol, 0.5 mM EDTA, 2 mM 2-mercapthoethanol) supplemented with 0.2 mM PMSF and loaded on a HiTrap Heparin HP column (GE Healthcare). The column was washed with buffer B + 300 mM NaCl and eluted with buffer B + 600 mM NaCl. Subsequently, the eluted proteins were dialysed to TST buffer (20 mM Tris pH 7.6, 150 mM NaCl, 0.05% Tween) and loaded on IgG SepharoseTM 6 Fast flow beads (GE Healthcare). After three washing steps with TST buffer, the RNAP was eluted by incubation with ProTEV protease (Promega) to remove the PrA-tag, concentrated by ultrafiltration and dialysed to a buffer of 20 mM Tris pH 8.0, 100 mM NaCl, 0.5 mM EDTA, 50% glycerol and 1 mM 2mercaptoethanol. Western blot Protein samples were subjected to 12% SDS-PAGE, and subsequently transferred onto a nitrocellulose membrane (Hybond-C Extra, Ge Healthcare). The membrane was blocked for 1h at room temperature with PBST buffer (PBS + 0.1% Tween, pH 7.5) containing 5% (w/v) powder milk. After incubation with a 1:5000 dilution of monoclonal anti-Strep-tag® II antibodies conjugated to Horse Radish Peroxidase (IBA) in PBST buffer for 1h at room temperature, the


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membrane was extensively washed with PBST. Revelation was carried out by enhanced chemiluminescence (ECL, GE Healthcare). Native gel mobility shift Combinations of 2 pmol of E. coli or P. aeruginosa RNAP, 4 pmol E. coli or P. aeruginosa σ70 and 12 pmol of gp12 were incubated in transcription buffer (30 mM Tris pH 8.0, 40 mM KCl, 10 mM MgCl2, 2 mM 2-mercaptoethanol) for 10 min at 37°C. The samples were loaded on a 4-15% native polyacrylamide gel (PhastGel gradient, GE Healthcare) for 30 min using PhastGel Native buffer strips (GE healthcare). The gel was Coomassie-stained, the shifted protein bands were excised from the gel and the protein composition was determined by SDS-PAGE. Mass spectrometry analysis Preceding the mass spectrometry analysis, protein samples were loaded on a 12% SDS-PAGE gel. The Coomassie-stained gel was cut into slices and subjected to trypsin digestion

34

. LC-

MS/MS analyses were carried out using a combination of two systems. Firstly, protein digests were analysed by ESI-MS/MS on a LCQ Classic (ThermoFinnigan) equipped with a nano-LC column switching system as described

35

. In a second method, an Easy-nLC 1000 liquid

chromatograph (Thermo Scientific) which was on-line coupled to a mass calibrated LTQOrbitrap Velos Pro (Thermo Scientific) was used

23

. MS/MS spectra were searched against a

database containing all P. aeruginosa PAO1 proteins and all ‘stop-to-stop’ protein sequences in all six frames of all phages. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium

36

(http://proteomecentral.proteomexchange.org) via the PRIDE

partner repository with the dataset identifier PXD001199.


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Expression of phage proteins in P. aeruginosa and E. coli Using the pENTR/SD/D-TOPO cloning kit (Invitrogen), the MS-identified phages genes were cloned into the Gateway entry vector. Using the Gateway cloning system (Invitrogen), the genes were subsequently transferred to pUC18-mini-Tn7T-Lac-GW construct and pTNS2 to P. aeruginosa PAO1

38

37

. Co-transformation of the

, allowed single-copy integration of the phage

genes into the P. aeruginosa genome . A dilution of an overnight culture was spotted on LB agar ± 1 mM IPTG and incubated overnight at 37°C. To follow growth on single-cell level, 2 µl of a thousand times diluted overnight culture was spotted on LB agar ± 1 mM IPTG. Growth was recorded at 37°C in real time for 5 h with a Nikon Eclipse Ti Time-Lapse Microscope using the NIS-Elements AR 3.2 software 39. Bacterial two-hybrid Bacterial two-hybrid was performed using the BACTH System kit (Bacterial adenylate cyclase two-hybrid system kit, Euromedex)

40

pUT18, pUT18C, pKT25 and pN-25

. The phage gene gp12 was cloned into the four vectors: 41

. Bacterial P. aeruginosa RNAP subunit α (RpoA), β

divided into three parts RpoB1 (sequence encoding amino acid 1-525), RpoB2 (463-1001), RpoB3 (954-1357) and β’ divided into three parts RpoC1 (sequence encoding amino acid 1-500), RpoC2 (456-954), RpoC3 (885-1399) were cloned into the vectors pUT18C and pN-25. To assess interaction, co-transformations of pairs of plasmids were spotted on synthetic minimal M63 medium supplemented with 50 µg/ml ampicillin, 25 µg/ml kanamycin, 0.5 mM IPTG and 40 µg/ml X-Gal and incubated for 24h-48h at 30°C. β-galactosidase activity was measured quantitatively using a Miller assay 42. As a negative control, each construct was co-transformed with its empty counterpart.


