Culturing of strain NJSZ-13 and pinewood nematode
The B. cereus strain NJSZ-13 was grown in NB medium (1% peptone, 0.3% beef extract, 0.5% NaCl, pH 7.2 ± 0.2) at 28 °C on a rotary shaker at 200 rpm. The test species was the pinewood nematode B. xylophilus which was cultured in Botrytis cinerea for 5–7 days at 25 °C and harvested using the Baermann funnel technique . An nematode suspension (5 nematodes/μL) in sterile PBS (pH 7.4) was used as working stock.
The bioassay was performed as previously described . Briefly, the NA medium (1% peptone, 0.3% beef extract, 0.5% NaCl, 2% agar, pH 7.2 ± 0.2) plates were covered with axenic cellophane paper to prevent movement of the nematodes into the medium. The bacteria were placed on the cellophane and cultivated in an incubator at 28 °C. When the entire plate was covered by the bacterial colony, 100 μL of the nematode suspension (containing about 500 nematodes) was transferred to the plate center. The plates were each divided into 20 sections, and every 24 h, five sections were randomly selected and the nematode mortality assessed using a light dissecting microscope. Experiments were conducted in triplicate, and at least 30 nematodes were observed each time. Non-pathogenic bacteria (Escherichia coli, ATCC 25922) were used as a control.
Nematodes treated with strain NJSZ-13 were picked up and gently mounted in sterile PBS containing 1.42 g/L Na2HPO4, 0.27 g/L KH2PO4, 0.2 g/L KCl, and 8 g/L NaCl, pH 7.4 before examination under light microscopy and scanning electron microscopy (SEM). If the nematodes did not move when examined under the light microscope or if they did not respond to light tapping with a needle, they were considered dead. The percentage of dead nematodes was described as the mortality rate.
Extraction of extracellular proteins from NJSZ-13
The strain NJSZ-13 was inoculated into a 250-mL Erlenmeyer flask containing 100 mL NB medium and incubated at 28 °C on a rotary shaker (200 rpm) for 4 days. The culture solution was centrifuged at 8500 g and 4 °C for 15 min, the supernatant retained, and sufficient (NH4)2SO4 added to 100% saturation. After standing at 4 °C overnight, the solution was centrifuged at 5500 g at 4 °C for 30 min and the precipitate was dissolved in 10 mL of sterile PBS (pH 7.4). The solution was loaded into a dialysis bag with a cut-off molecular weight of ~ 8000–15,000 Da. The dialysis bag was then immersed in 20 volumes of phosphate buffer, changed four times with each dialysis lasting approximately 3 h. The resulting component was the crude extracellular protein extract of the strain NJSZ-13. The crude extract was filtered through a 0.22-μm filter and used immediately.
Measurement of the effects of the protein extract against the pinewood nematode
Two hundred microliters of the protein extract were placed in sterile 1.5 mL Eppendorf tubes with 200 μL of the nematode suspension (approximately 1000 nematodes) and incubated at 25 °C. Each 24 h, a 20 μL aliquot of the protein-nematode suspension (approximately 50 nematodes) was placed on a glass slide and the nematode mortality assessed under light microscopy. Experiments were conducted in parallel with five replications and were repeated three times. Controls contained sterile PBS (pH 7.4), NB medium, NJSZ-13 culture supernatant, and heat-denatured (100 °C for 15 min) protein extract.
Changes in the cuticles of the nematodes were assessed under light microscopy and SEM.
Preparation of nematode samples for the light microscope and SEM
The treated nematodes were immersed in sterile PBS (pH 7.4). The nematodes were then observed and photographed under the light microscope (Axio Imager M2; Zeiss, Oberkochen, Germany).
For the SEM observation, the nematodes were first pre-fixed in glutaraldehyde (4%) in sterile PBS at 4 °C for 2 h then dehydrated in serial ethanol concentrations (50, 70%, two changes of 90%, and three changes of 100% ethanol for 10 min each) at room temperature. The dehydrated nematodes were then dried by a critical point drying instrument (K850; Emitech, East Sussex, UK) and sputter-coated with Au-Pd using ion-sputtering equipment (E-1010; Hitachi, Tokyo, Japan) before being examined and photographed with a “desktop SEM (NeoScope JCM-5000; Nikon, Tokyo, Japan)”.
Five hundred milliliters of NJSZ-13 culture was centrifuged at 8500 g and 4 °C for 15 min. Sufficient (NH4)2SO4 was added to the supernatant to achieve 20% saturation. After standing overnight at 4 °C, the solution was centrifuged (5500 g, 4 °C, 30 min), and the supernatants were pooled. Then, (NH4)2SO4 was added to reach 40, 60, 80%, and finally 100% saturation, solutions were centrifuged as above, and the nematicidal pellet was dissolved in PBS for chromatography.
Hydrophobic interaction chromatography (HIC). The dissolved precipitate with nematicidal activity was supplemented with 1 M (NH4)2SO4 and applied to a HiTrap™ phenyl FF column (high sub，1 mL; General Electric Company, Boston, USA) that had been equilibrated with sterile PBS containing 1 M (NH4)2SO4 (pH 7.4). The bound proteins were eluted with a linear gradient of 1–0 M (NH4)2SO4 in PBS with a flow rate of 1 mL/min. Fractions (1 mL) were collected, dialyzed, and the protease and nematicidal activity was measured.
Ion-exchange chromatography (IEC). Fractions positive for protease and nematicidal activities were pooled and applied to a HiTrap™ Capto Q column (1 mL; General Electric Company, Boston, USA) equilibrated with sterile PBS (pH 6.0). Bound proteins were eluted with a linear gradient of 0–1 M NaCl (pH 6.0) with a flow rate of 1 mL/min. Fractions (1 mL) were collected, dialyzed, and their protease and nematicidal activities evaluated.
SDS-PAGE. Fractions positive for protease and nematicidal activity were electrophoresed by SDS-PAGE (12% running gel and 4% concentration gel). Gels were “stained with Coomassie brilliant blue R-250” for 2 h.
Protease activity was measured with 1% casein solution as substrate. One hundred and twenty-five microliters of 1% casein in sterile PBS (pH 7.4) and the protease-containing sample were incubated in 1.5 mL-Eppendorf tubes at 37 °C for 10 min. The reaction was halted by the addition of 250 μL of 0.4 M trichloroacetic acid (TCA), and the solution was centrifuged at 4 °C and 8500 g for 15 min. The supernatant was transferred to tubes containing 2.5 mL of 0.4 M sodium carbonate and 0.5 mL Folin phenol reagent, incubated at 37 °C for 20 min, and the OD at 680 nm read in a spectrophotometer. A tyrosine-containing standard was used for calibration and the protease activity in the samples was assessed as the tyrosine increase over 1 min at 37 °C.
The protein content was measured using an enhanced BCA protein assay kit (Beyotime, Shanghai, China) with bovine serum albumin (BSA) as the standard.
Characterization of the protease
To determine the optimum temperature for proteolytic reaction, the activity of the protease was measured by incubating the reaction mixture at 20, 30, 37, 45, 50, 55, 60, 70, 80 and 90 °C as described above. The optimum pH was measured with the “Britton Robinson universal buffer system” at pH 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, 10.0, and 11.0, respectively.
Samples were incubated in the presence of different metals (Li+, K+, Na+, Mg2+, Ca2+, Mn2+, Zn2+, and Ba2+) to measure the effects of metals on the enzyme with protease activity without any metal ions set to100%.