ISOLATION AND CHARACTERIZATION OF PSEUDOMONAS SP. SF4C MUTANTS IMPAIRED IN BACTERIOCIN PRODUCTION

SONIA FISCHER; EDGARDO JOFRÉ; AGUSTINA GODINO; GLADYS MORI

Villa Carlos Paz, Córdoba, Argentina

Workshop; II Argentine Workshop in Current Topics in Pseudomonas and Burkholderia Research; 2009

Bacteria living in a competitive environment are able to secrete proteinaceous toxins, known as bacteriocins, which cankill 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. Pseudomonas sp.SF4C (native strain isolated from wheat rhizosphere) produces a high-molecular-weight bacteriocin (resistant toproteinase K) that is active against Pseudomonas fluorescens CTR 212. To obtain mutants impaired in bacteriocinproduction, random transposon mutagenesis of Pseudomonas sp SF4c was carried out. Kanamycin-resistant Tn5-B20mutants of strain SF4C that had lost the capacity to inhibit the growth of strain CTR 212 were isolated. The presence ofa single Tn5-B20 insertion in some mutants (496, 581, 634) was confirmed by Southern blot analysis. All thetransconjugants analyzed showed hybridizing fragments of similar sizes. Therefore, it is possible that the transposon hasbeen inserted at identical positions in the genomes of these mutants. An EcoRI fragment containing the Tn5-B20insertion present in the mutant strain 634 was cloned into vector pBluescript to generate the plasmid pSF16. DNAsequence analysis of pSF16 revealed that the transposon was inserted into a gene encoding a putative phage tapemeasureprotein involved in the determination of tail length in phage. This gene belongs to Prophage 01 cluster fromPseudomonas fluorescens Pf0-1. Closely related prophages 01 exist in the genomes of P. fluorescens Pf-5, P.fluorescens Q8r1-96 and P. fluorescens SBW25. The homologous prophage elements from Pf-5 and Q8r1-96 aresimilar to F-type pyocins. These pyocins were first discovered in P. aeruginosa and represent a class of high-molecularweight protease- and nuclease-resistant bacteriocins that resemble flexible and non-contractile tails of bacteriophages. Aprophage element found in the identical spot (between mutS and cinA) in the genome of P. fluorescens SBW25 has asimilar overall organization but resembling another class of phage tail-like bacteriocins, the R-type pyocins of P.aeruginosa. Furthermore, a homologous region from P. fluorescens Pf0-1 contains two clusters, similar to the hybridR2/F2 pyocin locus from P. aeruginosa PAO1. The differences in organization of the putative phage tail-like pyocinsamong these prophages clearly indicate that the corresponding loci are subject to extensive recombination.