Xanthomonas campestris pv. glycines 噬菌體 ϕXcg25 之分離與分析

Abstract

Xanthomonas campestris pv. glycines (Xcg) is a Gram-negative plant pathogenic bacterium causing pustules in susceptible soybean cultivars. Phages are virus that specifically infect bacteria, in which lytic phages can cause host cells death. So far, no Xcg phage has been reported. In this study, 4 lytic phages of Xcg, designated as ϕXcg6L, ϕXcg15L, ϕXcg25L and ϕXcg25S, were isolated from 28 environmental samples. Results of restriction analysis of the genomic DNA and profiling of the virion proteins in SDS-polyacrylamide gel indicated that these phages are of the same type. Phage ϕXcg25S which exhibited the strongest lysis ability was selected for further study. Electron microscopy revealed that ϕXcg25 possesses the typical morphology of a siphovirus, with a hexagonal head (65 X 60 nm) and a long non-contractile tail (193 nm in length). ϕXcg25 can infect Xcg12609 and Xcg12620 but not Xcg strain YR32, E. coli and P. aeruginosa. A phage titer reaching 1.0 x 1011 PFU/ml can be produced upon infecting rapidly growing Xcg12609. After being stored at 4 oC for 12 months, about 54% of the phage particles were found to retain infectivity. Testing of host lysis ability showed that complete lysis of Xcg12609 can be achieved by infecting at an MOI of 0.01, but no apparent lysis was observed on Xcg12620 by infecting at an MOI of 10. Host lysis was enhanced in the presence of 10 mM MgSO4 supplemented, indicating that Mg2+ is required for the lysis of host cells by ϕXcg25. In adsorption test, 10% of the phage particles was found to be adsorbed at 10 min, with the adsorption rate reaching 60% at 60 min. One step growth assay suggested that ϕXcg25 has a latent period and burst size of approximately 45 min and 150 PFU per infected cell, respectively. Sequencing revealed that the ϕXcg25 genome consists of 43,221 bp with a G+C content of 54.9%. Of the 56 ORFs identified, 33 are similar to known proteins which can be classified into four different functional categories (lysis, head, tail and DNA replication/repair), 18 are similar to hypothetical proteins, and 5 find no homologues in database. Similarity analysis showed that proteins of ϕXcg25 shares an average identity of 85 %, 77% and 65% with those from P. aeruginosa phage vB_Pae-Kakheti25 (PA25), P. aeruginosa phage 73 (PA73), and Burkholderia phage KL1 (KL1), respectively. In addition, the tail related proteins of ϕXcg25 showed an average identity of 34.5% and 35.3% to those of YuA-like phage YuA and M6, respectively. ϕXcg25 is similar to many P. aeruginosa phages but it cannot infect P. aeruginosa indicating that the major host anti-receptor is significantly different. Further efforts are required for developing ϕXcg25 into a biological agent for control of bacterial pustules in soybean.