Integrated Hfq-interacting RNAome and transcriptomic analysis reveals complex regulatory networks of nitrogen fixation in root-associated Pseudomonas stutzeri A1501.

The RNA chaperone Hfq acts as a global regulator of numerous biological processes, such as carbon/nitrogen metabolism and environmental adaptation in plant-associated diazotrophs; however, its target RNAs and the mechanisms underlying nitrogen fixation remain largely unknown. Here, we used enhanced UV cross-linking immunoprecipitation coupled with high-throughput sequencing to identify hundreds of Hfq-binding RNAs probably involved in nitrogen fixation, carbon substrate utilization, biofilm formation, and other functions. Collectively, these processes endow strain A1501 with the requisite capabilities to thrive in the highly competitive rhizosphere. Our findings revealed a previously uncharted landscape of Hfq target genes. Notable among these is nifM, encoding an isomerase necessary for nitrogenase reductase solubility; amtB, encoding an ammonium transporter; oprB, encoding a carbohydrate porin; and cheZ, encoding a chemotaxis protein. Furthermore, we identified more than 100 genes of unknown function, which expands the potential direct regulatory targets of Hfq in diazotrophs. Our data showed that Hfq directly interacts with the mRNA of regulatory proteins (RsmA, AlgU, and NifA), regulatory ncRNA RsmY, and other potential targets, thus revealing the mechanistic links in nitrogen fixation and other metabolic pathways. IMPORTANCE: Numerous experimental approaches often face challenges in distinguishing between direct and indirect effects of Hfq-mediated regulation. New technologies based on high-throughput sequencing are increasingly providing insight into the global regulation of Hfq in gene expression. Here, enhanced UV cross-linking immunoprecipitation coupled with high-throughput sequencing was employed to identify the Hfq-binding sites and potential targets in the root-associated Pseudomonas stutzeri A1501 and identify hundreds of novel Hfq-binding RNAs that are predicted to be involved in metabolism, environmental adaptation, and nitrogen fixation. In particular, we have shown Hfq interactions with various regulatory proteins' mRNA and their potential targets at the posttranscriptional level. This study not only enhances our understanding of Hfq regulation but, importantly, also provides a framework for addressing integrated regulatory network underlying root-associated nitrogen fixation.

Research status and development trend of extracorporeal membrane oxygenation based on bibliometrics.

Background: Using bibliometric method to analyze the research status and development trend of extracorporeal membrane oxygenation (ECMO), we aim to provide clinicians, scientists, and stakeholders with the most up-to-date and comprehensive overview of ECMO research. Materials and methods: Using Excel and VOSviewer, the literature on ECMO was systematically analyzed regarding publication trends, journal source, foundation, countries, institutions, core authors, research hotspots, and market distribution. Results: There were five important time nodes in the research process of ECMO, including the success of the first ECMO operation, the establishment of ELSO, and the outbreak of influenza A/H1N1 and COVID-19. The R&D centers of ECMO were the United States, Germany, Japan, and Italy, and the attention to ECMO was gradually increasing in China. The products most used in the literature were from Maquet, Medtronic, and LivaNova. Medicine enterprises attached great importance to the funding of ECMO research. In recent years, the literature has mainly focused on the following aspects: the treatment of ARDS, the prevention of coagulation system-related complications, the application in neonatal and pediatric patients, mechanical circulatory support for cardiogenic shock, and ECPR and ECMO during the COVID-19 pandemic. Conclusion: The frequent epidemic occurrence of viral pneumonia and the technical advancement of ECMO in recent years have caused an increase in clinical applications. The hot spots of ECMO research are shown in the treatment of ARDS, mechanical circulatory support for cardiogenic shock, and the application during the COVID-19 pandemic.

Development and Regulation of the Extreme Biofilm Formation of Deinococcus radiodurans R1 under Extreme Environmental Conditions.