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In vitro pull-down assay 20 pmol P. aeruginosa RNAP was incubated with 300 pmol gp12 of 14-1 in calmodulin binding (CB) buffer (10 mM Tris pH 8.0, 150 mM NaCl, 0.1% NP-40, 1 mM Mg(C2H3O2)2, 1 mM imidazole, 2 mM CaCl2 and 10 mM 2-mercaptoethanol) for 20 min at 37°C in a final volume of 60 µl. The proteins were mixed with 30 µl pre-washed Calmodulin-affinity beads (Stratagene) and incubated for 10 min at room temperature while gently agitating. Beads were washed with 1 ml CB buffer and proteins were eluted by adding 20 µl of loading buffer and boiling for 5 min at 95°C. The collected fractions were visualized by SDS-PAGE. As a negative control, gp12 was incubated without the presence of the RNAP. In vitro transcription assay For the abortive initiation assay, reactions of 10 µl were made by mixing 70 nM P. aeruginosa RNAP, 280 nM P. aeruginosa σ70 and 1.1 µM gp12 in transcription buffer. After incubation for 10 min at 37°C, 20 nM of a PCR fragment containing the selected promoter, 100 µM initiating dinucleotide CpA, 20 µM UTP and 0.5 µl of α-[32P]UTP were added. Incubation for 10 min at 37°C was stopped by the addition of 10 µl formamide loading buffer containing 7 M urea. The reaction products were loaded on a denaturing 7 M urea 20% (w/v) polyacrylamide gel and visualized using a PhosphorImager. Run off transcription assays were also performed in 10 µl reactions. Therefore, 70 nM P. aeruginosa RNAP, 280 nM P. aeruginosa σ70 and 1.1 µM gp12 were incubated in transcription buffer for 10 min at 37°C, after which 20 nM of a PCR fragment containing the selected promoter was added and incubated for 8 min at 37°C. Finally, a mix of 200 µM ATP, 200 µM GTP, 200 µM CTP, 20 µM UTP and 0.5 µl of α-[32P]UTP and 0.05 mg/ml heparine was added, incubated for 10 min 37°C and the reaction was stopped by an equal volume of


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denaturing loading buffer. The reaction products were loaded on a denaturing 7 M urea 6% (w/v) polyacrylamide gel and visualized using a PhosphorImager.

RESULTS AND DISCUSSION Affinity tags do not hinder bacterial growth, nor phage infection To study the bacterium-virus PPI network in P. aeruginosa, we selected key proteins of important bacterial intracellular processes (listed in Table 2), based on evolutionary conserved interactions collected in IntAct 43, STRING

44

and PortEco

45

databases. Since many phages are

known to encode proteins that interact with the RNA and DNA polymerases of their host to either inhibit or redirect bacterial transcription and genome replication 13, these complexes were targeted in this work (RpoA, RpoC, DnaN and DnaX). Moreover, we hypothesized that phages could impact other important metabolic proteins, involved in general transcriptional regulation (MvaT), posttranscriptional regulation (Hfq), fatty acid biogenesis (AcpP), cell division (FtsZ) and energy household (GlcB). For each target protein, a P. aeruginosa strain with the corresponding genes fused to a sequence coding for a C-terminal affinity tag was engineered, allowing in vivo exploration of the PPIs under physiologically relevant conditions

46

. Next, we verified that the tags did not

influence bacterial growth and/or the infection process of the bacteriophages. Under the conditions tested, we noticed no differences in growth and infection parameters for any of the engineered strains or phages used (Figure S2). The presence of each tagged protein under physiological conditions was confirmed by Western blot analysis using affinity tag-specific antibodies (Figure 1A). A signal corresponding to the expected molecular weight was visible for each target protein, with DnaX, AcpP and GlcB being apparently less abundantly present in the


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cell lysate than the others. Finally, pull-down experiments using cell lysate of non-infected cells demonstrated that each of the tagged proteins and corresponding bacterial interaction partners (when known) can be purified (Figure 1B and Table S1). While in case of AcpP, many bacterial proteins were co-purified, the purification of FtsZ and the DNA replication complex revealed fewer proteins than expected (see below). Co-purification of 37 non-structural phage proteins Next, cultures of each tagged P. aeruginosa strain were infected in parallel with each phage at high multiplicity of infection. Cells from infected cultures were collected at the early stage (approx. 1/3) of the infection cycle (Table 1), since many host-phage PPIs are hypothesized to occur at this stage 7. After affinity purification of the various complexes, the protein composition of the samples was revealed by SDS-PAGE and proteins were subjected to in-gel trypsin digestion and MS analysis (Table S1). The MS spectra were screened against a database that contains all host proteins and all ‘stop-to-stop’ protein sequences in all six reading frames of the used phages, which helps to avoid biases towards annotated genes 47–49. Using this experimental setup, 37 non-structural phage proteins were identified in samples infected with one of the seven selected phages (Table 3). As such, the existence of 23 proteins which were annotated as ‘hypothetical proteins’ of unknown function, could be experimentally confirmed. Proteins that were uniquely retrieved from cell cultures originating from a single engineered strain or strains targeting the same complex, were considered as possible interaction partners of the corresponding tagged protein or the protein complex of which the tagged protein is part. On the other hand, a number of phage and bacterial proteins were co-purified with more than one single complex (e.g. gp7 and gp17 of phage LKA1), and were considered to be false positive PPI hits. This could be due to secondary interactions, high abundance in the cell 50 (e.g.


