Native strain P. fluorescens SF4c produces a bacteriocin that is active against pytopathogenic Pseudomonas

SONIA FISCHER; AGUSTINA GODINO; JOSÉ MIGUEL QUESADA; PAULA CORDERO; EDGARDO JOFRÉ; GLADYS MORI; MANUEL ESPINOSA-URGEL

Congreso; XXV Reunión Latinoamericana de Rizobiología; 2011

Pseudomonas fluorescens SF4c is a strain isolated from the rhizosphere of wheat grown in fields of Argentina. This strain promotes the growth of wheat and tomato under greenhouse conditions (Fischer et al., 2007). Bacteria living in a competitive environment are able to secrete proteinaceous toxins, known as bacteriocins, which can kill closely related bacterial competitors while causing little harm to the bacteriocinogenic cells. On the other hand, bacteriocins have potential as biological control agents that can be used against bacterial pathogens (Parret et al., 2005). P. fluorescens SF4c secretes a protease-resistant bacteriocin that kills P. fluorescens CTR 212 and phytopathogens of the genus Pseudomonas (P. corrugata, P. syringae). The production of this bacteriocin is enhanced by mitomycin C, a DNA-damaging agent, suggesting that SOS response is activated. Previously, mutants of strain SF4c impaired in bacteriocin production were generated by Tn5-B20 random mutagenesis in our lab. In one of these mutants, the transposon was inserted into a gene that has homology with a gene enconding ?phage tail tape measure protein TP901? of P. fluorescens Pf0-1. This gene belongs to prophage 01 from strain Pf0-1, which is similar to the R2/F2 pyocin (phage like-bacteriocin from P. aeruginosa PAO1) (Mavrodi et al., 2009). We have named this gene ptm for Phage tail tape measure protein. Tn5-B20 insertion into ptm could have a polar effect on the expression of genes downstream from ptm. Therefore, we constructed a non-polar ptm mutant of P. fluorescens SF4. The clones obtained were checked by Southern hybridization, PCR, and sequencing to confirm replacement of ptm by ptm::Km. One of these clones was selected and named PTM50. This mutant did not present antibacterial activity against strains P. fluorescens CTR212. To elucidate the structure of high-molecular weight bacteriocin secreted by P. fluorescens SF4c, we purified the bacteriocin and performed transmission electron microscopy examination. This technique allowed visualization of particles resembling the bacteriophage tails in the culture supernatant of strain SF4c, confirming that this strain produced phage-like bacteriocin. Lytic system is necessary for a phage-like pyocin to be released from the cells. Genes encoding lytic system (hol and lys) are present in prophage 01 from P. fluorescens and pyocin from P. aeruginosa (Nakayama et al., 2000; Mavrodi et al., 2009). We demonstrated the presence of these genes in the genome of P. fluorescens SF4c. Finally, we analyzed the expression of pyocin under different condition through RT-PCR. Results show that UV light and hydrogen peroxide induce the expression of pyocin in P. fluorescens SF4c. Generally, bacteriocins are associated with bacterial competitiveness in the environment, but this topic is relatively unexplored. In the future, we will evaluate the ecology role of bacteriocin secreted by native strain P. fluorescens SF4c.