To grow in various harsh environments, extremophiles have developed extraordinary strategies such as biofilm formation, which is an extremely complex and progressive process. However, the genetic elements and exact mechanisms underlying extreme biofilm formation remain enigmatic. Here, we characterized the biofilm-forming ability of Deinococcus radiodurans in vitro under extreme environmental conditions and found that extremely high concentrations of NaCl or sorbitol could induce biofilm formation. Meantime, the survival ability of biofilm cells was superior to that of planktonic cells in different extreme conditions, such as hydrogen peroxide stress, sorbitol stress, and high UV radiation. Transcriptome profiles of D. radiodurans in four different biofilm development stages further revealed that only 13 matched genes, which are involved in environmental information processing, carbohydrate metabolism, or stress responses, share sequence homology with genes related to the biofilm formation of Escherichia coli, Pseudomonas aeruginosa, and Staphylococcus aureus. Overall, 64% of the differentially expressed genes are functionally unknown, indicating the specificity of the regulatory network of D. radiodurans. The mutation of the drRRA gene encoding a response regulator strongly impaired biofilm formation ability, implying that DrRRA is an essential component of the biofilm formation of D. radiodurans. Furthermore, transcripts from both the wild type and the drRRA mutant were compared, showing that the expression of drBON1 (Deinococcus radioduransBON domain-containing protein 1) significantly decreased in the drRRA mutant during biofilm development. Further analysis revealed that the drBON1 mutant lacked the ability to form biofilm and DrRRA, and as a facilitator of biofilm formation, could directly stimulate the transcription of the biofilm-related gene drBON1. Overall, our work highlights a molecular mechanism mediated by the response regulator DrRRA for controlling extreme biofilm formation and thus provides guidance for future studies to investigate novel mechanisms that are used by D. radiodurans to adapt to extreme environments.

Regulation of hierarchical carbon substrate utilization, nitrogen fixation, and root colonization by the Hfq/Crc/CrcZY genes in Pseudomonas stutzeri.

Bacteria of the genus Pseudomonas consume preferred carbon substrates in nearly reverse order to that of enterobacteria, and this process is controlled by RNA-binding translational repressors and regulatory ncRNA antagonists. However, their roles in microbe-plant interactions and the underlying mechanisms remain uncertain. Here we show that root-associated diazotrophic Pseudomonas stutzeri A1501 preferentially catabolizes succinate, followed by the less favorable substrate citrate, and ultimately glucose. Furthermore, the Hfq/Crc/CrcZY regulatory system orchestrates this preference and contributes to optimal nitrogenase activity and efficient root colonization. Hfq has a central role in this regulatory network through different mechanisms of action, including repressing the translation of substrate-specific catabolic genes, activating the nitrogenase gene nifH posttranscriptionally, and exerting a positive effect on the transcription of an exopolysaccharide gene cluster. Our results illustrate an Hfq-mediated mechanism linking carbon metabolism to nitrogen fixation and root colonization, which may confer rhizobacteria competitive advantages in rhizosphere environments.

Maize Growth Promotion by Inoculation with an Engineered Ammonium-Excreting Strain of Nitrogen-Fixing Pseudomonasstutzeri.

Diazotroph mutants designed using metabolic engineering to excrete surplus ammonium were used to enhance nitrogen fixation and plant growth, as the levels of nitrogen fixation attained with diazotrophs are insufficient for the plant's needs. In this study, wild-type (A1501) and engineered ammonium-excreting (1568/pVA3) strains of nitrogen-fixing Pseudomonas stutzeri strains were tested in vitro based on plant growth-promoting traits, such as phosphate solubilization ability, indole acetic acid (IAA) production and nitrogenase activities, as well as ammonium excretion as affected by mannitol-mediated osmotic stress. The maize plant growth-promoting effect of the A1501 and 1568/pVA3 strains was evaluated in pots and in the field, and the 15N-dilution technique was employed to assess the proportion of plant nitrogen derived from nitrogen fixation. The results demonstrate that the 1568/pVA3 strain displayed higher IAA production and nitrogenase activity than A1501 and released significant quantities of ammonium. After 50 days, in all of the conditions assayed, maize inoculated with 1568/pVA3 accumulated more plant biomass (3.3% on average) and fixed N (39.4% on average) than plants inoculated with A1501. In the field experiment, the grain yield of maize was enhanced by 5.6% or 5.9% due to the inoculation of seeds with 1568/pVA3 in the absence or presence of exogenous N fertilizer, respectively. Therefore, the engineered P. stutzeri strain tested in the greenhouse and field was shown to perform better than the wild-type strain with respect to maize growth parameters and biologically fixed nitrogen.