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F0F1 ATP synthase subunits) or to affinity for the resin used during the pull-downs (e.g. acetylCoA carboxylase biotin carboxyl carrier protein subunit, AccB). Some bacterial proteins like the ribosomal proteins, are known to be hubs and are therefore expected to be present in several pulldowns. For two chosen host targets, malate synthase G (GlcB) and transcription factor MvaT, no unique interacting phage proteins were identified. Tagging Hfq leads to the co-purification of an RNA-binding protein AP-MS analyses of the RNA-binding protein Hfq, a key protein in post-transcriptional regulation, led to the identification of components of the P. aeruginosa RNA degradosome (Table S1). This is consistent with previous observations that Hfq in complex with RNA is associated with the RNA degradosome during mRNA and sRNA degradation

51–54

. Moreover,

the catabolite repression control protein Crc, which is involved in translational repression of mRNA, was also identified. This supports the hypothesis that Hfq accounts for the RNA binding properties of Crc 55. In total 55% of the P. aeruginosa proteins predicted as possible interaction partner of Hfq by an in silico machine learning-based approach (probability >0.95)

22

, were

identified in at least one phage-infected sample. From the phage perspective, protein gp70 of phage 14-1 was co-purified with Hfq, which is noteworthy since no viral interaction partners for Hfq have been identified to date. Bioinformatic analyses revealed homology to the RNaseH family (PF00075, E-value 6.96e-3) at the C-terminal part of gp70, a family which digests the RNA strand of an RNA/DNA hybrid 56. As such, a function in RNA turnover during infection can be hypothesized for gp70, which was probably co-purified with RNA bound to Hfq.


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Giant phage φKZ targets the divisome The bacterial tubulin analogue FtsZ is a key component of the cell division machinery

57,58

.

Although this abundant protein recruits other components of the divisome, none of these bacterial proteins were identified during AP-MS of FtsZ. One reason could be that the divisome associates with the cell membrane

59

, while in our experimental setup only soluble proteins are

purified. A single protein of unknown function of giant phage φKZ (gp287) was purified in FtsZ pulldowns (Table 3). It was recently found that Phikzlikevirus produce a tubulin-like protein that forms a spindle-like array which positions phage DNA in the centre of the infected cell 60, which is combined with an arrest in cell division

23

. Therefore, it seem reasonable that φKZ could

produce divisome-inhibiting proteins, whereby further studies of gp287 are warranted. Phage proteins targeting the bacterial DNA replication apparatus Several phage proteins are known to target the DNA replication machinery during infection, mostly shutting down host DNA replication 11,12,61,62. Therefore, we equipped two subunits of the highly processive P. aeruginosa DNA polymerase III enzyme complex with an affinity tag: the β-clamp (DnaN) and the clamp loader (DnaX) Escherichia coli

16

63

. In contrast to similar studies made with

, only few of the other subunits of the DNA polymerase III complex were

identified in our pull-down experiments. This can be possibly explained by a combination of the low abundance of this complex and the dynamics of the clamp, which is transiently recruited to the complex by the clamp loader followed by the release of the latter 64–66. Proteins of four phages were specifically purified alongside the DNA polymerase subunits, including two hypothetical proteins of phage φKZ (gp58 and gp124), a putative single-stranded


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DNA-binding protein (gp61) of phage PEV2 and two proteins of phage YuA (gp4 and gp26). For this last phage, an extraordinary large number of phage proteins were found (Table 3), all encoded among the genes responsible for the DNA metabolism and replication 30. Most of these proteins were also co-purified alongside the acyl carrier protein (AcpP), suggesting that both samples share some bacterial binding proteins (Table S1). Furthermore, this might indicate that these proteins form a macromolecular complex involved in phage DNA replication. Finally, gp29.1 of podovirus LKA1 was purified during the pull-down of the β-clamp of the DNA polymerase III (DnaN). The gene encoding this 7.3 kDa protein overlaps with the last nucleotides of ORF29 (location 14958-15155), has TTG as start codon and was missed in the original genome annotation by our group in 2006

24

, illustrating the power of proteogenomic

analyses. No significant homologues or conserved domains were found in gp29.1 using Blast, Pfam, HHpred or Phyre2

3–6

. Since gp29 is the phage encoded DNA polymerase, a function of

gp29.1 in replication inhibition can be proposed. The broad bacterial protein network of the Acyl Carrier Protein (ACP) The acyl carrier protein ACP plays a central role in fatty acid biosynthesis and is attracting attention as possible drug target

