Exploring the Ancestral Mechanisms of Regulation of Horizontally Acquired Nitrogenases (original) (raw)
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2021
Pseudomonas stutzeri A1501 is the model strain for studying associative nitrogen fixation and possesses the nitrogen regulatory NtrC protein in the core genome. Nitrogen source is one of the important factors affecting the efficiency of biological nitrogen fixation in the natural environment. However, the regulation of NtrC in nitrogen metabolism of P. stutzeri A1501 is not clear. In this work, phenotypic analysis of the ntrC mutant characterized the roles of NtrC for the nitrogen metabolism and oxidative stress response of P. stutzeri A1501. To systematically identify NtrC-controlled gene expression, RNA-seq was performed to further analyze 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 the ntrC deletion, among which 147 associative genes had NtrC-binding sites, and pathways for nitrogen fixation regulation, the acquisition and catabolism of nitroge...
Microorganisms
The phylogeny of nitrogenase has only been analyzed using the structural proteins NifHDK. As nifHDKENB has been established as the minimum number of genes necessary for in silico prediction of diazotrophy, we present an updated phylogeny of diazotrophs using both structural (NifHDK) and cofactor assembly proteins (NifENB). Annotated Nif sequences were obtained from InterPro from 963 culture-derived genomes. Nif sequences were aligned individually and concatenated to form one NifHDKENB sequence. Phylogenies obtained using PhyML, FastTree, RapidNJ, and ASTRAL from individuals and concatenated protein sequences were compared and analyzed. All six genes were found across the Actinobacteria, Aquificae, Bacteroidetes, Chlorobi, Chloroflexi, Cyanobacteria, Deferribacteres, Firmicutes, Fusobacteria, Nitrospira, Proteobacteria, PVC group, and Spirochaetes, as well as the Euryarchaeota. The phylogenies of individual Nif proteins were very similar to the overall NifHDKENB phylogeny, indicating...
World Journal of Microbiology and Biotechnology
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 com...
BMC Genomics, 2010
Background: Biological nitrogen fixation is highly controlled at the transcriptional level by regulatory networks that respond to the availability of fixed nitrogen. In many diazotrophs, addition of excess ammonium in the growth medium results in immediate repression of nif gene transcription. Although the regulatory cascades that control the transcription of the nif genes in proteobacteria have been well investigated, there are limited data on the kinetics of ammonium-dependent repression of nitrogen fixation. Results: Here we report a global transcriptional profiling analysis of nitrogen fixation and ammonium repression in Pseudomonas stutzeri A1501, a root-associated and nitrogen-fixing bacterium. A total of 166 genes, including those coding for the global nitrogen regulation (Ntr) and Nif-specific regulatory proteins, were upregulated under nitrogen fixation conditions but rapidly downregulated as early as 10 min after ammonium shock. Among these nitrogen fixation-inducible genes, 95 have orthologs in each of Azoarcus sp. BH72 and Azotobacter vinelandii AvoP. In particular, a 49-kb expression island containing nif and other associated genes was markedly downregulated by ammonium shock. Further functional characterization of pnfA, a new NifA-σ 54 -dependent gene chromosomally linked to nifHDK, is reported. This gene encodes a protein product with an amino acid sequence similar to that of five hypothetical proteins found only in diazotrophic strains. No noticeable differences in the transcription of nifHDK were detected between the wild type strain and pnfA mutant. However, the mutant strain exhibited a significant decrease in nitrogenase activity under microaerobic conditions and lost its ability to use nitrate as a terminal electron acceptor for the support of nitrogen fixation under anaerobic conditions.
Distribution of nitrogen fixation and nitrogenase-like sequences amongst microbial genomes
BMC Genomics, 2012
Background The metabolic capacity for nitrogen fixation is known to be present in several prokaryotic species scattered across taxonomic groups. Experimental detection of nitrogen fixation in microbes requires species-specific conditions, making it difficult to obtain a comprehensive census of this trait. The recent and rapid increase in the availability of microbial genome sequences affords novel opportunities to re-examine the occurrence and distribution of nitrogen fixation genes. The current practice for computational prediction of nitrogen fixation is to use the presence of the nifH and/or nifD genes. Results Based on a careful comparison of the repertoire of nitrogen fixation genes in known diazotroph species we propose a new criterion for computational prediction of nitrogen fixation: the presence of a minimum set of six genes coding for structural and biosynthetic components, namely NifHDK and NifENB. Using this criterion, we conducted a comprehensive search in fully sequenc...