Post-transcriptional control of bacterial nitrogen metabolism by regulatory noncoding RNAs.

Nitrogen metabolism is the most basic process of material and energy metabolism in living organisms, and processes involving the uptake and use of different nitrogen sources are usually tightly regulated at the transcriptional and post-transcriptional levels. Bacterial regulatory noncoding RNAs are novel post-transcriptional regulators that repress or activate the expression of target genes through complementarily pairing with target mRNAs; therefore, these noncoding RNAs play an important regulatory role in many physiological processes, such as bacterial substance metabolism and stress response. In recent years, a study found that noncoding RNAs play a vital role in the post-transcriptional regulation of nitrogen metabolism, which is currently a hot topic in the study of bacterial nitrogen metabolism regulation. In this review, we present an overview of recent advances that increase our understanding on the regulatory roles of bacterial noncoding RNAs and describe in detail how noncoding RNAs regulate biological nitrogen fixation and nitrogen metabolic engineering. Furthermore, our goal is to lay a theoretical foundation for better understanding the molecular mechanisms in bacteria that are involved in environmental adaptations and metabolically-engineered genetic modifications.

The Sigma Factor AlgU Regulates Exopolysaccharide Production and Nitrogen-Fixing Biofilm Formation by Directly Activating the Transcription of pslA in Pseudomonas stutzeri A1501.

Pseudomonas stutzeri A1501, a plant-associated diazotrophic bacterium, prefers to conform to a nitrogen-fixing biofilm state under nitrogen-deficient conditions. The extracytoplasmic function (ECF) sigma factor AlgU is reported to play key roles in exopolysaccharide (EPS) production and biofilm formation in the Pseudomonas genus; however, the function of AlgU in P. stutzeri A1501 is still unclear. In this work, we mainly investigated the role of algU in EPS production, biofilm formation and nitrogenase activity in A1501. The algU mutant ΔalgU showed a dramatic decrease both in the EPS production and the biofilm formation capabilities. In addition, the biofilm-based nitrogenase activity was reduced by 81.4% in the ΔalgU mutant. The transcriptional level of pslA, a key Psl-like (a major EPS in A1501) synthesis-related gene, was almost completely inhibited in the algU mutant and was upregulated by 2.8-fold in the algU-overexpressing strain. A predicted AlgU-binding site was identified in the promoter region of pslA. The DNase I footprinting assays indicated that AlgU could directly bind to the pslA promoter, and ß-galactosidase activity analysis further revealed mutations of the AlgU-binding boxes drastically reduced the transcriptional activity of the pslA promoter; moreover, we also demonstrated that AlgU was positively regulated by RpoN at the transcriptional level and negatively regulated by the RNA-binding protein RsmA at the posttranscriptional level. Taken together, these data suggest that AlgU promotes EPS production and nitrogen-fixing biofilm formation by directly activating the transcription of pslA, and the expression of AlgU is controlled by RpoN and RsmA at different regulatory levels.

Master regulator NtrC controls the utilization of alternative nitrogen sources in Pseudomonas stutzeri A1501.