67,68

. Many enzymes linked to the biogenesis of fatty acids like

the fab-gene products (FabA, FabB, FabD, FabG, FabF1, FabF2 and FabZ) and 3-oxoacyl-(acyl carrier protein) synthase III (PqsD) and previously reported interacting proteins like GlmU (glucosamine-1-phosphate

acetyltransferase/

N-acetylglucosamine-1-phosphate

uridyltransferase) and SpoT (guanosine-3',5'-bis(diphosphate) 3'-pyrophosphohydrolase), were co-purified with AcpP. Overall, 63% of the predicted P. aeruginosa PPIs with AcpP (probability >0.95) were confirmed by our pull-downs

22

. Furthermore, several proteins which were not

predicted or found in other studied bacterial PPI networks, were detected in remarkably high


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numbers. Some of them are clearly involved in lipid metabolism (e.g. a probable beta-ketoacyl synthase (PA5174) and a probable 3-oxoacyl-(acyl carrier protein) synthase III (PA3286)), thus likely being true interactors. This corresponds to the previous observation that ACP and its interaction partners show significant divergence among different bacteria 16. In the pull-down experiment with AcpP, unique phage proteins were identified for phage 14-1, YuA and PEV2 (Table 3). Given the broad interaction network of AcpP, it is hard to make direct functional predictions for these phage proteins. As mentioned before, several other proteins of phage YuA were co-purified for both DnaX and AcpP. Remarkably, two YuA hypothetical proteins, gp12 and gp14, were at least 10 times more abundant in the pull-down with AcpP. This suggests that these proteins might participate in the fatty acid biosynthesis regulation. Phage proteins targeting the bacterial transcriptional machinery Several host proteins were purified from extracts of cells containing tagged bacterial RNA polymerase (RNAP) subunits α (RpoA) and β’ (RpoC). These included the expected other RNAP subunits β (RpoB), ω (RpoZ), the principal sigma factor for σ70 (RpoD) and the transcription elongation factor NusA. Depending on the infecting phage, other host transcription proteins were also identified, including alternative sigma factors RpoH, AlgU, RpoS and SigX, transcription elongation factors GreB and transcription antitermination factor NusG. In contrast, the RpoA pull-down of the LUZ19 infected sample yielded only a fragment of the α subunit itself (Table S1). This is caused by a previously observed cleavage of the RNAP α subunit during LUZ19 infection 69, leading to the release of the untagged N-terminal fragment which is connected to the rest of the RNAP.


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In total, five phage proteins from four different phages were co-purified alongside the bacterial RNAP. Two of them, gp25.1 of LUZ19 and gp36 of LKA1 have already been shown to interact with the downstream jaw domain of the β’ subunit, thereby inhibiting the P. aeruginosa RNAP 31

. A result which supports the reliability of the AP-MS approach. Besides these two, the

hypothetical proteins gp12 of phage 14-1, gp2 of LKA1 and gp23 of LUZ24 were detected. Since phages 14-1 and LUZ24 do not encode viral RNAPs (Table 1), it is likely that these proteins direct the activity of the host RNAP to phage promoters. In contrast, a phage encoding its own RNAP (like LKA1) may rather shut down the host RNAP at a certain time point during infection 13. Several interacting phage proteins impact bacterial growth To test possible inhibitory effects on host growth, the genes encoding for most of the 37 identified phage proteins could be introduced in single-copy into the P. aeruginosa genome under control of an IPTG-inducible lac promoter

37

(Figure S3). In addition to gp36 of phage

LKA1 and gp25.1 of LUZ19 which were previously shown to affect cell growth

31

, six other

phage proteins were found to have an inhibitory effect on cell growth. The expression of the possible RNAP-binding protein gp23 of LUZ24 and gp70 of phage 14-1 (co-purified with Hfq) had a strong inhibitory effect on growth on solid medium, whereas the expression of a second potential RNAP-binding protein gp12 of 14-1 and the YuA proteins gp4, gp11 and gp13 caused less pronounced inhibitory effects (Figure 2A+S4). Subsequent multicopy expression in E. coli cells demonstrated inhibitory effects on the growth for four proteins (gp11 (YuA), gp12 (14-1), gp23 (LUZ24), gp70 (14-1)), suggesting conservation of the bacterial target between these Gram-negative bacteria (Figure 2B).


