Characterization of a Lytic Bacteriophage against Pseudomonas syringae pv. actinidiae and Its Endolysin

Abstract

Pseudomonas syringae pv. actinidiae (Psa) is a phytopathogen that causes canker in kiwifruit. Few conventional control methods are effective against this bacterium. Therefore, alternative approaches, such as phage therapy are warranted. In this study, a lytic bacteriophage (PN09) of Psa was isolated from surface water collected from a river in Hangzhou, China in 2019. Morphologically, PN09 was classified into the Myoviridae family, and could lyse all 29 Psa biovar 3 strains. The optimal temperature and pH ranges for PN09 activity were determined as 25 to 35 ∘C and 6.0 to 9.0, respectively. The complete genome of PN09 was found to be composed of a linear 99,229 bp double-stranded DNA genome with a GC content of 48.16%. The PN09 endolysin (LysPN09) was expressed in vitro and characterized. LysPN09 was predicted to belong to the Muraidase superfamily domain and showed lytic activity against the outer-membrane-permeabilized Psa strains. The lytic activity of LysPN09 was optimal over temperature and pH ranges of 25 to 40 ∘C and 6.0 to 8.0, respectively. When recombinant endolysin LysPN09 was combined with EDTA, Psa strains were effectively damaged. All these characteristics demonstrate that the phage PN09 and its endolysin, LysPN09, are potential candidates for biocontrol of Psa in the kiwifruit industry.

Keywords: Pseudomonas syringae pv. actinidiae; endolysin; kiwifruit; lytic bacteriophage; phage therapy.

Figure 1

Morphology of phage PN09 observed using transmission electron microscopy.

Figure 2

One-step growth curve of phage PN09 on the bacterial host Pseudomonas syringae pv. actinidiae (Psa) strain SCJY02-1 at 27 °C. The means and standard deviations of three independent assays are shown.

Figure 3

Stability of phage PN09. (A) Thermostability (B) pH stability. The means and standard deviations of three independent assays are shown. Shared letters above bars indicate no statistical difference among groups (p > 0.05) and different letters above bars indicate a statistically significant difference (p < 0.05). Phage PN09 showed a relatively high survival rate at pH 6.0–9.0.

Figure 4

BLASTn comparison of the complete genome sequence of the phage PN09 with the closet homolog phage phiPsa315 using Easyfig. The blue arrows indicate the predicted genes of both phages. The homologous regions between the phages are indicated by gray shading.

Figure 5

Expression and characterization of LysPN09, the endolysin in the lytic phage PN09. (A) The domain organization of LysPN09. (B) SDS-PAGE analysis of LysPN09. Lane M, molecular weight marker. Lane 1, un-induced lysate extract of BL21(DE3). Lane 2, IPTG-induced bacterial lysate. Lane 3, purified recombinant LysPN09.

Figure 5

Expression and characterization of LysPN09, the endolysin in the lytic phage PN09. (A) The domain organization of LysPN09. (B) SDS-PAGE analysis of LysPN09. Lane M, molecular weight marker. Lane 1, un-induced lysate extract of BL21(DE3). Lane 2, IPTG-induced bacterial lysate. Lane 3, purified recombinant LysPN09.

Figure 6

The lytic activity of different concentrations of LysPN09 on the Pseudomonas syringae pv. actinidiae (Psa) strain SCJY02-1 used as the host bacterium. LysPN09 concentrations of 12.5, 25.0, 50.0, 100.0, 200.0, and 400.0 μg/mL were used. The means and standard deviations of three independent assays are shown. Shared letters above bars indicate no statistical difference among groups (p > 0.05) and different letters above bars indicate a statistically significant difference (p < 0.05).

Figure 7

Stability of LysPN09. (A) Thermostability of the phage PN09 endolysin. LysPN09 (final concentration: 400.0 μg/mL) was incubated at different temperatures for 30 min. (B) pH stability of PN09 endolysin. LysPN09 (final concentration: 400.0 μg/mL) was incubated at the indicated pH conditions for 30 min. Shared letters above bars indicate no statistical difference among groups (p > 0.05) and different letters above bars indicate a statistically significant difference (p < 0.05).

Figure 8

Antibacterial activity of LysPN09 in combination with EDTA. (A) Pseudomonas syringae pv. actinidiae (Psa) strain SCJY02-1 was treated with LysPN09 (final concentration: 400.0 μg/mL), EDTA (final concentration: 1.0 mM), and LysPN09 + EDTA (final concentration: 400.0 μg/mL and 1.0 mM, respectively). The control contained 50.0 μL of PBS buffer (pH 7.4) instead of LysPN09 or EDTA. The OD₆₀₀ values of Psa were measured every 5 min for 60 min. (B) After 60 min of incubation, the residual viable cells were plated and measured. Shared letters above bars indicate no statistical difference among groups (p > 0.05) and different letters above bars indicate a statistically significant difference (p < 0.05).