Pseudomonas stutzeri A1501 is a model strain used to study associative nitrogen fixation, and it possesses the nitrogen regulatory NtrC protein in the core genome. Nitrogen sources represent one of the important factors affecting the efficiency of biological nitrogen fixation in the natural environment. However, the regulation of NtrC during nitrogen metabolism in P. stutzeri A1501 has not been clarified. In this work, a phenotypic analysis of the ntrC mutant characterized the roles of NtrC in nitrogen metabolism and the oxidative stress response of P. stutzeri A1501. To systematically identify NtrC-controlled gene expression, RNA-seq was performed to further analyse the gene expression differences between the wild-type strain and the ∆ntrC mutant under nitrogen fixation conditions. A total of 1431 genes were found to be significantly altered by ntrC deletion, among which 147 associative genes had NtrC-binding sites, and the pathways for nitrogen fixation regulation, nitrogenous compound acquisition and catabolism and nitrate assimilation were discussed. Furthermore, the oxidative stress-related gene (katB), which was upregulated by ntrC deletion, was suggested to be a potential target gene of NtrC, thus highlighting the importance of NtrC in nitrogenase protection against oxygen damage. Based on these findings, we propose that NtrC is a high-ranking element in the regulatory network of P. stutzeri A1501 that controls a variety of nitrogen metabolic and oxidative stress responsive traits required for adaptation to complex rhizosphere environments.

A regulatory network involving Rpo, Gac and Rsm for nitrogen-fixing biofilm formation by Pseudomonas stutzeri.

Biofilm and nitrogen fixation are two competitive strategies used by many plant-associated bacteria; however, the mechanisms underlying the formation of nitrogen-fixing biofilms remain largely unknown. Here, we examined the roles of multiple signalling systems in the regulation of biofilm formation by root-associated diazotrophic P. stutzeri A1501. Physiological analysis, construction of mutant strains and microscale thermophoresis experiments showed that RpoN is a regulatory hub coupling nitrogen fixation and biofilm formation by directly activating the transcription of pslA, a major gene involved in the synthesis of the Psl exopolysaccharide component of the biofilm matrix and nifA, the transcriptional activator of nif gene expression. Genetic complementation studies and determination of the copy number of transcripts by droplet digital PCR confirmed that the regulatory ncRNA RsmZ serves as a signal amplifier to trigger biofilm formation by sequestering the translational repressor protein RsmA away from pslA and sadC mRNAs, the latter of which encodes a diguanylate cyclase that synthesises c-di-GMP. Moreover, RpoS exerts a braking effect on biofilm formation by transcriptionally downregulating RsmZ expression, while RpoS expression is repressed posttranscriptionally by RsmA. These findings provide mechanistic insights into how the Rpo/Gac/Rsm regulatory networks fine-tune nitrogen-fixing biofilm formation in response to the availability of nutrients.

Berberine Suppresses Mice Depression Behaviors and Promotes Hippocampal Neurons Growth Through Regulating the miR-34b-5p/miR-470-5p/BDNF Axis.

BACKGROUND: Berberine has been found to inhibit the progression of depression disorder, but its specific mechanism is still unclear. MicroRNA (miRNA) is considered to play an important role in the progression of depression. However, it is unclear whether Berberine is involved in the regulation of depression progression through miRNA. METHODS: The chronic unpredictable mild stress (CUMS) mice model was constructed. Mice depression behaviors were evaluated by sucrose preference test (SPT) and forced swim test (FST). Quantitative real-time PCR was employed to assess the expression of miR-34b-5p, miR-470-5p and brain-derived neurotrophic factor (BDNF). The protein expression of BDNF was examined using Western blot analysis. In addition, the viability and apoptosis of hippocampal neurons were determined using cell counting kit 8 assay, flow cytometry and TUNEL assay. The interaction between BDNF and miR-34b-5p or miR-470-5p was verified by dual-luciferase reporter assay and RNA immunoprecipitation assay. RESULTS: Our data indicated that Berberine could inhibit CUMS mice depression behaviors and enhance hippocampal neurons growth by targeting miR-34b-5p and miR-470-5p. In addition, we found that BDNF was a target of miR-34b-5p and miR-470-5p. Overexpressed BDNF could reverse the regulation of miR-34b-5p and miR-470-5p on CUMS mice depression behaviors and hippocampal neurons growth. Furthermore, Berberine could promote BDNF expression to regulate CUMS mice depression behaviors and hippocampal neurons growth. CONCLUSION: Berberine might inhibit the progression of depression disorder by regulating the miR-34b-5p/miR-470-5p/BDNF axis.

