A codon-optimized orf63 gene (NCBI ID: 2703507) for high level expression E. coli was synthesized by ATUM (Menlo Park, CA) and supplied in plasmid pD441-NH for direct transformation of a BL21 host strain (Novagen). Amino terminal 6xHis and Flag (DYKDDDDK) tags were included to facilitate affinity purification and detection. Milligram quantities of isotopically labeled 6xHis-Flag-Orf63 for NMR spectroscopy were obtained from a 1.0 L fermentation in a minimal medium containing 1 g ¹⁵NH₄Cl, 3 g of ¹³C-glucose, and 1 g of ¹⁵N-¹³C Celtone algal extract (CIL; Cambridge, MA). The cell pellet was dissolved in T300 buffer (20 mM Tris-HCl, 300 mM NaCl, 0.05% NaN₃) and lysed by French press and sonication. The Orf63 protein purified from the bacterial soluble fraction by Nickel-NTA affinity chromatography (Qiagen) that included a 10 mM imidazole wash step and a 20 mM EDTA elution step, all in T300 buffer. A subsequent gel filtration chromatography step (Sephacryl-100, HiLoad 16/60; GE Life Sciences) was employed to further purify the Orf63 protein and exchange it into NMR spectroscopy buffer (5 mM Tris-HCl, 0.15 M NaCl, 0.05% NaN₃).

Chromatograms of eleven proteins on the same gel filtration column used to purify Orf63 were compiled to produce a standard curve describing the relationship between retention volume and molecular weight. Specific proteins used for the standard curve included the HACS1 SH3 domain (10.6 kDa), the AIDA1 PTB domain (22.0 kDa), the monomeric and dimeric states CASKIN2 SAM domain tandem (20.2 / 40.5 kDa), the CASKIN2 SAM1 domain (10.3 kDa), the SHP2 adaptor SH2 domain (14.1 kDa), the AIDA1 SAM domain tandem (16.4 kDa), two deletion mutants of the La RRM domain (14.6 / 15.9 kDa), the Crk2 adaptor SH2 domain (15.5 kDa), dimeric glutathione S-transferase (52 kDa), and calmodulin (18.8 kDa).

A 0.5 mM sample of ¹³C,¹⁵N-labeled Orf63 was prepared for NMR spectroscopy in NMR buffer supplemented with 10% D₂O. All experiments were performed at 310 K using a Bruker Avance 700 MHz NMR spectrometer equipped with a cryogenically cooled 5 mm probe at the York University Life Sciences Building Central Facility. Backbone (HN, N, CA, CB, C') assignments were achieved using a set of conventional triple resonance experiments (HNCA, HNCACB, CBCAcoNH, HNCO, HNcaCO) incorporating sparse sampling for the optimum sensitivity and resolution. Datasets were processed with NMRpipe (Delaglio et al., 1995) and istHMS (Hyberts et al., 2012) and interpreted with CCPN Analysis (Skinner et al., 2015).

