Functional Study Of A Molecular Chaperone Hfq In Pseudomonas Protegens FD6 And Its Effect On The Control Ability Against Tomato Bacterial Wilt

Tomato bacterial wilt（TBW）,caused by Ralstonia solanacearum,is an important bacterial disease in tomato production.Pseudomonas protegens FD6 produces a variety of secondary metabolites,including the antibiotics 2,4-DAPG,PLT,orfamide,protease,and pyoverdine,which provided good protection against tomato gray mold and peach brown rot diseases.To determine whether P.protegens FD6 could inhibit the expansion of TBW,dual-cultural assay,test-tube seedling experiment and pot experiment were performed to assay its potential inhibitory activities.The results indicated that strain FD6 efficiently inhibited the development of R.solanacearum.Pot experiments showed that the control efficacy of strain FD6 against TBW can reach more than 60%,as well as FD6 significantly reduced the population of R.solanacearum in tomato rhizosphere and stem base.Antibiotic production is usually affected by a variety of regulatory factors.RNA molecular chaperone Hfq is a conserved bacterial RNA-binding protein,and has a significant regulatory effect on protein translation efficiency and RNA attenuation in Gram-negative bacteria through binding to mRNA or small regulatory non-coding RNA（sRNA）,Hfq is an important post-transcriptional regulatory factor and has various regulatory functions in bacteria.A 261 bp hfq gene was found from FD6 genomic DNA sequence,encodes 86 amino acids and has a conserved Hfq domain which includes Sm1 and Sm2 motifs involved in RNA binding.The hfq mutant Mhfq was constructed by homologous recombination.RT-PCR data showed that there was no polarity effect in hfq gene mutation.The results of biological phenotype showed that hfq mutation resulted in a significant reduction in bacterial growth rate,swimming capacity,type VI secretion system（T6SS）,the production of protease,pyoverdine and HCN,and an increase in biofilm formation ability.However,hfq mutation did not affect the antibacterial activity of FD6 and the control effect on TBW.HPLC data showed that Hfq did not affect the synthesis of orfamide,and caused 1.5 times increase of PLT production in Mhfq compared with the wild type.qRT-PCR results revealed that the expression levels of PLT synthesis genes pltA,pltB,pltC,pltD and pltE in Mhfq were significantly up-regulated,suggesting that Hfq negatively controls the PLT production at the transcriptional level.In addition,the yield of 2,4-DAPG decreased significantly or even hardly produced in Mhfq compared with the wild type.qRT-PCR detection demonstrated that hfq mutation caused an increase of the expression level of phlABC,and a significant down-regulation of the expression level of phlD,The expression level of regulatory gene phlH increased while that of phlF decreased significantly.The above results indicate that Hfq regulates differentially the synthesis of these two antibiotics.The 5’ untranslated region of the mRNA of the 2,4-DAPG synthesis gene cluster was searched for Hfq binding motifs,and the possible target mRNAs phlH,phlA,and phlD for Hfq binding were postulated base on the bioinformatics analysis.First,the prokaryotic expression vector of Hfq protein was constructed,and we successfully expressed 13 kDa Hfq protein fused with His-tag in Escherichia coli BL21 in a small portion soluble protein and most of it existed in the form of inclusion bodies.Next,the protein expression conditions were optimized,and finally the Hfq protein was purified by using nickel affinity packing with 0.5 mM IPTG induction for 8 h at 16℃.The purified protein was concentrated to 891 ng/μL after 200 mM imidazole elution.RNA-EMSA experiments confirmed that phlA and phlD had strong binding ability to Hfq,and phlH could bind to Hfq under higher concentration of Hfq protein.In summary,Hfq in P.protegens FD6 has a multi-effect regulation on the biosynthesis of antibiotics,biofilm formation,pyoverdine and T6SS.Hfq positively regulates the synthesis of 2,4-DAPG by binding to the mRNA5’ untranslated region of phlA,phlD and regulatory gene phlH.Whether other sRNAs are involved in the regulation of antibiotic synthesis by Hfq remains to be further studied.