Cloning and expression of a toxin gene from Pseudomonas fluorescens GcM5-1A

Abstract

Pseudomonas fluorescens GcM5-1A was isolated from the pine wood nematode (PWN), Bursaphelenchus xylophilus, obtained from wilted Japanese black pine, Pinus thumbergii, in China. In this paper, a genomic library of the GcM5-1A strain was constructed and a toxin-producing clone was isolated by bioassay. Nucleotide sequence analysis revealed an open reading frame of 1,290 bp encoding a protein of 429 amino acids with N-terminal putative signal peptide of 36 amino acids, which shared a similarity of 83, 82 and 80% identity with hypothetical protein PFLU2919 from P. fluorescens SBW25, Dyp-type peroxidase family protein from P. fluorescens Pf-5 and Tat-translocated enzyme from P. fluorescens Pf0-1, respectively. The gene encoding a full-length protein or without the putative signal peptide was cloned and expressed as a soluble protein in E. coli. The recombinant protein was purified to electrophoretic homogeneity by affinity chromatography using a Ni2+ matrix column. Its relative molecular weight was estimated to be 48.5 kDa by SDS-PAGE for full-length protein, and 45.0 kDa for the recombinant protein without putative signal peptide. Bioassay results showed that the recombinant protein with or without the putative signal peptide was toxic to both suspension cells and P. thunbergii seedlings. HPLC analysis demonstrated that components in branch extracts of P. thunbergii were significantly changed after addition of the recombinant full-length protein and hydrogen peroxide, which indicated that it is probably a peroxidase. This study offers information that can be used to determine the mechanism of pine wilt disease caused by the PWN.

Nematicidal Bacteria Associated to Pinewood Nematode Produce Extracellular Proteases

“…Some authors are convinced that those bacteria might play an important role in PWN pathogenicity [25,27] although the role of the bacteria carried by the nematode in PWD is not clear. The literature (reviewed in 28) supports the idea that bacteria isolated from nematodes from PWD trees, interact in symbiosis with the nematode to cause the disease but also points to the fact that there is no data from USA or Europe to support that idea.…”

Bacterial role in pine wilt disease development – review and future perspectives

“…Guo and co‐workers () showed that two dipeptides (cyclo ‐Pro‐Val‐ and cyclo ‐Pro‐Tyr‐) produced by Pseudomonas fluorescens GcM5‐1A caused calli necrosis within 48 h, and rapid wilting (5 days after inoculation) of black pine seedlings. Another toxin protein from P. fluorescens GcM5‐1A, identified as Dyp‐type peroxidase, showed high toxicity to calli and seedlings of P. thunbergii by oxidizing phenolic and other smaller molecular weight compounds in the presence of hydrogen peroxide (Kong et al ., ). Guo and colleagues () documented that P. fluorescens GcM5‐1A flagellin was able to increase PWN and associated bacteria population.…”

DyP-type Peroxidases: A Promising and Versatile Class of Enzymes

“…This indicates that DyP peroxidases could be promising, novel anti-microbial (pro) drug targets. This notion is supported by a recent study, which showed that a DyP peroxidase from Pseudomonas fluorescens GcM5-1A is toxic to cells of the Japanese black pine [36].…”