Functional Characterization Of An Ammonium-induced Small Non-coding RNA, AmiR, Required For Nitrogen Fixation And Denitrification In Pseudomonas Stutzeri A1501

Bacterial small non-coding RNAs?sRNA?are generally untranslated,and range from 50 to500 n ucleotides in length.These sRNAs generally function as regulators by base-pairing with target m RNAs,or by modulating protein activity,involved in gene transcription or translation.Antisense sRNAs can be categorized as cis-encoded sRNAs,where there is an overlap between the antis ense sRNA gene and the target gene,and trans-encoded sRNAs,where the antisense sRNA gen e is separate from the target gene.Numerous sRNAs have been identified using both computati onal analysis and laboratory-based techniques such as Northern blotting,microarrays and RNA-S eq in a number of bacterial species including Escherichia coli,the model pathogen Salmonella,the nitrogen-fixing alphaproteobacterium Sinorhizobium meliloti,marine cyanobacteria,Francisella tularensis,Streptococcus pyogenes,and the plant pathogen Xanthomonas oryzae.Recent studies have revealed that in prokaryotes,sRNAs exert important regulatory functions in nutrient meta bolism,quorum sensing,bacterial virulence and adjust bacterial physiology in response to enviro nmental stress.However,itremainsunclearwhetherbacterialsRNAinvolvedinbiologicalnitrogenfixation.The Pseudomonas stutzeriA1501,isolated from rice,is capable of colonizing on the root surface or invade into root tissue from lesion and exhibits excellent properties such as nitrogen fixation,growth promoting effect and salt tolerance.In order to uncover whether sRNA involved in regulation of biological nitrogen fixation,the transcripts of P.stutzeri A1501 under nitrogen fixation?0.1mM NH₄⁺,0.5%O₂?and ammonium repression?20 mM NH₄⁺,0.5%O₂?conditions were sequenced by Illumina Solexa's high-throughput sequencing platform.A total of 53 small RNAs were identified,17were remarkably up-regulated and 6 were down-regulated.According to data from transcriptome sequencing,a small RNA?119 nt in length?was found to be highly expressed in nitrogen fixation conditions.The small RNA is located in between two conserved proteins PST₁955 which encoding“nitrite reductase[NAD?P?H]subunit”and PST₁956 which encoding the“Cobalamin biosynthetic protein”.This transcript was up-regulated by 93 fold under nitrogen fixation conditions.Analysis of the nucleotide sequence homology by BLAST?Basic Local Alignment Search Tool?showed that it was a novel bacterial small non-coding RNA.Since the transcript was ammonium-responsive sRNA,we finally suggested calling the small RNA as ammonium-responsive AmiR.AmiR was validated by northern blotting analysis.5'-RACE experiment suggested that the transcription direction of AmiR was same as downstream gene and the transcription start site was also further determined.Real-time qPCR data demonstrated that the expression of Ami R was remarkably up-regulated under ammonium-limiting conditions.Compared to the wild-type,expression of AmiR transcript was both dramatically down-regulated in?ntrcand?rpoNmutant strains.In order to investigate the biological function of AmiR,deletion mutant?amiR,knock-down,overexpression,and complement strains of AmiRgene were constructed.The following phenotypic properties had been determined and analyzed:growth rate in LB and minimal medium,nitrogenase activity,denitrification activity,bacterial motility,oxidant and osmotic stress,salt tolerance,biofilm formation.The result showed that the nitrogenase activity in the AmiR deletion mutant??amiR?has decreased by 30%whichindicated that AmiR could be playing an important role in biological nitrogen fixation.Resistance of?amiR strain to oxidative,osmotic stress and salt tolerance were not change from wild type pattern,which suggested that AmiR could not be playing any physiological roles in stress resistance in regards to the phenotypes that been investigated.The denitrification of?amiR mutnt strainwas reduced about30%compared with wild type when nitrate as the electron acceptor,while the AmiRhad little denitrification when nitrite as the electron acceptor.These data indicated that AmiR involved in the process of denitrification.Deletion of AmiRmade no impact on the swimming or swarming mobility of A1501.Whether sRNA AmiR plays roles in other cellular processes,still needs to be further studied.To elucidate the molecular mechanism of nitrogen fixation regulation and the mode of action by AmiR in P.stutzeri A1501,we have done the transcriptome analyses for the wild-type strain with?amiR mutantunder nitrogen fixation conditions.The RNA-seq data showed that a total of 416 transcripts were up and down regulated from total 4278 in the mutant as compared to the wild-type.Most of the genes were involved in the denitrification process and energy transfer through the ABCs transporters,suggesting that AmiR played an important role in these processes.Dramatically down regulation in nitrite reducatse nirD large-subunitappeared in the deletion mutant of?amiR.None of nif genes were affected.Further analyses using?nirD deletion mutant were done to figure the similarity between the two mutants??amiR and?nirD?,which results in the same phenotype as?amiR mutant,indicating that the phenotype of the deletion AmiR is due to the defect in the expressional pattern of the downstream gene nirD.Further sequence analyse for the AmiR sequence were done to investigate if it's or not a cis-acting element for the downstream gene nirD,the data showed balanced evidence without confirmation.Target prediction using online algorithm?TARGET-RNA2 program?was used to find out the possible targets for the small non-coding RNA AmiR,the result showed there are several active loops region in the secondary structured AmiR,most are found in the reagion 25-63 nt on thesequence.The loops'locations were confirmed by the structure analyses of the AmiR.Those predicted loops could directly complement with several cellular mRNAs.However,further investigations are needed to find out if one or more of those predictions are linked to any phenotypes occur due to the deletion of the AmiRfrom A1501 wild type.Structure analyses were done for the AmiR transcribed sequence started with structure prediction via RNAFold program which used to predict and mimic RNA secondary structures,followed by small RNA transcription and purification,then folding analyses using circular dichroism with different buffers.The buffers were designed based on the simulation of the intracellular salt composition,thus,several salt concentrations were involved in the analyses of the small RNA folding.Subsequently,the result showed the ability of AmiR changing the folding pattern under different salt concentration.