The E. coli strains, bacteriophages and plasmids used in in vivo work are presented in Table 1. Work with these strains was approved by the Ministry of Environment (decision no. 189/2016). The E. coli lysogens were obtained using the following lambdoid phages: λ, λΔorf63, Φ24_B or Φ24_BΔorf63 (Bloch et al., 2013; Licznerska et al., 2016b). In the first step, phage lysates were prepared. Bacterial cultures were grown at 37°C to A₆₀₀ = 0.1. Then, mitomycin C (Sigma—Aldrich) was added to all flasks to a final concentration of 1 μg/ml. The incubation with shaking was continued for about 12 h. To obtain lysates, bacterial debris were centrifuged (2,000 × g for 10 min at 4°C) and supernatants were filtered through the 0.22-μm-pore-size filters (Sigma—Aldrich). In the next step, the lysogenization procedure was carried out. Briefly, E. coli strain C600 was cultivated at 37°C to A₆₀₀ = 0.2. Then, 4 ml of bacterial culture was centrifuged (2,000 × g for 5 min at RT), the pellet was washed with TCM buffer (10 mM Tris-HCl, 10 mM MgSO₄, 10 mM CaCl₂, pH 7.2; Sigma—Aldrich) and suspended in LB medium (Sigma—Aldrich) supplemented with MgSO₄ (phages λ and λΔorf63) or with MgSO₄ and CaCl₂ (phages Φ24_B and Φ24_BΔorf63) to a final concentration of 10 mM. Bacteriophages were added to the suspensions to m.o.i. of 10. Following incubation at 37°C, the mixtures were spread on LB agar plates. After overnight incubation at 37°C, bacterial colonies were tested for the presence of prophages by using UV irradiation (this procedure is described in detail in the next section). For construction of the plasmid pSB_orf63_λ, nucleotide sequence of orf63 from phage λ was amplified by PCR with primers: Fλorf63_EcoRI (5′GGA GAA TTC GGC TGT ATG CAC AAA GC) and Rλorf63_BamHI (5′ GAG GAT CCT GCA TTC CGT GGT TGT C), and phage DNA as a template, which was isolated by using MasterPure™ Complete DNA and RNA Purification Kit (Epicenter). Then, the λorf63 was ligated with fragment of plasmid pUC18 (insert and vector were digested with EcoRI and BamHI restrictions endonucleases; Thermo Scientific), bearing an ampicillin resistance gene and sequence of p_lac promoter. The plasmid pSB_orf63_Φ24_B was constructed according to similar procedure. To amplify a DNA fragment containing vb_24B_8c sequence (the homolog of λorf63) by PCR method, two primers: FΦ24_Borf63_EcoRI (5′GGA GAA TTC GGC TGT ATG CAC AAA GC) and RΦ24_Borf63_BamHI (5′GTA GGA TCC TTG TCA TGC CGG GTC) were used. Next, plasmid pUC18 and insert were cut with EcoRI and BamHI enzymes and ligate by the T4 DNA ligase (Thermo Scientific). The construction of pUC18 derivatives was confirmed by DNA sequencing (Genomed).

All in vivo experiments were performed in LB liquid medium (Sigma—Aldrich) supplemented with 10 mM MgSO₄ (phage λ or phage λΔorf63) or with 10 mM MgSO₄ and 10 mM CaCl₂ (phage Φ24_B or phage Φ24_BΔorf63), and with 50 μg/ml ampicillin (if necessary) (Sigma—Aldrich). To stimulate Orf63 protein production from the recombinant pUC18 derivatives, overnight bacterial cultures were diluted 1:100 in fresh LB medium and treated with IPTG (A&A Biotechnology) to a final concentration of 1 mM. Then, host bacteria were grown in aeration condition, achieved by shaking, at 30°C to A₆₀₀ = 0.1 or 0.2 (the optical density of bacterial cells was dependent on the experimental conditions described in the following chapters).

Bacteriophage titration was performed on the standard Petri dishes (Alchem) filled with 25 ml of LB agar (1.5% agar; Sigma—Aldrich), according to a procedure described by Sambrook and Russell (2001), with some modification. The top layer was prepared by mixing 2 ml of LB agar (0.7% agar; Sigma—Aldrich) with 1 ml of the overnight bacterial cell culture. To obtain visible plaques formed by Stx phages, the bottom agar was supplemented with sublethal concentration of chloramphenicol (Sigma—Aldrich). This antibiotic was effective in increasing of size of plaques of phage Φ24_B and its derivative, which possessed in genomes chloramphenicol resistance gene (Table 1). As described previously (Loś et al., 2008a), the cm gene expression, especially after phage infection of E. coli bacteria, may have the positive influence on cellular productivity by decreasing the inhibitory effects of the antibiotic on protein synthesis. To determine the number of phages per ml of suspension (PFU/ml), serial 10-fold dilutions were prepared in TM buffer (10 mM Tris–HCl, 10 mM MgSO₄; pH 7.2). Then, appropriate volume of each dilution of phage lysate was spotted onto double agar layer. The plates were incubated at 37°C overnight, plaques were counted, and the phage titer was calculated.