Nitrogen fixation genetics and regulation in a Pseudomonas stutzeri strain associated with rice
The Pseudomonas stutzeri strain A1501 (formerly known as Alcaligenes faecalis) fixes nitrogen under microaerobic conditions in the free-living state and colonizes rice endophytically. The authors characterized a region in strain A1501, corresponding to most of the nif genes and the rnf genes, involved in electron transport to nitrogenase in Rhodobacter capsulatus. The region contained three groups of genes arranged in the same order as in Azotobacter vinelandii: (1) nifB fdx ORF3 nifQ ORF5 ORF6; (2) nifLA-rnfABCDGEF-nifY2/nafY; (3) ORF13 ORF12-nifHDK-nifTY ORF1 ORF2-nifEN. Unlike in A. vinelandii, where these genes are not contiguous on the chromosome, but broken into two regions of the genome, the genes characterized here in P. stutzeri are contiguous and present on a 30 kb region in the genome of this organism. Insertion mutagenesis confirmed that most of the nif and the rnf genes in A1501 were essential for nitrogen fixation. Using lacZ fusions it was found that nif and rnf gene expression was under the control of ntrBC, nifLA and rpoN and that the rnf gene products were involved in the regulation of the nitrogen fixation process.
Exploring the Intrinsic Limits of Nitrogenase Transfer from Bacteria to Eukaryotes
Journal of Molecular Evolution, 2013
Biological nitrogen fixation is widespread among the Eubacteria and Archae domains but completely absent in eukaryotes. The lack of lateral transfer of nitrogen fixation genes from prokaryotes to eukaryotes has been partially attributed to the physiological requirements necessary for the function of the nitrogenase complex. However, symbiotic bacterial nitrogenase activity is protected by the nodule, a plant structure whose organogenesis can be trigged in the absence of bacteria. To explore the intrinsic potentiality of this plant organ, we generated rhizobium-independent nodules in alfalfa by overexpressing the MsDMI3 kinase lacking the autoinhibitory domain. These transgenic nodules showed similar levels of leghemoglobin, free oxygen, ATP and NADPH to those of efficient Sinorhizobium meliloti B399-infected nodules, suggesting that the rhizobium-independent nodules can provide an optimal microenvironment for nitrogenase activity. Finally, we discuss the intrinsic evolutionary constraints on transfer of nitrogen fixation genes between bacteria and eukaryotes.
Applied and Environmental Microbiology, 2019
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 n itrogen f ixation condition- i nducible nc R NA), 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 si...
Nitrogen Fixation by New Strains of Pseudomonas pseudoflava and Related Bacteria
Microbiology, 1989
Five representative bacterial strains resembling Pseudomonas pseudoflava, chosen from among forty strains isolated by three different procedures, were characterized by phenotypic and genotypic analyses. They were shown to belong to the species P. pseudoflava, or to a distinct, but closely related, cluster. In contrast to the initial isolates of this species, most of the new strains were able to fix N2, as shown by their growth in nitrogen-free medium and by the acetylene reduction test. Moreover, DNA regions of these new strains were homologous to Klebsiella pneumoniae nijHDK genes, confirming the presence of nitrogenase genes. The nifgenes are probably chromosomally located, since no plasmid was detected in these strains. The presence of complete but defective nifgenes in P. pseudoflava GA3 (the type strain, which does not fix N2) was excluded, as no homology was found between K. pneumoniae nifgenes and P. pseudoflava GA3 total DNA. The isolation conditions seem to determine the selection of P. pseudo_fEava N2fixing strains, since selection for H2-chemolithoautotrophs gave no N2-fixing isolates, whereas H2-chemolithoheterotrophic selection conditions (with combined nitrogen) yielded a majority of N2 fixers. METHODS Bacterial strains and isolation methods. Pseudomnas pseudoflava GA3 (type strain, DSM 1034 = NEU 21 11) was isolated by Auling et al. (1978) from water by liquid enrichment in mineral medium containing NH4C1 under H2, C 0 2 and O2 (conditions for the selection of H2-chemolithoautotrophs). Strain SA27
PLoS ONE, 2013
Nitrogen is the second most critical factor for crop production after water. In this study, the beneficial rhizobacterium Pseudomonas protegens Pf-5 was genetically modified to fix nitrogen using the genes encoding the nitrogenase of Pseudomonas stutzeri A1501 via the X940 cosmid. Pf-5 X940 was able to grow in L medium without nitrogen, displayed high nitrogenase activity and released significant quantities of ammonium to the medium. Pf-5 X940 also showed constitutive expression and enzymatic activity of nitrogenase in ammonium medium or in nitrogen-free medium, suggesting a constitutive nitrogen fixation. Similar to Pseudomonas protegens Pf-5, Pseudomonas putida, Pseudomonas veronii and Pseudomonas taetrolens but not Pseudomonas balearica and Pseudomonas stutzeri transformed with cosmid X940 showed constitutive nitrogenase activity and high ammonium production, suggesting that this phenotype depends on the genome context and that this technology to obtain nitrogen-fixing bacteria is not restricted to Pf-5. Interestingly, inoculation of Arabidopsis, alfalfa, tall fescue and maize with Pf-5 X940 increased the ammonium concentration in soil and plant productivity under nitrogen-deficient conditions. In conclusion, these results open the way to the production of effective recombinant inoculants for nitrogen fixation on a wide range of crops.