The Novel ncRNA OsiR Positively Regulates Expression of katE2 and Is Required for Oxidative Stress Tolerance in Deinococcus radiodurans.

Deinococcus radiodurans is a polyextremophilic bacterium well known for its extreme resistance to irradiation, oxidative stress, and other damaging conditions. Many small noncoding RNAs (ncRNAs) in D. radiodurans have been identified by deep sequencing analysis and computational predictions. However, the precise roles of ncRNAs and their target genes in the oxidative stress response have not been investigated. Here, we report the identification and characterization of a novel ncRNA named OsiR (for oxidative stress-induced ncRNA). Oxidative stress tolerance analysis showed that deleting osiR significantly decreased viability, total antioxidant capacity, and catalase activity in D. radiodurans under oxidative stress conditions. Comparative phenotypic and qRT-PCR analyses of an osiR mutant identify a role of OsiR in regulating the expression of the catalase gene katE2. Microscale thermophoresis and genetic complementation showed that a 21-nt sequence in the stem-loop structure of OsiR (204-244 nt) directly base pairs with its counterpart in the coding region of katE2 mRNA (843-866 nt) via a 19 nt region. In addition, deletion of katE2 caused a significant reduction of catalase activity and oxidative stress tolerance similar to that observed in an osiR mutant. Our results show that OsiR positively regulates oxidative stress tolerance in D. radiodurans by increasing the mRNA stability and translation efficiency of katE2. This work provides a new regulatory pathway mediated by ncRNA for the oxidative stress response that most likely contributes to the extreme tolerances of D. radiodurans.

Genome-Wide Analysis of Sugar Transporters Identifies the gtsA Gene for Glucose Transportation in Pseudomonas stutzeri A1501.

Pseudomonas stutzeri A1501 possesses an extraordinary number of transporters which confer this rhizosphere bacterium with the sophisticated ability to metabolize various carbon sources. However, sugars are not a preferred carbon source for P. stutzeri A1501. The P. stutzeri A1501 genome has been sequenced, allowing for the homology-based in silico identification of genes potentially encoding sugar-transport systems by using established microbial sugar transporters as a template sequence. Genomic analysis revealed that there were 10 sugar transporters in P. stutzeri A1501, most of which belong to the ATP-binding cassette (ABC) family (5/10); the others belong to the phosphotransferase system (PTS), major intrinsic protein (MIP) family, major facilitator superfamily (MFS) and the sodium solute superfamily (SSS). These systems might serve for the import of glucose, galactose, fructose and other types of sugar. Growth analysis showed that the only effective medium was glucose and its corresponding metabolic system was relatively complete. Notably, the loci of glucose metabolism regulatory systems HexR, GltR/GtrS, and GntR were adjacent to the transporters ABCMalEFGK, ABCGtsABCD, and ABCMtlEFGK, respectively. Only the ABCGtsABCD expression was significantly upregulated under both glucose-sufficient and -limited conditions. The predicted structure and mutant phenotype data of the key protein GtsA provided biochemical evidence that P. stutzeri A1501 predominantly utilized the ABCGtsABCD transporter for glucose uptake. We speculate that gene absence and gene diversity in P. stutzeri A1501 was caused by sugar-deficient environmental factors and hope that this report can provide guidance for further analysis of similar bacterial lifestyles.

LncRNA FGD5-AS1 can be predicted as therapeutic target in oral cancer.

