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

GODINO, A.; PRÍNCIPE, A.; FERNANDEZ, M.; JOFRÉ, E.; FISCHER, S.E.

La Falda

Workshop; PGPR Latinoamérica 2014; 2014

In the rhizosphere, bacteria are constantly competing for nutrients and space. Competition is oftendriven by the production of bacteriocins, which are proteins or peptides that kill closely relatedspecies, providing the producer better access to limited resources. The rhizospheric isolate P.fluorescens SF39a secretes a low-molecular-mass bacteriocin. Previously, a miniTn5Km1 mutantaffected in bacteriocin production was obtained. In this mutant, the transposon was inserted intocbrA gene which encodes the sensor of the two-component system CbrAB. CbrAB system isnecessary for the growth of pseudomonads on numerous C and N sources. When bacteria grow ona less preferred source the CbrAB system is actived and the response regulator CbrB activates thetranscription of the sRNA gene crcZ. CrcZ sequesters the protein Crc and the catabolic pathways forless preferred substrates become functional. In this work, we study the implication of the CbrABsystem in the regulation of the bacteriocin of P. fluorescens SF39a. Primers were designed from thegenome of P. fluorescens Pf01 and F113 to amplify cbrA, cbrB, crcZ and crc genes of P. fluorescensSF39a. 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 mutantderivative of P. fluorescens SF39a was constructed by the replacement of an internal 1000 bpfragment with a Km-resistance cassette. The bacteriocin production was analyzed in the strainSF39a, cbrA mutant, cbrB mutant and cbrA mutant complemented with pBBR5cbrA plasmid.Pyoverdin production and growth on minimal medium supplemented with arginine as carbon andnitrogen source were also analyzed in these strains. The cbrA, cbrB, crcZ and crc genes from strainSF39a were amplified by PCR and the PCR products were sequenced. Therefore, we have thecomplete sequence of the cbrA and cbrB genes, which are part of an operon, the crcZ gene, which isdownstream of cbrB but outside the operon, and the crc gene, which is in a more distant region ofthe genome. Moreover, we have complemented the cbrA mutant and have obtained a cbrBmutant. The cbrA and cbrB mutants showed an increase in bacteriocin production with respect tothe wt strain. The cbrA mutant complemented with the plasmid pBBR5CbrA was able to restore thewild-type phenotype, confirming that the increase in the zone of inhibition in this mutant wascaused by inactivation of the cbrA gene. In addition, the cbrA and cbrB mutants were unable togrow on minimal medium supplemented with arginine as sole carbon and nitrogen source andshowed a decrease in pyoverdin production. We have identified the genes from the twocomponentsystem CbrAB, the sRNA CrcZ and the protein Crc from P. fluorescens SF39a. Mutationsin cbrA and cbrB genes affect the bacteriocin production in this strain; thereforewe propose thatthe 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 andprotein Crc in the bacteriocin production in this plant growth promoting Pseudomonas.