To investigate the intracellular lytic development of lambdoid phages the one-step-growth experiment was prepared using the method described by Wegrzyn et al. (1995), with a minor modification (Bloch et al., 2014; Nejman-Falenczyk et al., 2015). Host bacteria were grown in LB medium at 30°C to A₆₀₀ = 0.2. In the next step, 10 ml of a bacterial culture was centrifuged (2,000 × g for 10 min at 4°C). The pellet was suspended in 1 ml of LB medium supplemented with 3 mM sodium azide (Sigma—Aldrich). Bacteriophages were added to E. coli cells to m.o.i. of 0.05. After 10 min incubation at 30°C, unadsorbed phages were removed by three times washing in LB medium with 3 mM sodium azide (2,000 × g for 10 min at 4°C). Then, 25 μl of the suspension was added to 25 ml of LB medium prewarmed to 30°C (time 0) and cultivated in an incubator shaker. The number of infection centers were determined at times: 5, 10, 15 min after infection by mixing 0.1 ml of the sample with 0.9 ml of an overnight culture of appropriate indicator bacteria and 2 ml of top agar. Next, the mixture was poured onto LB agar plate (phages λ and λΔorf63) or LB agar plate with 2.5 μg/ml chloramphenicol (phage Φ24_B and Φ24_BΔorf63). Samples taken at later times were treated with chloroform (POCH), shaken vigorously and cleared by centrifugation (2,000 × g for 5 min at RT). The phage lysate was diluted in TM buffer and titrated under permissive condition. Plates were incubated at 37°C overnight. The number of viruses released from each infected cell (burst size) was calculated as a ratio of phage titer to the titer of infection centers.

Bacteria lysogenic for lambdoid phages were grown in LB medium at 30°C to A₆₀₀ = 0.1. Next, the culture was divided into two aliquots. One of them was treated with 1 mM hydrogen peroxide (Sigma—Aldrich) to provoke the prophage induction. The second one was a control without an induction agent. The cultivation was continued at 30°C. At indicated times samples were harvested, mixed with chloroform and vortexed for 1 min. The suspension was centrifuged for 5 min in a microfuge at RT. The supernatant was diluted in TM buffer and 2.5 μl of each serial dilution was dropped onto a freshly prepared double-layer LB agar in plastic Petri dishes. Plates were incubated at 37°C overnight. The relative phage titer was estimated by subtracting the phage titer determined in non-induced cultures from the phage titer estimated in induced cultures.

To estimate the percentage of surviving cells after bacteriophage infection the procedure created by Sambrook and Russell (2001) was used, with a minor modification. A bacterial culture was grown at 30°C to A₆₀₀ = 0.2. Then, 4 ml of the sample was centrifuged (2,000 × g for 10 min at 4°C). The supernatant was discarded and the pellet was washed with 0.85% sodium chloride (POCH) (2,000 × g for 10 min at 4°C). Finally, the bacterial pellet was suspended in 1 ml of LB medium supplemented with MgSO₄ (phage λ and phage λΔorf63) or with MgSO₄ and CaCl₂ (phage Φ24_B and Φ24_BΔorf63) to a final concentration of 10 mM. The suspension was incubated for 30 min at 30°C and then phage particles were added to m.o.i. of 1, 5, 10. The mixture was kept for 15 min (phage λ and phage λΔorf63) or 30 min (phage Φ24_B and Φ24_BΔorf63) at 30°C. In the next step, serial dilutions in 0.85% sodium chloride were prepared and 40 μl of each dilution was spread on LB agar plates. After overnight incubation at 37°C, percentage of surviving E. coli bacteria was calculated relative to bacterial culture in which TM buffer was added instead of phage particles.

Efficiency of lysogenization was estimated according to Arber et al. (1983) and Wegrzyn et al. (1992), with some modification. Host bacteria were cultured at 30°C to A₆₀₀ = 0.2. Next, 1 ml of the sample was centrifuged (2,000 × g for 10 min at 4°C). Bacterial culture was washed with TCM buffer twice, and then pellet was suspended in the same buffer. Bacteriophages were added to bacterial cells to m.o.i. of 1, 5, 10. The mixture was incubated at 30°C. Then, serial dilutions were prepared and 20 μl of each suspension was spread on LB agar plates prior to overnight incubation at 37°C. The next day, 96 colonies were passaged in each well of a 96-well plate with 200 μl of LB medium and shaken at 37°C to A₆₀₀ = 0.1. To estimate a percent of lysogens among survivors, bacterial cultures were treated with UV light at 50 J/m² (the dose used routinely for lambdoid prophage induction) and incubated at 37°C for 2 h. Following induction, putative lysogens were mixed with chloroform, centrifuged (2,000 × g for 10 min at 4°C) and the water phase was spotted onto a double-layer LB agar (phage λ and phage λΔorf63) or a double-layer LB agar supplemented with chloramphenicol to a final concentration of 2.5 μg/ml (phage Φ24_B and Φ24_BΔorf63). Efficiency of lysogenization was calculated as a percent of lysogens relative to all tested bacterial cells. Lysogens were also infected with the same phage to check their resistance to superinfection, as described previously (Wegrzyn et al., 1992).