BACKGROUND: Long non-coding RNAs (lncRNAs) have been extensively studied to participate in the carcinogenesis of various tumors. LncRNA FGD5-AS1 has been studied as an oncogene in several cancers; however, the role it plays in oral squamous cell carcinoma (OSCC) still remains unclear. METHODS: Quantitative real-time polymerase chain reaction (qRT-PCR) was used to assess relevant RNAs expression. CCK-8 and colony formation were combined to investigate cell proliferation. Flow cytometry was implemented to test the apoptosis of cell. Wound healing assay and transwell assays were conducted to investigate cell migration and invasion. Western blot assay was conducted to measure relevant protein expression. RESULTS: FGD5-AS1 expression was aberrantly up-regulated in OSCC tissue and cells. FDG5-AS1 up-regulation induced USP21 overexpression advances OSCC development. Knockdown of FGD5-AS1 inhibited cell growth, migration, and invasion, yet promoted apoptosis. CONCLUSION: FGD5-AS1 regulates OSCC via competitively binding to miR-520b against USP21. It could become a potential diagnostic biomarker for OSCC.

Effects of glutamate-related drugs on anxiety and compulsive behavior in rats with obsessive-compulsive disorder.

Objective: Altered glutamatergic neurotransmission has been implicated in the pathogenesis of obsessive-compulsive disorder (OCD). We examined the effects and potential mechanism of glutamate-related drugs on compulsive behavior in quinpirole (QNP)-sensitized rats, to deepen our understanding of the link between OCD and glutamate.Method: This study systematically compared the effects of the partial NMDA agonist D-Cycloserine and the NMDA antagonist NVP-AAMO77, Ro25-6981 on compulsive behavior using the elevated zero maze, open field, and marble burying tests in QNP-induced OCD model.Results: The competitive N-methyl-D-aspartate glutamate receptor (NMDAR) antagonists NVP-AAMO77 (5 mg/kg) and Ro25-6981 (5 mg/kg) significantly inhibited anxiety-like and compulsive behavior in rats. And D-Cycloserine at all doses showed significant suppression on anxiety-like and marble-burying behavior. Glutamic acid (Glu) levels, reflecting changes in the glutamatergic neurotransmission, were significantly decreased in rat hippocampus of the NVP-AAMO77 and D-Cycloserine-treated group compared to the saline-treated group. The levels of other amino acids were unaffected. Moreover, NVP-AAMO77 significantly decreased the expression of the subunit NR2A of the NMDAR, and Ro25-6981 suppressed the level of the subunit NR2B of the NMDAR, while D-Cycloserine decreased both the subunit NR2A and NR2B of the NMDAR.Conclusion: Collectively, these findings suggest a functional role of NMDARs in anxiety and compulsive behaviors, with NMDARs inhibition promoting anxiolytic-like and anti-compulsive responses. These findings suggest that D-cycloserine, NVP-AAMO77, and Ro25-6981 could be useful drugs for the treatment of OCD, which may be due to the suppression of NR2A- or NR2B- containing NMDAR.

Quantitative and Qualitative Analyses of Psychological Experience and Adjustment of In Vitro Fertilization-Embryo Transfer Patients.

BACKGROUND The aim of this study was to quantitatively analyze the psychosocial characteristics of in vitro fertilization-embryo transfer (IVF-ET) couples and normal couples, and to identify the influencing factors of psychological characteristics and pregnancy outcomes. MATERIAL AND METHODS There were 260 infertile couples undergoing IVF-ET and 277 healthy couples of childbearing age in Shengjing Hospital of China Medical University recruited into 2 groups. Psychosocial characteristics were compared to analyze the influencing factors of pregnancy outcomes after IVF-ET. In-depth interviews (n=11) and infertility-related forum posts (n=12) were adopted to obtain the data related to the psychological experience and adjustment. Nvivo 11 software was utilized to collect and analyze the data. RESULTS The levels of anxiety and depression in the IVF-ET group were significantly higher (both P<0.01), the total scores of marital quality scale and social support scale were significantly lower (both P<0.05), immature defense mechanism score was significantly higher (P<0.05), and mature defense mechanism score (P<0.05) was significantly lower than those in the control group. Bod mass index (BMI), family's rural residence, marital quality, and immature and mature defense mechanisms were influencing factors of depression in IVF-ET female patients (all P<0.05). Marital quality, mature and immature defense mechanisms, concealment factors, and Harm Avoidance (HA) score were influencing factors of depression in IVF-ET males (all P<0.05). The age of the female patient was an independent influencing factor of IVF-ET pregnancy success rate (P<0.01). CONCLUSIONS The mental health levels of IVF-ET patients were worse than those of fertile couples. The younger the female infertile patient, the higher pregnancy rate of IVF-ET.