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In the case of expression of gp70 of phage 14-1, cell division is arrested, followed by cell death (Figure S4). Since this strong inhibitor putatively contains a domain of the RNase H protein family, RNA was extracted from P. aeruginosa cells expressing gp70. A smear of degraded RNA was observed after gel electrophoresis, suggesting a role of this protein in host RNA destabilization with a tremendous effect on cell fitness (Figure S5). Gp12 of phage 14-1 interacts with the RNAP α subunit and inhibits transcription Since all PPI methods are prone to false positive identifications, we performed complementary PPI assays for the possible RNAP interacting protein gp12 of phage 14-1, to validate the AP-MS strategy and illustrate its power in providing functional information on the identified proteins. First, in an in vitro pull-down using the P. aeruginosa RNAP as a bait and gp12 as a prey, gp12 was eluted together with the RNAP while it was not present in the control with gp12 alone (Figure 3A). A native gel mobility assay with the recombinant phage protein and the P. aeruginosa or E. coli RNAP holoenzyme, yielded comparable results, since a clear shift in migration of the RNAP was visible in both cases (Figure 3B). When this shifted protein bands were excised from the native gel and loaded on an SDS-PAGE gel, all subunits of the RNAP as well as the phage protein appeared to be present (Figure 3C). This assay confirms the ability of gp12 to bind the RNA complex of different bacteria and, thus, the conservation of the binding site between P. aeruginosa and E. coli RNAPs. Since both assays confirm the interaction of gp12 of phage 14-1 with the RNAP complex, the binding site was further mapped. A native gel mobility assay with the σ70 subunit showed no shift in migration of σ70 (Figure S6), suggesting an interaction with a component of the core RNAP enzyme. Therefore, a bacterial two-hybrid assay was designed where two possible interacting proteins are C- or N-terminally fused to the T18 and T25 domain of the Bordetella


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pertussis adenylate cyclase, respectively

70

. Upon interaction of the proteins, functional

complementation of the enzyme is achieved, yielding the activation of the reporter genes

40

.

Gp12 was fused to one domain whereas the core subunits of the P. aeruginosa RNAP (the β and β’ subunit divided in 3 parts and the complete α subunit) were fused to the second adenylate cyclase domain. A spot assay on selective medium yielded a positive signal for the interaction of gp12 with the α subunit, which was confirmed as being significant compared to the controls in a Miller assay (Figure 4). The RNAP contains two α subunits which consist of an N-terminal domain (αNTD) that coordinates the assembly of the RNAP complex and a C-terminal domain (αCTD) involved in transcription regulation by specifically binding upstream promoter (UP) elements and activators as CAP (catabolite activating protein)

71,72

. Some proteins are already

known to influence the function of the α subunit 13,26. Mod and Alt of E. coli phage T4 inactivate the UP-dependent and CAP-dependent transcription by ADP-ribosylation

73

, whereas gp67 of

Staphylococcus aureus phage G1 binds the σA subunit of the host RNAP but selectively interferes with the binding of the αCTD with UP elements 74. Indicative tests were done to study the consequences of the binding of gp12 of phage 14-1 to the α subunit of the RNAP at a functional level. An in vitro abortive initiation assay on the well characterized σ70-dependent promoters T7 A1 belonging to the -10/-35 class and galP1 belonging to the extended -10 class was performed (Figure 5A). The P. aeruginosa RNAP holoenzyme and gp12 were mixed prior to the addition of the template DNA. A reduction in transcription initiation was observed, especially in the case of the galP1 promoter, in which the transcription activity was reduced to 43%. Run off in vitro transcription assays of the host σ70-dependent promoters downstream of the housekeeping gene oprF (major porin and structural outer membrane porin) and the sigma-factor encoding gene rpoD also led to a reduced activity, 53%


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and 40% residual activity, respectively (Figure 5B). Although the precise function of gp12 remains to be determined including promoter specificity and mode of action, these data imply an inhibitory function of the phage protein on the activity of the P. aeruginosa RNAP. Since phage 14-1 does not encode a viral RNAP and therefore is dependent on the host RNAP, we hypothesize that gp12 is involved in a specificity switch from host to phage transcription or from early phase to late phase transcription.

GENERAL CONCLUSIONS Upon injection of their genome in the host cell, lytic bacteriophages often produce small proteins which reprogram the bacterial metabolism towards phage production by phage-host PPIs. With the exception of a handful of well-studied model phages, the functions of these small, early expressed proteins remains unknown or unverified. In this work, we demonstrate the use of AP-MS to study the intracellular phage-host interaction network and subsequently derive the potential functions of hypothetical phage proteins. By analyzing the varying composition of bacterial complexes in the early stage of the phage’s infection cycle, we co-purified 37 nonstructural phage proteins encoded by seven unrelated P. aeruginosa phages. In addition, we retrieved new information on the P. aeruginosa PPI network itself. The strength of this approach lies in (a) its unbiased analyses towards gene annotation, illustrated by the identification of the unannotated gp29.1 of phage LKA1, (b) the number of samples/complexes investigated, facilitating the distinction between true and false positive PPI hits, (c) the increasing sensitivity of MS methods, enabling the purification of lowly abundant proteins, and (d) the investigation of whole complexes which is not restricted to binary interactions.


