The Post-transcriptional Regulation Of The Oxidative Stress Resistance By NcRNA NfiS In Nitrogen-fixing Pseudomonas Stutzeri

The Pseudomonas genome contains four major catalase genes?katA,katB,katE and katG?,and the expression of these genes are induced by oxidative stress condition and regulated by oxidative stress response protein OxyR.Besides,non-coding RNAs also play an important role in the oxidative stress response of bacteria.Nitrogen-fixing Pseudomonas stutzeri A1501 was isolated from the rice rhizosphere,the nitrogenase activity of which is highly affected by the abiotic stress factors in the rhizophere,especially the oxidative stress factor.Our previous studies indicated that A1501 possesses a novel non-coding RNA NfiS that is involved in the regulation of optimal nitrogenase activity and oxidative stress resistance.NfiS regulates the nitrogenase activity through the interaction with nifK mRNA,while the target gene and molecular mechanism of NfiS involved in oxidative stress resistance regulation remain unclear.In this study,the mutation strain construction,gene truncation,MST measurement and other assays were performed,and the results indicated that NfiS was involved in the regulation of oxidative stress resistance at the post-transcriptional level,with the identification of target gene katB.The main research processes are described as follows:1.Oxidative stress-response genes of A1501 such as oxyR,katA,katB and katG were up-regulated under H₂O₂ shock condition,among which the expression of katB was remarkably enhanced,showing a ninefold increase.The katB insertion mutant strain and its complementary strain were constructed,and the inactivation of katB caused significant decreases in oxidative stress resistance and catalase activity of A1501 under H₂O₂ shock condition,while the complementary strain could recover the phenotypes,indicating that KatB might play an important role in the oxidative stress response of A1501.2.Bioinfoimatic analysis showed that there are two possible katB mRNA binding sites on the stem-loop structure of NfiS,located at 6-23 bp and 141-153 bp,respectively.The single site mutant strain could partially restore the oxidative stress resistance of nfiS mutant,while the double sites mutant strain has completely lost the tolerance,demonstrating that both of the two regions may be the target binding sites of NfiS involved in the oxidative stress response.3.RNA oligonucleotide fragments containing the wt and mutant binding sites sequences of katB mRNA were synthesized in vitro.The MST measurement assay results showed that katB mRNA could bind with the two sites on NfiS stem-loop structure.The effect of nfiS mutation on the half-life of katB mRNA was further checked and it was found that the transcript half-life of katB was 6 min in the WT and 3 min in the nfiS mutant strain under H₂O₂ shock condition.The above results showed that NfiS enhances the stability of katB mRNA by direct binding.4.The nitrogenase activity of wild type strain,katB mutant and its complementary strain,nfiS mutant and its complementary strain under different oxygen concentration was determined.At the optimum oxygen concentration of 1.0%,the nitrogenase activity of the katB mutant was equivalent to67%of the wild type.With the increase of oxygen concentration,the activity of all the strains decreased significantly,among which the decline was the most significant in katB mutant:when oxygen concentration rose to 2.0%,its activity was only about 13%of the wild type.Besides,katB complementary strain can restore the nitrogenase activity at any oxygen concentration.These results indicated that the mutation of katB increased the oxygen sensitivity of the nitrogenases.In addition,it's noting that the nitrogenase activity of nfiS mutant was equivalent to about 50%of wild type under different oxygen concentration,leading to the speculation that there may be other regulatory factors involved in the regulation of nitrogenase activity in A1501 through interaction with katB mRNA.In summary,NfiS is involved in the regulation of oxidative stress resistance and optimal nitrogenase activity in A1501 through enhancing the stability of katB transcript and thus increasing the expression of catalase via the interaction between NfiS and katB mRNA.