Characterization Of Transcriptional And Posttranscriptional Regulators Of2,4-Diacetyphloroglucinol Biosynthesis Genes In Pseudomonas Fluorescens2P24

Pseudomonas fluorescens2P24is a biocontrol agent that isolated from suppressive soil of wheat take-all disease in Shandong Province. It has remarkable biocontrol activity against plant diseases caused by soilborne pathogens. The polyketide metabolite2,4-diacetylphloroglucinol (2,4-DAPG) plays a major role in the biological control of diseases by this strain. Biosynthesis proteins of2,4-DAPG in strain2P24were encoded by a genetic locus phlACBD, whose expression was under the control of a number of transcriptional and post-transcriptional regulators. Among these regulators, RsmA and RsmE are important post-transcriptional repressors. In this study, we first analysed the promoter of phlA gene and constract the transcriptional report plasmid system of phlA gene promoter. Then, we screened the regulatory factors of rsmA/rsmE genes to further study the regulatory mechanism of the factors affecting the production of2,4-DAPG.In the first part, the promoter of the first gene phlAinphlA CBD operon was analysed and cut to11different length fragment. Then these fragments containing different length promoters of phlA gene were linked to plasmid p970Km to form11transcriptional report plasmids of phlA gene. These report plasmids were imported into the mutants of six regulator genes and the wild-type2P24by electroporation to explore the possible binding sites on phlA promoter by these regulators. The results indicated that the pho site which is bound by PhlF repressor was the major acting site of all the six regulators. Then, the regulatory mechanism of GacA was selected for further study. The results suggested that low production of2,4-DAPG could not relieve the repression by PhlF on pho site in gacA gene mutant. Therefore we speculated that PhlF was the major transcriptional regulator ofphlA gene and the other transcriptional regulators seemed to regulate the phlA transcription through affecting the function of PhlF.In the second part of this work, the strains with rsmA or rsmE transcriptional reporter were subjected to the random transposon mutagenesis. About86mutants were selected from-30000insertion colonies. Among these86mutants, the rsmA/E transcriptions in18mutants were increased while the other68mutants were decreased. A mutant defective in the vacB gene, which encoded the Ribonuclease R, drastically increased the rsmA gene expression. The effect of VacB on other biocontrol characters implied that VacB was a globe regulator in strain2P24.In the last part of this work, we studied the regulatory mechanism of DsbA on the production of2,4-DAPG. DsbA is a major periplasmic disulfide-bond-forming protein. The dsbA mutant produced more2,4-DAPG compared with the wild strain. Genetic evidences showed that DsbA did not affect transcription of the phlA gene but observably increased the phlA gene translation. Further study showed that the DsbA probably affected the2,4-DAPG production by promoting the expression of repression protein RsmA at both transcriptional level and translation level while this transcriptional regulation seemed to be indirect.