| Pseudomonas fluorescence 2P24 isolated from wheat take-all decline soil,which can control many root diseases caused by soil-borne pathogens.The antibiotic 2,4-diacetylphloroglucinol(2,4-DAPG)produced by strain 2P24 is the major determinant for its biocontrol capacity.The Gac/Rsm signal transduction pathway is highly conserved in many Gram-negative bacteria,which is composed of Gac S/Gac A two-component regulatory system,Rsm Z-like small non-coding RNAs(s RNAs),and Rsm A family proteins.By sensing an unknown signal,the membrane sensor Gac S autophosphorylates and activates the Gac A by phosphorylation.The phosphorylated Gac A directly influences the transcription of Rsm Z-like s RNA genes,which sequester the Rsm A family proteins.Previous studies have shown that the production of 2,4-DAPG was positively regulated by the Gac S/Gac A two-component regulatory system in strain 2P24,but the molecular mechanism remains unknow.In this study,we identified Gac Aregulated s RNAs and Rsm A family proteins in strain 2P24 and elucidated the mechanism of their regulation of 2,4-DAPG production.In addition,an upstream regulator of the Gac/Rsm pathway,RNase E,was identified,and its pathway regulating 2,4-DAPG production was also elucidated.The results of these are as follows:1.The Gac S/Gac A two-component regulatory system positively regulates2,4-DAPG production through two different pathways in strain 2P24.Four Rsm Zlike s RNAs(Rsm X,Rsm X1,Rsm Y,and Rsm Z)were identified in strain 2P24.Based on sequence analysis,a conserved Gac A box sequence(5′-TGTAAGNNATNNCTTACA-3′)was founded in the rsm X,rsm X1,rsm Y,and rsm Z promoter regions.Genetic studies showed that the expression of these four genes was positively regulated by Gac A.Our data showed that mutations of single or double s RNAs did not significantly influence the production of 2,4-DAPG.However,the 2,4-DAPG production was significantly decreased in the Δrsm XYZ triple mutant or the Δrsm XX1 YZ quadruple mutant when compared to the wildtype strain.Interestingly,the production of 2,4-DAPG in Δrsm XX1 YZ quadruple mutant was still higher than that of Δgac A mutant.A transcriptional reporter plasmid library of s RNAs was constructed to screen the novel Gac A-regulated s RNAs,and one s RNA-coding gene,rgs A,showed a significantly increased in theΔgac A mutant compared with that in the wild-type strain.Sequence analysis revealed that the rgs A gene is conserved in Pseudomonas sp.strains.However,there is no Gac A box sequence in the promoter region of rgs A.Genetic analysis showed that the expression of rgs A was negatively regulated by Gac A and the production of 2,4-DAPG was negatively regulated by Rgs A.2.Rsm A and Rsm E may negatively regulate the production of 2,4-DAPG by binding the putative GGAI motif in the phl A m RNA leader.Three Rsm A family proteins(Rsm A,Rsm E,and Rsm I)were identified in strain 2P24.Genetic studies showed that the production of 2,4-DAPG was significantly increased in theΔrsm A mutant and the Δrsm E mutant,but not in the Δrsm I mutant.The secondary structure of phl A m RNA leader sequence was predicted by MFOLD program,and two putative Rsm A-binding motifs(GGAI and GGAII)were founded near the ribosome-binding site(RBS)of phl A m RNA.Translational fusion assays showed that mutating the GGAI motif abolishes the regulation of phl A gene by Rsm A and Rsm E proteins.3.Rsm A and Rsm E proteins are important regulators in strain 2P24 that operate a sophisticated mechanism for fine-tuning the concentration of 2,4-DAPG in the cells.To further understand the function of Rsm A and Rsm E proteins,transcriptome sequencing(RNA-seq)was employed to define the Rsm A and Rsm E regulon in strain 2P24.We identified 621 up-regulated genes and 304down-regulated genes in Δrsm AE double mutant compared to the wild-type strain.These genes were implicated in secondary metabolism,cell motility,carbon metabolism,and the type VI secretion system.Genetic analysis demonstrated that Rsm A and Rsm E proteins negatively regulate the expression of T6 SS and the synthesis of quorum sensing(QS)signaling,and positively regulate swimming motility and biofilm formation.Furthermore,we demonstrated that the overproduction of 2,4-DAPG in the Δrsm AE double mutant affected the growth of the strain.This delay of growth could be partially reversal when the phl D gene was deleted in the Δrsm AE double mutant.In addition,the expression of the phl F and phl G genes was similarly dramatically increased in the Δrsm AE double mutant,indicating that the Rsm A and Rsm E proteins can finely regulate intracellular 2,4-DAPG levels in strain 2P24.4.RNase E regulates the production of 2,4-DAPG through distinct pathways in strain 2P24.Random mutagenesis using mini-Tn5 transposon was performed to screen for the upstream regulators of the Gac/Rsm signal pathway,and three upstream regulatory genes(gid A,grx D and rne)were screened.The rne gene encoding ribonuclease E(RNase E).Genetic analysis showed that deletion of the carboxy-terminal half(CTH)of RNase E significantly influence the expression of rsm A and rsm E genes at the post-transcriptional level.Further studies showed that the production of 2,4-DAPG in the Δrsm AE CTH triple mutant was identical to that of the Δrsm AE double mutant,implying that the regulation of 2,4-DAPG by RNase E is dependent on both Rsm A and Rsm E proteins.RNase E hydrolyzes RNA substrates through both the 5′-end-dependent pathway and the direct-entry pathways,and replacing the Thr168 residue with Val(ΔT168V)in RNase E impairs the 5′-end-dependent pathway in vivo.The quantitative reverse transcriptional PCR(q RT-PCR)and Western blot analysis revealed that the expression of rsm A and rsm E genes were not influenced by the T168 V alteration in RNase E,indicating that the regulation of rsm A and rsm E by RNase E may depend on the direct-entry pathways.Interestingly,the production of 2,4-DAPG was also dramatically reduced in the ΔT168V mutant compared to the wild-type strain,implying that RNase E regulates 2,4-DAPG production via a pathway independent of Rsm A and Rsm E proteins. |
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