Mechanism Of SRNA Regulating Orfamide A Synthesis In Pseudomonas Protegens Pf-5

Pseudomonas protegens Pf-5 is a model strain for the study of plant growth promoting rhizobacteria(PGPR),it can produce various antimicrobial substances that play a key role in the prevention and control of plant diseases.Among them,the cyclic lipopeptide orfamide A is an important biocontrol factor which not only has antimicrobial activity,but also regulates swarming motility,biofilm formation,and rhizosphere colonization of Pf-5.Therefore,studying it’s regulation mechanismwould provide theoretical support for the development of high effeiciancy microbial agents.The two-component system Gac S/Gac A,as a significant global regulatory system,exists in most Pseudomonas species and is able to participate in regulating the synthesis of secondary metabolite genes.This study showed that the transcription level of three small RNAs(rsmX,rsmY,and rsmZ)in the mutant Δgac A decreased significantly compared with the wild type,while the complement was able to recover.To study the downstream targets regulated by these three small RNAs,single,double,and triple mutants of these sRNA were constructed.The detection of swarming motility demonstrated that knocking out one of the three sRNAs alone had no effect on the swarming motility,while that of ΔrsmYZ andΔrsmXYZ is significantly reduced.It is reported that orfamide A plays a decisive role in the swarming motility ability of Pf-5 strains.So that detection of orfamide A production was conducted to show that among these mutants,only the orfamide A production of ΔrsmYZ and ΔrsmXYZ decreased significantly.Our result indicated that in the Pf-5 strain,the global regulatory factor Gac A positively regulates the transcription of three sRNAs and the synthesis of orfamide A,which in turn affects the strain’s ability to swarming motility.In order to study the molecular mechinasmof three sRNAs regulating orfamide A synthesis positively,which in turn affects swarming motility of Pf-5,we mutated the repressor gene rsmA and rsmE in ΔrsmYZ and ΔrsmXYZ,respectively.In Pseudomonas,sRNA could relieve the inhibitory activity of the repressor protein on the post-transcriptional translation of target gene,and then positively regulate it’s expression.The experimental results showed that the swarming motility and orfamide A yield of the mutants ΔrsmAYZ,ΔrsmEYZ and ΔrsmAEYZ are restored to varying degrees,which suggested that in the absence of rsmY and rsmZ,rsmX cannot simultaneously release the inhibitory activity of both two repressor proteins,only one of them can be recoverd.When we mutated one of the repressor genes on the basis of ΔrsmXYZ,the swarming motility ofΔrsmAXYZ and ΔrsmEXYZ and the production of orfamide A were failed to restore,only knocking out these two genes at the same time,releated phenotypes of the mutantΔrsmAEXYZ can be restored.As a result,in the absence of three sRNAs,the presence of any repressor protein can inhibit the production of orfamide A,while only when these two repressor proteins are knocked out simultaneously can the production of orfamide A be restored.These results indicate that in the Pf-5 strain the Gac-Rsmcascade system composed of Gac S/Gac A system,sRNA and repressor protein can regulate the synthesis of orfamide A.In order to further reveal the downstream targets of the Gac-Rsmcascade in Pf-5,we carried out following research.Previous research showed that there are two Lux R-type transcriptional regulators in the upstream and downstream of the ofa gene cluster in the Pf-5,called Lux R1 and Lux R2.They could activate the expression of ofa and lux R1 respectively,then in turn to regulate the synthesis of orfamide A.This study showed that compared with the wild type strain Pf-5,the transcription level of ofa,lux R1,lux R2 was significantly decreased in ΔrsmYZ.In the mutant ΔrsmAYZ,ΔrsmEYZ and ΔrsmAEYZ,the transcription levels of the three target mRNAs were differently recovery.Furthermore,expression of ofa,lux R1 and lux R2 in ΔrsmXYZ showed a significant decline compared with Pf-5,and they were restored in ΔrsmAEXYZ and ΔrsmAE.Microscale thermophoresis(MST)and RNA-EMSA(gel migration arrest)experiments demonstrated in vitro that the repressor proteins RsmA and RsmE can bind to the 5’untranslated regions of ofa operon,lux R1 and lux R2,respectively.Results in vivo and in vitro above illustrated that the three sRNAs(rsmX,rsmY,rsmZ)have a synergistic effect to dismiss the post-transcriptional inhibitory of the repressor proteins RsmA and RsmE on lux R1,lux R2 and ofa operon,thereby positively regulating production of orfamide A.In conclusion,we identified the Gac-Rsmcascade system which is composed of Gac S/Gac A,sRNA(rsmX,rsmY,rsmZ)and repressor protein(RsmA,RsmE)in Pf-5.The mechanismof Gac-Rsmregulating the synthesis of the antimicrobial substance orfamide A via affecting three downstream target mRNA(ofa operon,lux R1,lux R2)was clarified as well.