The Pseudomonas stutzeri-Specific Regulatory Noncoding RNA NfiS Targets <i>katB</i> mRNA Encoding a Catalase Essential for Optimal Oxidative Resistance and Nitrogenase Activity

Abstract

<i>Pseudomonas stutzeri</i> A1501 is a versatile nitrogen-fixing bacterium capable of living in diverse environments and coping with various oxidative stresses. NfiS, a regulatory noncoding RNA (ncRNA) involved in the control of nitrogen fixation in A1501, was previously shown to be required for optimal resistance to H₂O₂; however, the precise role of NfiS and the target genes involved in the oxidative stress response is entirely unknown. In this work, we systematically investigated the NfiS-based mechanisms underlying the response of this bacterium to H₂O₂ at the cellular and molecular levels. A mutant strain carrying a deletion of <i>nfiS</i> showed significant downregulation of oxidative stress response genes, especially <i>katB</i>, a catalase gene, and <i>oxyR</i>, an essential regulator for transcription of catalase genes. Secondary structure prediction revealed two binding sites in NfiS for <i>katB</i> mRNA. Complementation experiments using truncated <i>nfiS</i> genes showed that each of two sites is functional, but not sufficient, for NfiS-mediated regulation of oxidative stress resistance and nitrogenase activities. Microscale thermophoresis assays further indicated direct base pairing between <i>katB</i> mRNA and NfiS at both sites 1 and 2, thus enhancing the half-life of the transcript. We also demonstrated that <i>katB</i> expression is dependent on OxyR and that both OxyR and KatB are essential for optimal oxidative stress resistance and nitrogenase activities. H₂O₂ at low concentrations was detoxified by KatB, leaving O₂ as a by-product to support nitrogen fixation under O₂-insufficient conditions. Moreover, our data suggest that the direct interaction between NfiS and <i>katB</i> mRNA is a conserved and widespread mechanism among <i>P. stutzeri</i> strains.<b>IMPORTANCE</b> Protection against oxygen damage is crucial for survival of nitrogen-fixing bacteria due to the extreme oxygen sensitivity of nitrogenase. This work exemplifies how the small ncRNA NfiS coordinates oxidative stress response and nitrogen fixation via base pairing with <i>katB</i> mRNA and <i>nifK</i> mRNA. Hence, NfiS acts as a molecular link to coordinate the expression of genes involved in oxidative stress response and nitrogen fixation. Our study provides the first insight into the biological functions of NfiS in oxidative stress regulation and adds a new regulation level to the mechanisms that contribute to the oxygen protection of the MoFe nitrogenase.