The Regulation Of Antibiotic Production By OprF And SigX Gene In Pseudomonas Fluorescens 2P24

Pseudomonas fluorescens 2P24 was isolated from the wheat take-all infected filed in Shandong province.The 2,4-diacetylphloroflucinol(2,4-DAPG)is the major antibiotic compound produced by strain 2P24 to protect crops from several soilbome diseases.In this study,two novel 2,4-DAPG regulators,the genes oprF and sigX,were identified via the mini-Tn5 transposon mutagenesis,and the regulatory mechanism of these genes on 2,4-DAPG production was explored.Eight mutants with obviously increased 2,4-DAPG production were selected out from approximately 10000 Tn5 mutants.Sequence analysis revealed that the transposon-disrupted genes were phlF,oprF,ass,nirF,waaQ,vacB,oprL and emhA in these mutants,among which the mutant PM650(oprF::Tn5)showed the biggest improvement of 2,4-DAPG production.The oprF gene encoding an outer membrane protein was involved in forming non-specific channels and the sigX gene encoding an ECF(extracytoplasmic functions)sigma factor localized immediately upstream of oprF.The oprF mutant PM651,sigX mutant PM652 and double mutant PM653 were constructed by the two-step homologous recombination.Antibiotics quantification revealed that the oprF gene negatively regulated production of 2,4-DAPG and phloroglucinol(PG),whereas the sigX gene showed a positive regulation.Double mutant PM653 produced less antibiotics similar to the sigX mutant.The antifungal activities of above mutants in dual culture test were consistent with their antibiotic production.Genetic complementation of SigX,but not OprF,in the double mutant PM653recovered the PG and 2,4-DAPG production as well as the antifungal activity,indicating that the regulation of 2,4-DAPG production by oprF depends on sigX.Both AoprF and AsigX showed the impaired abilities to suppress tomato bacterial wilt and to colonize on wheat rhizosphere.Furthermore,the oprF gene negatively affected the quorum-sensing signal(AHL)production,but the sigX gene positively regulated the AHL.Both oprF and sigX genes positively affected the swimming mobility and the biofilm formation.All these tests showed that both two genes were important in environmental adaptation in biocontrol agent 2P24.OprF transcription was positively regulated by SigX and the deficiency of OprF led to the overexpression of SigX.Although neither OprF nor SigX affected the expression of PhlA and PhlD,an increased PG production was observed in the oprF mutant.It was speculated that the amounts of substrates for 2,4-DAPG biosynthesis,malonyl-CoA and acetyl-CoA,might be changed.HPLC-MS analysis found that the amount of malonyl-CoA was obviously decreased in sigX mutant,indicating a positive regulation of SigX on malonyl-CoA production.Further analysis of four acetyl-CoA carboxylase subunits(AccA,AccB,AccC,and AccD)suggested that the expression of accA and accC were increased at both transcriptional and translational levels in oprF mutant and decreased in sigX mutant.All these results concluded that both oprF and sigX genes were novel regulators of 2,4-DAPG production by changing the substrate supply.Phenotype Microarray(Biolog,America)system provides nearly 2000 cultural conditions for bacterial metabolism,which were employed in our screening to find out the environmental factors triggered sigX expresson.By using sigX::bla-TEM transcriptional fusion reporter,we identified dozens of inducing conditions,including salts starvation,glycine addition and antibiotic addition.Further investigation showed that the PG production of the wild type 2P24,but not of the sigX mutant,was improved in the sigX-inducing conditions,such as a low concentration of NaCl and a high concentration of glycine.All these findings concluded that SigX as a sensor of the outer membrane pressures and the regulator of 2,4-DAPG production is critical for defending bacteria from the harmful microorganisms,repairing the cell damage and adapting the challenging environment.