ArsZ,a Novel Ammonium-Responsive SRNA Involved In Regulation Of Nitrogen Fixation In Pseudomonas Stutzeri

Bacterial small non-coding RNAs （sRNA）, are 40-500 nucleotides in length, locate in the intergenic regions of bacterial chromosomes, most are highly structured and contain several stem-loops and usually do not encode proteins but function as RNA molecules. Recent studies have revealed that in prokaryotes, sRNAs exert important regulatory functions in nutrient metabolism, quorum sensing, bacterial virulence and adjust bacterial physiology in response to environmental stress. Numerous sRNAs in nitrogen-fixing bacteria have been identified and analyzed using both computational analysis and laboratory-based techniques.Functions of these well-known sRNAs focused on house-keeping, environmental signal, stress responses, heterocyst formation and symbiotic nodulation. However, it remains unclear whether bacterial sRNA involved in biological nitrogen fixation.The Pseudomonas stutzeri A1501, isolated from rice paddy, is capable of colonizing on the root surface or invade into root tissue from lesion and exihibits excellent properties of associative nitrogen fixation, growth promotingeflect 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 （20mM NH4+,0.5%O2） conditions were sequenced by Illumina Solexa’s high-throughput sequencing platform. A total of 53 small RNAs were identified,17 were remarkably up-regulated and 6 were down-regulated.According to data from transcriptome sequencing, a small RNA （54nucleotides in length） which located in the intergenic region （IGR） between the PST₂408 （hypothetical protein） and PST₂409 （assimilatory nitrite reductase subunit） genes was identified. This transcript was up-regulated by 23 times 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 designating ammonium-responsive small RNA （ArsZ）.ArsZ was validated by northern blotting analysis.5’-RACE experiment suggested that the transcription direction of ArsZ was same as downstream gene and the transcription start site was also further determined. Real-time qPCR data demonstrated that the expression of ArsZ was remarkably up-regulated under ammonium-limiting conditions. Compared to the wild-type,expression of ArsZ transcript was both dramatically down-regulated in AntrC and ArpoN mutant strains. Results showed that the of ArsZ was induced by ammonium-limiting conditions. ArsZ transcription was dependent on σ54-dependent NtrC protein. Moreover, the EMSA analysis and Dnase I foot printing assay both revealed that NtrC could directly bind to the promoter region of arsZ gene. NtrC and σ54, acted in concert to regulate the transcription of ArsZ in P. stutzeri A1501. In order to investigate the biological function of ArsZ, deletion mutant and complement strain of arsZ gene were constructedand the following phenotypic properties had been determined and analyzed:growth in medium, nitrogenase activity, denitrification, carbon source use ability, nitrogen source use ability, bacterial motility, oxidant and osmotic stress, salt tolerance, biofilm formation.The results showed that inactivation of arsZ led to a mutant strain （AarsZ） decrease by half in nitrogenase activities, which indicated that ArsZ played important roles in biological nitrogen fixation.Resistance of AarsZ to oxidativeand osmotic stress was significantly reduced and salt tolerance was also declined, which suggested that ArsZ could play physiological roles in stress resistance. The denitrification of △arsZ was reduced about 50% compared with wild type when nitrate as the electron acceptor, while the AarsZ had little denitrification when nitrite as the electron acceptor. These data indicated that △rsZ involved in the process of denitrification. Deletion of arsZ made P. stutzeri A1501 almost lost the swimming ability while had no effect on the swarming mobility. As for carbon source （GN2） or nitrogen source （PM3b） utilization ability by BIOLOG plates and biofilm formation, results showed that no apparent changes for any of these phenotypes were observed in AarsZ compared to the wild type, indicating that ArsZ was probably not involved in these cellular processes. Whether sRNA ArsZ plays roles in other cellular processes still needs to be further studied.To elucidate the molecular mechanism of regulation of nitrogen fixation and the mode of action by ArsZ in P. stutzeri A1501, we have compared the proteome expression profiles of wild-type strain with AarsZ under nitrogen fixation conditions.According to2-DE maps and MS analysis, collected datas showed that a total of 110 spots were found to be differentially expressed. And 38 proteins were up-regulated,72 proteins were down-regulated in the mutant as compared to the wild-type. Most of the proteins involved in nitrogen metabolism and regulation of nitrogen fixation, stress resistance, chemotaxis and motility, suggesting that ArsZ played important roles in these processes. NifDK as the molybdenum-iron （MoFe） protein （dinitrogenase or component I） of nitrogenase was dramatically down-regulated in the mutant strain, which was coincident with the result of Western blot analysis.Based on prediction and analysis by IntaRNA program, there exsited a stem-loop region in the secondary structure of ArsZ which could directly complement with the 3’end of the nifD and nifK mRNA targets forming a kissing complex through base-pairing and then maintained the stability of the target mRNAs. To obtain more insight into ArsZ regulatory function, several key nucleotides were completely and then separately mutated in the expressing plasmid （pLAFR3）. The RNAFold program was used to predict and mimic RNA secondary structures. It is noteworthy that complementation experiments of nitrogenase activities showed that the G15G16 in the frist stem-loop of ArsZ might play crucial roles in this interactions between sRNA-mRNA.