Induction of tested prophages was provoked in lysogenic bacteria by addition of hydrogen peroxide to a final concentration of 1 mM. At the appropriate time, 10⁹ bacterial cells were harvested, treated with 10 mM sodium azide and deep frozen in liquid nitrogen (this procedure was necessary to inhibit the growth of host bacteria). Total RNA from all samples were isolated with the High Pure RNA Isolation Kit (Roche Applied Science). To remove DNA from RNA preparations the TURBO DNA-free™ Kit (Life Technologies) was used. The quality and quantity of total isolated RNA were analyzed by a NanoDrop spectrophotometer and agarose gel electrophoresis. The contamination of DNA from RNA samples was also tested by routine PCR and qRT-PCR.

To synthesize cDNA from an RNA template, the Transcriptor Reverse Transcriptase and random hexamer primers (Roche Applied Science) were used, according to the protocol supplied from the provider. 1.25 μg of the total RNA was taken for each reaction. Finally, mixture was diluted 10-fold and tested in qRT-PCR.

The pattern of genes expression after prophage induction was performed by using the LightCycler® 480 Real-Time PCR System (Roche Applied Science), LightCycler® 480 SYBR Green I Master (Roche Applied Science) and cDNA samples. Transcription rates of genes of lambdoid bacteriophages were compared in parallel to the 16S rRNA housekeeping gene (according to a procedure described by Strauch et al. (2008), which expression was stable during prophage induction provoked by hydrogen peroxide. All primers were created by Primer3web version 4.0.0 and are listed in Table 2. Each reaction mixture consisted of: 2x SYBR Green I Master Mix, 6.25 ng/μl cDNA and 200 nM specific primers. qRT-PCR amplifications were performed for 55 cycles. To confirm the specificity of primers, melting curve for each product was analyzed. The relative changes in gene expressions were determined by E-Method and calculated by the following formula: Normalized relative ratio = E_t ^{CT (target) calibrator−CT (target) sample} / E_r ^{CT (reference) calibrator−CT (reference) sample}, where E_t is the PCR efficiency of target and E_r means the PCR efficiency of reference. The sample before the addition of the inductor (the time point “zero”) was a calibrator. The raw run data for tested lambdoid phages were transferred using the “LC480 Conversion: conversion of raw LC480 data” software and then, PCR efficiency for each gene was calculated by LinRegPCR program, which was successfully used previously (Bloch et al., 2014, 2015; Nejman-Falenczyk et al., 2015; Licznerska et al., 2016a).

Each experiment was repeated three times and variation among replicates was presented as the error bars indicating the standard deviation (SD). All data comparisons were made by using Student's t-test. Significant differences were marked by asterisks when P < 0.05 (^*) or P < 0.01 (^**).

Samples from four independent preparations of 6xHis-Flag tagged Orf63 (15 aa. tag + 63 aa. protein) eluted as one peak on a preparative gel filtration column with an average retention volume of 59.5 mL corresponding to an apparent molecular weight of ~26 kDa (Figure 2A). Since affinity-tagged Orf63 is only 9 kDa, the gel filtration results suggest that Orf63 is oligomeric with a trimer as the most plausible configuration. This estimate is most accurate if Orf63 has the characteristics of a globular protein to match the standards used.

Consistent with the observation that Orf63 is oligomeric in solution, NMR spectra of Orf63 at 298 K (25°C) suffered from considerable resonance line broadening that was characteristic of proteins >20 kDa in overall molecular weight. Consequently, a higher temperature of 310 K (37°C) was chosen for all NMR studies to increase the tumbling time of the protein that, in turn, improves the sensitivity of triple resonance experiments. In Figure 2B, a ¹H-¹⁵N HSQC spectrum is presented. The amide resonances in this two-dimensional spectrum are disperse indicating that the protein is folded. The combined analysis of several triple resonance (¹H, ¹³C, ¹⁵N) spectra lead to the determination of backbone (HN, N, CA, CB, C') chemical shift assignments for residues 14–52 of Orf63. Resonances for the amino terminal affinity tags and from residue 53 onwards to the carboxy-terminus were either not observed or unassignable. Thus, the NMR data suggest that the folded region of Orf63 includes from residues 14–52.