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Among the key findings are a protein of phage 14-1 with a possible function in RNA destabilisation, a potential macromolecular replication-related complex formed by phage YuA, a possible interaction partner of the cell division component FtsZ produced by giant phage φKZ and the identification of a phage protein of 14-1 which attaches to the α subunit of the bacterial RNA polymerase. These AP-MS identifications provide strong indications towards the biological function of the given protein, after which confirmatory assays are necessary for each individual case. This was demonstrated by the further analyses on gp12 of myovirus 14-1, which was confirmed to interact with the α subunit of the RNA polymerase. Importantly, in total eight proteins of five different phages were found to have an inhibitory effect on growth of the host. This demonstrates that the AP-MS platform is not only useful to determine the function of uncharacterized proteins, but can also be helpful in bacteriophagebased drug discovery. Especially interesting are proteins which show similar effects on growth of E. coli, indicating a mode of action which is possibly conserved in a broader range of organisms. Narrowing down the host target and subsequent site of interaction of an inhibitory phage protein can help focusing the search and rational design of small molecules that can mimic the effect of the phage protein

11

. As such, unravelling hypothetical phage protein functions might provide a

fruitful strategy to identify many more novel and useful targets of P. aeruginosa.


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ACKNOWLEDGEMENTS We would like to thank Marleen Voet (Division of Gene Technology, KU Leuven, B-3001 Leuven, Belgium) and Erik Royackers (Biomedical Research Institute and Transnational University Limburg, Hasselt University, 3950 Diepenbeek, Belgium) for the technical support and Boris Görke (Max F. Perutz Laboratories, Department of Microbiology, Immunobiology and Genetics, University of Vienna, Vienna, Austria) for providing the plasmids for the bacterial two-hybrid analyses. We acknowledge the PRIDE team for the deposition of our data to the ProteomeXchange Consortium. AV and PC are pre- and postdoctoral fellows, respectively, supported by the ‘Fonds voor Wetenschappelijk Onderzoek’ (FWO, Belgium). JD and JW hold a predoctoral fellowships of the ‘Agentschap voor Innovatie door Wetenschap en Technologie in Vlaanderen’ (IWT, Belgium). This research was supported by Grant G.0599.11 from the FWO, the SBO-project 100042 of the IWT, the JPN project R-3986 of the Herculesstichting and the grants CREA/09/017 and IDO/10/012 from the KU Leuven Research Fund. AUTHOR INFORMATION Corresponding author * Rob Lavinge: Tel: (+32) 16 37 95 24; Fax: (+32) 16 32 19 65; Mail: [email protected] Present addresses †

Hannelore Bellon, Lab of Pneumology, University Hospital Gasthuisberg, KU Leuven, B-3000

Leuven, Belgium †

Nadja Leimer, Divisions of Infectious Diseases and Hospital Epidemiology, University

Hospital Zurich, University of Zurich, Zurich, Switzerland.


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Author contribution AV, JD, HH, HB, NL, JW, AD and PC were involved in the amplification and characterization of the bacteriophages, construction of the plasmids and P. aeruginosa strains, affinity purifications and mass spectrometry samples preparation. AV, PC and JN performed mass spectrometry and further analyses. AV, PC, LM and KS designed and performed experiments involving the RNA polymerase and transcription. WC and AA were responsible for the Nikon Eclipse Ti Time-Lapse Microscopy experiments. RL, PC, AD, LM, KS, BL and JN helped in experimental design and supervising the research project. The manuscript was written by AV, PC and RL and edited and corrected by all authors. CONFLICT OF INTEREST The authors declare that they have no conflict of interest.


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FIGURES LEGENDS Figure 1. Characterization of the engineered P. aeruginosa strains. A. Western blot analyses of cell lysates of the affinity tagged P. aeruginosa strains using monoclonal anti-Strep-tag® II antibodies conjugated to HRP (IBA). Each cell lysate was loaded on an 8%, 10% or 12% SDS-PAGE gel depending on the size of the target protein. Each lane represents an independent experiment. B. Pull-down of non-infected affinity tagged P. aeruginosa cells. The eluted fractions were loaded on a 12% SDS-PAGE gel and visualized by Coomassie stain. The tagged proteins are indicated with an asterisk. Figure 2. The expression of inhibitory phage proteins in wild type P. aeruginosa and E. coli cells. Dilution series of (A.) P. aeruginosa cells and (B.) E. coli cells spotted on solid medium with and without induction of the expression of the respective phage proteins, which are genomicallylocated in P. aeruginosa and plasmid-located in E. coli. Figure 3. Analysis of the interaction between gp12 of phage 14-1 and bacterial RNA polymerases. A. Pull-down of gp12 of phage 14-1 with the affinity tagged P. aeruginosa RNAP. Lane 1: PageRuler Prestained Protein Ladder (Fermentas). Lane 2: A mix of the RNAP with the phage protein was loaded on the Calmodulin affinity beads. Lane 3: Wash fraction after loading the mix. Lane 4: Proteins eluted from the beads. Lane 5: Gp12 alone was loaded on the beads as a control. Lane 6: Wash fraction after loading gp12. Lane 7: Eluted fraction for the control with gp12. The asterisk indicates the co-eluted gp12. B. Native gel mobility assay using P. aeruginosa (Lane 1-2) or E. coli (Lane 3-4) RNAP without (Lane 1-3) or with (Lane 2-4) protein gp12 of