NfiS, a species-specific regulatory noncoding RNA of Pseudomonas stutzeri, enhances oxidative stress tolerance in Escherichia coli.

Noncoding RNAs (ncRNAs) can finely control the expression of target genes at the posttranscriptional level in prokaryotes. Regulatory small RNAs (sRNAs) designed to control target gene expression for applications in metabolic engineering and synthetic biology have been successfully developed and used. However, the effect on the heterologous expression of species- or strain-specific ncRNAs in other bacterial strains remains poorly understood. In this work, a Pseudomonas stutzeri species-specific regulatory ncRNA, NfiS, which has been shown to play an important role in the response to oxidative stress as well as osmotic stress in P. stutzeri A1501, was cloned and transferred to the Escherichia coli strain Trans10. Recombinant NfiS-expressing E. coli, namely, Trans10-nfiS, exhibited significant enhancement of tolerance to oxidative stress. To map the possible gene regulatory networks mediated by NfiS in E. coli under oxidative stress, a microarray assay was performed to delineate the transcriptomic differences between Trans10-nfiS and wild-type E. coli under H2O2 shock treatment conditions. In all, 1184 genes were found to be significantly altered, and these genes were divided into mainly five functional categories: stress response, regulation, metabolism related, transport or membrane protein and unknown function. Our results suggest that the P. stutzeri species-specific ncRNA NfiS acts as a regulator that integrates adaptation to H2O2 with other cellular stress responses and helps protect E. coli cells against oxidative damage.

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

Pseudomonas stutzeri 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 nfiS showed significant downregulation of oxidative stress response genes, especially katB, a catalase gene, and oxyR, an essential regulator for transcription of catalase genes. Secondary structure prediction revealed two binding sites in NfiS for katB mRNA. Complementation experiments using truncated nfiS 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 katB mRNA and NfiS at both sites 1 and 2, thus enhancing the half-life of the transcript. We also demonstrated that katB 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 katB mRNA is a conserved and widespread mechanism among P. stutzeri strains.IMPORTANCE 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 katB mRNA and nifK 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.

NfiR, a New Regulatory Noncoding RNA (ncRNA), Is Required in Concert with the NfiS ncRNA for Optimal Expression of Nitrogenase Genes in Pseudomonas stutzeri A1501.