Several statistical methods are available to predict secondary structure from backbone chemical shift data with a high degree of accuracy. As shown in Figure 2C, two helices are predicted (α1: 13–21; α2: 33–50). The secondary structure determined from chemical shift data is consistent with the secondary structure of Orf63 predicted from sequence information alone, although the helical boundaries are different.

Since experiments shown in Figure 2 indicated that orf63 encodes a protein, we have tested similarity of the putative proteins encoded by orf63 of different lambdoid bacteriophages. Thus, scores of pairwise alignments of the predicted amino acid sequences of orf63 from six such phages have been calculated. As demonstrated in Table 3, all these putative proteins are similar to each other. This indicate that the high similarity is kept at the protein level of Orf63 of lambdoid bacteriophages.

Since previous studies suggested that genes from the exo-xis region might be involved in the regulation of bacteriophage development (Bloch et al., 2013, 2014; Licznerska et al., 2016a), we have tested two crucial controlled steps in the lambdoid phage life cycle, the lysis-vs.-lysogenization decision, and prophage induction. We found that lysogenization efficiency was significantly increased in bacteriophages λ and Φ24_B devoid of orf63 (Figures 3A,B, respectively) though this phenomenon was more pronounced in λ (the effects were seen at all tested m.o.i.) (Figure 3A) than in Φ24_B (significant effects observed only at m.o.i. = 10) (Figure 3B). Also, survival rates of bacterial cells (i.e., cells lysogenized and not infected) in populations infected with Δorf63 or Φ24_B Δorf63 were higher than those in experiments with wild-type λ or Φ24_B (Figures 4A,B, respectively), supporting the conclusion that lysogenization is more effective for the mutant, indeed.

To test efficiency of prophage induction, we have estimated the number of phages appearing after prophage induction with hydrogen peroxide (one of natural prophage inducers occurring in human intestine, the common habitat of E. coli). Deletion of orf63 caused a lower phage titer after prophage induction for both λ and Φ24_B (Figures 5A,B, respectively). However, when measured kinetics of phage development following infection of E. coli cells at low m.o.i. (0.05), we found that phages λ and Φ24_B devoid of orf63 gave even more progeny per infected cell than their wild-type counterparts (Figures 6A,B, respectively). Therefore, combining results of experiments presented in Figures 5, 6, one can conclude that deletion of orf63 influences efficiency of prophage induction in both λ and Φ24_B. Since formation of progeny phages is definitely not impaired in the absence of orf63 when lytic development starts after infection, we suggest that lower phage titer after prophage induction indicates lower efficiency of this process in phages devoid of this gene.

Since experiments described above indicated that orf63 function is involved in the regulation of lysogenization and prophage induction in both λ and Φ24_B, we aimed to measure expression of selected bacteriophage genes in E. coli cells after hydrogen peroxide-provoked prophage induction. Reverse transcription quantitative real time PCR (RT-qPCR) was used to assess abundance of particular transcripts. We have measured expression levels of genes from the exo-xis region (ea8.5, ea22, orf73, orf61, orf60a) and some key regulatory genes of λ and Φ24_B, i.e., N, cro, cII, Q, R. We found that expression of all tested genes was significantly impaired in Δorf63 mutants of both λ and Φ24_B relative to wild-type phages at all tested times after prophage induction (Figures 7A,B, respectively). These results confirm that prophage induction is significantly impaired in the absence of orf63, and suggest a regulatory role for the orf63 gene product in the control of expression of phage genes. In the case of phage λ, complementation of the Δorf63 mutation by overexpression of wild-type orf63 from a plasmid was successful, at least at certain times after prophage induction (Figure 7A). However, we failed to obtain such a complementation in phage Φ24_B (Figure 7B). This might suggest that specific ratio(s) of Orf63 is/are required for accurate regulation of phage development.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The reviewer RO and handling Editor declared their shared affiliation, and the handling Editor states that the process nevertheless met the standards of a fair and objective review.