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phage 14-1. The boxes indicate the regions used in C. C. SDS-PAGE of the shifted protein bands on the mobility assay for P. aeruginosa (Lane 1) and E. coli (Lane 2). Figure 4. Bacterial two-hybrid analyses of gp12 of phage 14-1 and the α subunit of the P. aeruginosa RNAP. The T25 (25) or T18 (18) domain of the adenylate cyclase CyaA is fused to the N-terminal (N) or C-terminal (C) side of a target protein (X or Y). As a negative control, non-fused T25 or T18 domains were used (No insert). The leucine zipper of GCN4 was used as a positive control. Interaction was visualized by a drop test on selective medium and β-galactosidase activity was measured quantitatively in Miller units. Data information: Error bars represent SD. P‐values were calculated using Student's t‐test (n = 3), *P < 0.005. Figure 5. In vitro transcription analyses on the P. aeruginosa RNA polymerase in the presence of gp12. A. Abortive initiation assays on the model -35/-10 promoter T7A1 and the extended -10 promoter galP1. The amount of reaction product (CpApU*) synthetized by 70 nM RNAP (supplemented with 280 nM P. aeruginosa σ70) was compared to the amount of product synthetized in the presence of 1.1 µM gp12 (%A). B. Run off transcription assays on the promoters of the P. aeruginosa genes rpoD and oprF using 70 nM RNAP (+ 280 nM P. aeruginosa σ70) and 1.1 µM gp12. The run-off transcripts (RO) were visualized similarly.


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TABLES Table 1. P. aeruginosa phages selected for the pull-down analyses. a

Time after infection, where infection was stopped and samples were collected.

Phage

Family

Genus

Genome

ORFs

GC%

RNAP

tsamplea

14-1

Myoviridae

Pbunalikevirus

66 238 bp

90

55.6%

/

5 min

φKZ

Myoviridae

Phikzlikevirus

280 334 bp

369

36.0%

multisubunit RNAP

15 min

LUZ19

Podoviridae

Phikmvlikevirus

42 548 bp

54

62.2%

viral RNAP

5 min

LKA1

Podoviridae

Phikmvlikevirus

41 593 bp

56

60.9%

viral RNAP

10 min

LUZ24

Podoviridae

Luz24likevirus

45 625 bp

68

52.0%

/

15 min

PEV2

Podoviridae

N4likevirus

72 697 bp

90

54.9%

2 viral RNAPs

10 min

YuA

Siphoviridae

YuAlikevirus

58 663 bp

78

64.3%

/

25 min

Table 2. P. aeruginosa proteins selected for the pull-down analyses.

Protein DNA-directed RNA polymerase subunit alpha DNA-directed RNA polymerase subunit beta' DNA polymerase III subunit beta DNA polymerase III subunits gamma and tau

Strain (P.aeruginosa PAO1)

rpoA::StrepII rpoC::PrA dnaN::StrepII dnaX::StrepII

acyl carrier protein

acpP::StrepII

RNA-binding protein Hfq

hfq::StrepII

transcriptional regulator MvaT, P16 subunit

mvaT::StrepII

cell division protein FtsZ

ftsZ::StrepII

malate synthase G

glcB::StrepII

Gene rpoA (PA4238) rpoC (PA4269) dnaN (PA0002) dnaX (PA1532) acpP (PA2966) Hfq (PA4944) mvaT (PA4315) ftsZ (PA4407) glcB (PA0482)

Mass (kDa)

Intracellular process

36,6

Transcription

154,4

Transcription

40,7 73,4 8,7 9,1

14,2

41,2 78,6

DNA replication, DNA strand elongation and processivity DNA replication, DNA strand elongation and processivity Fatty acid biosynthetic process, cellular lipid metabolic process RNA metabolic process Transcription regulation: secondary metabolite biosynthetic process, singlespecies biofilm formation… Cell division, cytokinesis by binary fission Glyoxylate cycle


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Table 3. Phage proteins identified by mass spectrometry after pull-down of bacterial target proteins. Structural protein are indicated in italics and proteins with an inhibitory effect on bacterial growth upon expression in P. aeruginosa in bold. The number of ‘Total spectral counts’ are indicated in parentheses. Proteins encoded by hypothetical genes of unknown function at the start of the project, are indicated with an asterisk. Target RpoA RpoC

14-1

φKZ

*gp12 (2)

LUZ19 *gp25.1 (1)

DnaN

gp32 (4)

LKA1

LUZ24

*gp2 (2) *gp2 (3) *gp4 (2) *gp7 (3) *gp17 (1) *gp36 (3) *gp7 (2) *gp17 (5) *gp29.1 (5)

*gp23 (28) *gp23 (12)

gp61 (2)

AcpP

*gp2 (2) *gp8 (2)

*gp4 (21) *gp7 (2) *gp17 (2)

Hfq

*gp8 (10) gp70 (5) *gp77 (3) *gp77 (3) *gp8 (2)

*gp7 (2) *gp17 (1) gp43 (2) *gp4 (1) *gp17 (1)

MvaT FtsZ

*gp287 (5)

