The CbrAB two-component system affects the bacteriocin production in Pseudomonas fluorescens SF39a

AGUSTINA GODINO; ANALÍA PRÍNCIPE; MARICRUZ FERNANDEZ; EDGARDO JOFRÉ; SONIA FISCHER

Workshop; II Workshop Latinoamericano sobre Rizobacterias Promotoras del Crecimiento Vegetal; 2014

In the rhizosphere, bacteria are constantly competing for nutrients and space. Competition is often driven by the production of bacteriocins, which are proteins or peptides that kill closely related species, providing the producer better access to limited resources. The rhizospheric isolate P. fluorescens SF39a secretes a low-molecular-mass bacteriocin. Previously, a miniTn5Km1 mutant affected in bacteriocin production was obtained. In this mutant, the transposon was inserted into cbrA gene which encodes the sensor of the two-component system CbrAB. CbrAB system is necessary for the growth of pseudomonads on numerous C and N sources. When bacteria grow on a less preferred source the CbrAB system is actived and the response regulator CbrB activates the transcription of the sRNA gene crcZ. CrcZ sequesters the protein Crc and the catabolic pathways for less preferred substrates become functional. In this work, we study the implication of the CbrAB system in the regulation of the bacteriocin of P. fluorescens SF39a. Primers were designed from the genome of P. fluorescens Pf01 and F113 to amplify cbrA, cbrB, crcZ and crc genes of P. fluorescens SF39a. cbrA gene was cloned into pBBR1-msc5 vector and the recombinant plasmid (pBBR5cbrA) was incorporated into cbrA mutant for complementation studies. A non-polar cbrB mutant derivative of P. fluorescens SF39a was constructed by the replacement of an internal 1000 bp fragment with a Km-resistance cassette. The bacteriocin production was analyzed in the strain SF39a, cbrA mutant, cbrB mutant and cbrA mutant complemented with pBBR5cbrA plasmid. Pyoverdin production and growth on minimal medium supplemented with arginine as carbon and nitrogen source were also analyzed in these strains. The cbrA, cbrB, crcZ and crc genes from strain SF39a were amplified by PCR and the PCR products were sequenced. Therefore, we have the complete sequence of the cbrA and cbrB genes, which are part of an operon, the crcZ gene, which is downstream of cbrB but outside the operon, and the crc gene, which is in a more distant region of the genome. Moreover, we have complemented the cbrA mutant and have obtained a cbrB mutant. The cbrA and cbrB mutants showed an increase in bacteriocin production with respect to the wt strain. The cbrA mutant complemented with the plasmid pBBR5CbrA was able to restore the wild-type phenotype, confirming that the increase in the zone of inhibition in this mutant was caused by inactivation of the cbrA gene. In addition, the cbrA and cbrB mutants were unable to grow on minimal medium supplemented with arginine as sole carbon and nitrogen source and showed a decrease in pyoverdin production. We have identified the genes from the two-component system CbrAB, the sRNA CrcZ and the protein Crc from P. fluorescens SF39a. Mutations in cbrA and cbrB genes affect the bacteriocin production in this strain; thereforewe propose that the CbrAB two-component system directly or indirectly regulates the bacteriocin production in P. fluorescens SF39a. Further studies are in progress to evaluate the implication of sRNA CrcZ and protein Crc in the bacteriocin production in this plant growth promoting Pseudomonas.