Phage manipulations

2.2.1. Phage isolation, amplification and purification

Phages were isolated from soil samples taken in 2011 and 2012, from the same fields from which bacterial isolates were obtained. To enrich for the desired phages, a bacterial host was grown in 25 ml LB with reduced salt concentration (LBrs) (0.5 g/l NaCl) at 26°C to exponential

growth phase and 5 g of a soil sample was added. After overnight incubation, 250 µl chloroform was added and incubation was continued for 1 h to lyse all cells. The bacterial culture was then centrifuged for 30 min at 4500 rpm in a Sorvall Legend RT+ centrifuge with swing-out 4-place rotor, type 75006445 (Thermo Scientific, Waltham, MA, USA). The supernatant was filtered using a Millex® syringe filter with a pore size of 0.45 µm (Merck Millipore Ltd.) and 10 µl was spotted on a soft agar layer supplemented with the bacterial host. Lysis zones were picked up with sterile toothpicks and suspended in phage buffer (10 mM Tris-HCl; pH 7.5; 10 mM MgSO4; 150 mM NaCl). These suspensions were plated by

addition of 250 µl overnight host culture and 100 µl phage suspension to 4 ml LBrs overlay

agar, according to the overlay agar technique (Adams, 1959). After overnight incubation at 26°C, single plaques were picked up again. Three successive single plaque isolations were performed to achieve pure phage isolates. Based on their DNA restriction pattern phages were selected for further analysis.

One bacteriophage (KIL3b) was obtained after a co-cultivation experiment. This experiment consisted of the infection of an exponentially growing liquid culture of P. syringae pv. porri strain CFBP 1687 with phage KIL3 at a multiplicity of infection (MOI) of 0.01. For four weeks, 40 µl of the liquid culture was transferred to a tube with 4 ml fresh LBrs medium every two to

three days and incubated at room temperature. Supernatant of the two day old liquid culture was spot tested against the bacterial strain P55 to assess the lytic activity.

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Phages were amplified by plating 105 _{pfu (plaque forming units) per plate together with their}

bacterial host in a soft agar overlay. After overnight incubation at 26°C, the soft agar was scraped off and suspended in phage buffer. Soft agar and cell debris was subsequently removed by centrifugation for 20 min at 4000 rpm in the Sorvall Legend RT+ centrifuge. The supernatant was filtered over a 0.22 µm membrane Millex® syringe filter (Merck Millipore Ltd.), after which polyethylene glycol (PEG) 8000 (Sigma-Aldrich) containing 1 M NaCl (Acros Organics) was added to the suspension to a final concentration of 8%. After minimum three hours incubation at 4°C, phages were precipitated by centrifugation at 4600 rpm for 30 min and the pellet was dissolved in 1 ml phage buffer.

Phage purification was performed using CsCl-gradient ultracentrifugation. A phage suspension (15 ml) with a minimum concentration of 1010 _{pfu/ml was layered on a CsCl step}

gradient (1.33, 1.45, 1.50 and 1.70 g/cm3_{) and centrifuged at 140,000 x g for 3 h with a}

Beckman Coulter Optima L-90K ultracentrifuge (rotor type SW28) (Brea, CA, USA). All phages were collected at the interface between the gradients of 1.45 and 1.50 g/cm3 _{and dialyzed}

three times for 30 minutes against 300 volumes of phagebuffer.

2.2.2. Electron microscopy

To obtain transmission electron microscopy (TEM) images of the bacteriophages, dilutions of the samples were spotted on carbon coated grids (Quantifoil, Großlöbichau, Germany) after glow-discharge, and negatively stained with 2% uranyl acetate. A Philips CM12 microscope was used at 120 kV acceleration voltage. Images were produced using a Gatan Orius 1k camera.

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2.2.3. Host range and general characterization

Phage host range was tested by spotting a phage suspension on a soft agar layer supplied with a possible host. For all the phages, three different concentrations ranging from 102_-106

pfu/ml were tested to determine infectivity. The bacterial strains used in the host range assay are listed in Table 6.1 and Table 3.1. This collection consists of field isolates supplemented with reference strains. Related pathovars were tested to determine the specificity of the investigated phages.

Adsorption experiments were performed according to Adriaenssens et al, (2012b). Briefly, bacteria and phage were mixed at an MOI of 0.0001. Next, samples were taken every minute and bacteria were immediately lysed by chloroform. The supernatant was titred to determine the number of non-adsorbed phages.

Killing curve at different MOIs were generated by infecting a bacterial culture at an optical density at 600 nm (OD600) of 0.3. The OD600 of the infected cultures was monitored every 20

min for 6 h and compared with that of an uninfected culture. OD results are the average of three independent biological repeats.

Phage stability was tested by incubating a phage suspension of 106 _{pfu/ml in phage buffer at}

different temperatures or at different pH ranging from 1 to 13. Infectivity of the phages at different temperatures was tested by incubating soft agar plates with spots of different phage concentrations at room temperature, 26°C and 30°C.

Resistance frequency was determined with an adaptation of a previously developed method (Beale, 1948). Using the agar overlay method, phage and bacteria were plated at MOI 1 and MOI 10, lysing the entire surface of the plate. After 72h of incubation, emerging colonies were counted and 20 colonies were cultured and tested for resistance against the other phages. Phage-resistant isolates of X. campestris pv. campestris and P. syringae pv. porri were plated on Pseudomonas agar F(PAF, Becton Dickinson) supplemented with sucrose or glycerol to check for respectively, yellow mucoid colony morphology or fluorescence under UV-light. Isolates of X. campestris pv. campestris were analyzed with a multiplex PCR consisting of three primer sets to confirm their identity. The UpBac primers binding on the 16S rDNA gene,

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present in all bacteria, served as internal control producing a 1511 bp amplification product. Second, the DLH-primers developed by Berg et al, (2005) binding on the hrpF gene and producing a PCR product of 619 bp with all X. campestris pathovars. And the third set consisting of ZUP-primers developed by Rijlaarsdam et al, (2004) on an AFLP-fragment of X.

campestris pv. campestris resulting in a PCR product of 446 bp with pvs. campestris and incanae. Isolates of P. syringae pv. porri were analyzed with another multiplex PCR containing

specific primers to confirm their identity (Ineke Wijkamp, Rijkzwaan, personal communication).

Tests for lysogeny in these resistant bacteria were performed as previously described (Petty

et al, 2006). Supernatant from overnight cultures of resistant bacteria were collected and

tested for the spontaneous release of phage particles by spotting 5 µl drops onto softagar containing the different bacterial hosts. The supernatant from CFBP1687, LMG 28496, LMG 28773, LMG 28774, LMG 28775 and LMG 28776 was used as a negative controls and phage lysates were used as positive control for plaque formation.