*gp2 (9)

*gp2 (4) gp62 (1) gp19 (1)

YuA

gp66 (4)

*gp23 (4) gp19 (1)

*gp58 (3) gp124 (2)

DnaX

PEV2

gp27 (8) *gp37 (1) gp43 (2)

*gp4 (5) *gp5 (13) gp7 (15) *gp10 (9) gp11 (4) gp12 (8) *gp14 (5) gp17 (11) gp21 (8) gp26 (1) *gp33 (2) gp41 (4) *gp42 (2) gp55 (13) gp56 (17) gp66 (29) gp67 (5) *gp5 (6) gp7 (32) *gp10 (35) gp11 (3) gp12 (96) gp13 (5) *gp14 (330) *gp15 (2) gp17 (6) gp21 (3) *gp33 (15) gp41 (2) gp56 (11) gp66 (21) *gp5 (6)

*gp5 (2) *gp15 (4) *gp33 (1) *gp42 (1) gp66 (1)

GlcB


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SUPPORTING INFORMATION This material is available free of charge via the Internet at http://pubs.acs.org. Figure S1. Overview of the genomes of the P. aeruginosa phages used in this research. The arrows indicate the open reading frames (ORF): Black arrows refer to structural proteins, grey arrows to proteins with a predicted or known function, white arrows to hypothetical proteins of unknown function, green arrows to proteins identified in this work and red arrows to proteins identified in this work and with an inhibitory effect on growth of P. aeruginosa. The numbers indicate the corresponding gene product number of the marked proteins. Figure S2. Characterization of the growth and infection parameters of the engineered P. aeruginosa strains. A. Optical density (OD600nm) in function of time of all constructed P. aeruginosa strains. WT is referring to the wild type PAO1 strain. For the other strains, the tagged target protein is indicated. B. Efficiency of plating (EOP) of six phages on the engineered strains. The EOP is determined by the ratio of the number of plaques on the mutant strain to the number of plaques on the wild type strain. Error bars represent SD. Figure S3. The expression of the identified phage proteins in P. aeruginosa. The expression of all (cloned) phage proteins (or a perfectly conserved homologue) in wild type P. aeruginosa cells. Growth was visualized by spotting dilution series on solid medium. Proteins with an inhibitory effect are marked with red boxes. Figure S4. The expression of inhibitory phage proteins in wild type P. aeruginosa cells. A snapshot was taken 5h after induction using a Nikon Eclipse Ti Time-Lapse Microscope.


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A. Wild type cells. B. gp23 of LUZ24 expression resulted in filamentary growth. C. gp12 of 14/1 expression resulted in small filamentary growth. D. gp70 of 14/1 expression resulted in cell death after one cell division. E. gp4 of YuA expression resulted in cell death for a fraction of the cells. F. gp11 of YuA expression resulted in filamentary cell growth followed by cell death. G. gp13 of YuA resulted in cell death for a fraction of the cells. Figure S5. RNA extraction from P. aeruginosa cells expressing of gp70 of phage 14-1. RNA was extracted by the use of TRIzol® reagent

23

in early exponential (1), late exponential

(2) and stationary (3) growth phase of induced and non-induced cells. 4 µg RNA was loaded on (A.) a denaturing 7 M urea 8% (w/v) polyacrylamide gel and (B.) a 1% agarose gel. The arrows indicate differences between non-induced and induced cells. Figure S6. Native gel mobility assay on gp12 and σ70 A native gel mobility assay using P. aeruginosa (Lane 1-2) or E. coli (Lane 3-4) σ70 without (Lane 1-3) or with (Lane 2-4) protein gp12 of phage 14/1.

Table S1. MS results of the affinity purifications by tagged bacterial protein, infected with one of the seven Pseudomonas phages. The numbers indicate the ‘Total spectral Count’ identified for a specific protein. +

Analyses done on a LCQ Classic (ThermoFinnigan)

#

Analyses done on a LTQ-Orbitrap Velos Pro (Thermo Scientific)

* Proteins predicted by a machine learning-based approach (probability >0.95) as an interaction partner of the targeted protein (Zhang et al, 2012).


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TABLE OF CONTENTS/ABSTRACT GRAPHIC


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Figure 1. Characterization of the engineered P. aeruginosa strains. 177x57mm (300 x 300 DPI)


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Figure 2. The expression of inhibitory phage proteins in wild type P. aeruginosa and E. coli cells. 172x105mm (300 x 300 DPI)



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Figure 3. Analysis of the interaction between gp12 of phage 14-1 and bacterial RNA polymerases. 177x70mm (300 x 300 DPI)


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Figure 4. Bacterial two-hybrid analyses of gp12 of phage 14-1 and the α subunit of the P. aeruginosa RNAP. 177x93mm (300 x 300 DPI)



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Figure 5. In vitro transcription analyses on the P. aeruginosa RNA polymerase in the presence of gp12. 81x98mm (300 x 300 DPI)


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Systematic Identification of Hypothetical Bacteriophage Proteins

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