Molecular and functional analysis of a putative pyocin S9, with endonuclease activity from P. chlororaphis subsp aurantiaca PB-St2

Abstract

Pyocins are bacteriocins which are explicitly associated with pseudomonads. In this study, the genome mining and in- depth sequence analysis identified three similar S9-like (a, b, and c), an S3-like (d) and one R-type pyocin systems from

P. chlororaphis subsp aurantiaca PB-St2. The phenotypic screening of bacteriocin production by PB-St2 indicated narrow- spectrum bactericidal activity against closely related Pseudomonas species i.e., Pseudomonas aeruginosa PAi, PAc1,

PAc3, PAc4; Pseudomonas fluorescens Psi-RS1 and Pseudomonas kilonensis OSRS3. Herein, the proposed pyocin S9c was further selected for molecular and functional characterization. The presumptive N-terminal receptor binding domain of candidate system lacks significant similarity with any characterized HNH-type pyocin S DNases from P. aeruginosa. In contrast, the cytotoxic domain showed 53% sequence similarity with pyocin S8 and 70% to pyocin S9. Thus, pyocin S9c was suggested as an isoform under the Class I DNase (H-N-H) family in pyocin S9 cluster, commonly found in P. chlororaphis subsp. aurantiaca and P. chlororaphis subsp aureofaciens strains. Molecular screening of the pyocin S9c system revealed its presence in 6 out of 7 tested strains of P. chlororaphis subsp. aurantiaca GS1, GS3, GS4, GS6, ARS38, FS2 and one P. chlororaphis RP4 relative strains, isolated from diverse plant hosts. The 1.59 kb fragment consisting of two structural genes of pyocin-immunity operon (S9c) in P. aurantiaca PB-St2 were cloned in pET28a(+) and expressed in Escherichia coli BL21 DE3 (pLysS) strain as a fusion protein with histidine tag. The recombinant cytotoxic protein of pyocin S9c operon was purified with N-term His-tag with a molecular weight of ≈50 kDa. The identity of target protein was affirmed by tandem mass spectrometry analysis. The purified cytotoxic protein was active against P. chlororaphis subsp. aurantiaca GS7, with a minimum inhibitory concentration of 12.5 μg/ml. The mechanism of cytotoxicity was affirmed as a metal-dependent endonuclease by evidence of non-specific hydrolysis of pTZ57R plasmid isoforms. These results indicate that pyocin S9c can contribute to the rhizo-competence of this strain in plant-associated natural habitats, occupied by related Pseudomonas strains.