Clone And Functional Analysis Of Pyrrolnitrin Regulators Gene From Pseudomonas Fluorescens FD6

Antagonistic bacterium FD6was isolated from the canola rhizosphere in Fujian Province. The strain with broad antimicrobial spectrum can inhibit many plant pathogenic fungi. The bacterial culture of strain FD6significantly inhibited the spore germination with an inhibition rate of99%for B.cinerea HY2-1and59.7%efficiency to control grey mould on detached tomato leaves. The strain FD6was identified as P. fluorescens based on16S rDNA BLAST combined with the analysis of physiological and biochemical characterization. Strain FD6could produce multiple antifungal metabolites, including pyrrolnitrin,2,4-diacetylphloroglucinol, pyoluterorin, siderphore, hydrogen cyanide and extracellular proteinase, but not phenazine-1-carboxylic acid. The pyrrolnitrin inhibited directly the spore germination and mycelial growth of B. cinerea HY2-1.The two-component regulatory system including sensor kinase GacS and response regulator GacA is widespread in bacteria. In order to confirm the effect of gacS gene on biocontrol ability of FD6, we constructed a gacS insertion mutant FD6-MS and a complementary strain FD6-S. The mutant strain FD6-MS couldn’t produce pyrrolnitrin,2,4-diacetylphloroglucinol, pyoluterorin and hydrogen cyanide, whereas generation of siderphore and biofilm were markedly reduced. The results showed that gacS gene was required for the production of a couple of antifungal metabolites, but has little effect on the strain growth rate. The biological experiment of B. cinerea and Monilinia sp. shows that the gacS mutant lost the inhibitory effect against these two plant pathogenic fungi. The lesion diameter of FD6-MS treatment is similar to the control group, and the complementary strain FD6-S could restore the inhibitory effect. These results indicated that gacS gene is an important regulatory element in biocontrol bacteria FD6.In order to find specific transcriptional regulators of pyrrolnitrin, the β-galactosidase activity for screening marker, we constructed FD6mutant library using Tn5transposon. Three mutants were obtained including prnA, hemK and ATP-binding protein by Tn5random mutagenesis. In P. fluorescens FD6, prnA gene is one of PRN structural genes and closely related to the synthesis of PRN. The deletion and complementation of prnA experiment indicated that that the prnA had little effect on the bacteria growth and antagonistic ability. The results of lacZ enzyme activity showed prnA regulated negativly on PRN production at the transcriptional level. Maybe prnA gene had feedback regulation on the production of PRN...