Expression of nitrogenase genes (nifHDK) is strictly regulated at both transcriptional and posttranscriptional levels. Efficient nitrogenase activity requires maintaining sufficient levels of nif mRNAs, yet the underlying mechanism is not fully understood due to its complexity. We have previously shown that a novel regulatory noncoding RNA (ncRNA), NfiS, optimizes nitrogen fixation through targeting nifK mRNA in Pseudomonas stutzeri A1501. Here, we report the identification and characterization of a second ncRNA inducible under nitrogen fixation conditions (nitrogen-free and microaerobic conditions), termed NfiR (for nitrogen fixation condition-inducible ncRNA), the expression of which is dependent on two global regulators, NtrC and Hfq. Comparative phenotypic and proteomic analyses of an nfiR mutant identify a role of NfiR in regulating the expression of nitrogenase genes. Further microscale thermophoresis and genetic complementation showed that an 11-nucleotide (nt) sequence in the stem-loop structure of NfiR (nucleotides 12 to 22) pairs with its counterpart in the coding region of nifD mRNA (nucleotides 1194 to 1207) by eight nucleotides. Significantly, deletion of nfiR caused a 60% reduction of nitrogenase activity, and the half-life of nifD mRNA was reduced from 20 min for the wild type to 15 min for the ΔnfiR mutant. With regard to nitrogenase activity and stability of the nifD and nifK transcripts, phenotypes were more severe for the double deletion mutant lacking nfiR and nfiS, suggesting that NfiR, in concert with NfiS, optimizes nitrogenase production at the posttranscriptional level.IMPORTANCE Biological nitrogen fixation is an energy-expensive process requiring the hydrolysis of 16 ATPs. Consequently, the expression of nif genes is highly regulated at both transcriptional and posttranscriptional levels through complex regulatory networks. Global regulation involves a number of regulatory proteins, such as the nif-specific activator NifA and the global nitrogen regulator NtrC, as well as various regulatory ncRNAs. We show that the two P. stutzeri ncRNAs, namely NfiS and NfiR (for nitrogen fixation condition-inducible ncRNA), optimize nitrogen fixation and environmental stress responses. NfiS and NfiR respond differently to various environmental signals and differ in their secondary structures. In addition, the two ncRNAs target the mRNAs of nifK and nifD, respectively. Such ncRNA-based posttranscriptional regulation of nitrogenase expression might be an evolved survival strategy, particularly in nitrogen-limiting environments. This study not only highlights the significant roles of regulatory ncRNAs in the coordination and fine tuning of various physiological processes but also provides a new paradigm for posttranscriptional regulation in nitrogen-fixing bacteria.

Global investigation of an engineered nitrogen-fixing Escherichia coli strain reveals regulatory coupling between host and heterologous nitrogen-fixation genes.

Transfer of nitrogen fixation (nif) genes from diazotrophs to amenable heterologous hosts is of increasing interest to genetically engineer nitrogen fixation. However, how the non-diazotrophic host maximizes opportunities to fine-tune the acquired capacity for nitrogen fixation has not been fully explored. In this study, a global investigation of an engineered nitrogen-fixing Escherichia coli strain EN-01 harboring a heterologous nif island from Pseudomonas stutzeri was performed via transcriptomics and proteomics analyses. A total of 1156 genes and 206 discriminative proteins were found to be significantly altered when cells were incubated under nitrogen-fixation conditions. Pathways for regulation, metabolic flux and oxygen protection to nitrogenase were particularly discussed. An NtrC-dependent regulatory coupling between E. coli nitrogen regulation system and nif genes was established. Additionally, pentose phosphate pathway was proposed to serve as the primary route for glucose catabolism and energy supply to nitrogenase. Meanwhile, HPLC analysis indicated that organic acids produced by EN-01 might have negative effects on nitrogenase activity. This study provides a global view of the complex network underlying the acquired nif genes in the recombinant E. coli and also provides clues for the optimization and redesign of robust nitrogen-fixing organisms to improve nitrogenase efficiency by overcoming regulatory or metabolic obstacles.

An enhanced vector-free allele exchange (VFAE) mutagenesis protocol for genome editing in a wide range of bacterial species.

Vector-free allele exchange (VFAE) is a newly developed protocol for genome editing in Pseudomonas species. Although several parameters have been determined to optimize the procedures for obtaining a stable and high-frequency mutation, numerous false-positive clones still appear on the plate, which increases the difficulty of finding the desired mutants. It has also not been established whether this protocol can be used for genome editing in other bacterial species. In the current study, the protocol was modified to dramatically decrease the occurrence of false-positive colonies using Pseudomonas stutzeri A1501 as a model strain. This improvement was reached by increasing the occurrence of circular-DNA cassettes of the correct size. Furthermore, the enhanced protocol was used to construct mutants in both the gram-negative Escherichia coli BL21 and gram-positive Bacillus subtilis 168 strains. The protocol works well in both strains, yielding ideal results with a low percentage of false-positive colonies. In summary, the enhanced VFAE mutagenesis protocol is a potential tool for use in bacterial genome editing.