Nif Expression Research Articles

Metal oxide nanoparticles inhibit nitrogen fixation and rhizosphere colonization by inducing ROS in associative nitrogen-fixing bacteria Pseudomonas stutzeri A1501

The potential effects of engineered metal oxide nanoparticles (MONPs) on bacterial nitrogen fixation are of great concern. Herein, the impact and mechanism of the increasing-used MONPs, including TiO2, Al2O3, and ZnO nanoparticles (TiO2NP, Al2O3NP, and ZnONP, respectively), on nitrogenase activity was studied at the concentrations ranging from 0 to 10 mg L−1 using associative rhizosphere nitrogen-fixing bacteria Pseudomonas stutzeri A1501. Nitrogen fixation capacity was inhibited by MONPs in an increasing degree of TiO2NP < Al2O3NP < ZnONP. Realtime qPCR analysis showed that the expressions of nitrogenase synthesis-related genes, including nifA and nifH, were inhibited significantly when MONPs were added. MONPs could cause the explosion of intracellular ROS, and ROS not only changed the permeability of the membrane but also inhibited the expression of nifA and biofilm formation on the root surface. The repressed nifA gene could inhibit transcriptional activation of nif-specific genes, and ROS reduced the biofilm formation on the root surface which had a negative effect on resisting environmental stress. This study demonstrated that MONPs, including TiO2NP, Al2O3NP, and ZnONP, inhibited bacterial biofilm formation and nitrogen fixation in the rice rhizosphere, which might have a negative effect on the nitrogen cycle in bacteria-rice system.

Regulation and Characterization of Mutants of fixABCX in Rhizobium leguminosarum.

Symbiosis between Rhizobium leguminosarum and Pisum sativum requires tight control of redox balance in order to maintain respiration under the microaerobic conditions required for nitrogenase while still producing the eight electrons and sixteen molecules of ATP needed for nitrogen fixation. FixABCX, a cluster of electron transfer flavoproteins essential for nitrogen fixation, is encoded on the Sym plasmid (pRL10), immediately upstream of nifA, which encodes the general transcriptional regulator of nitrogen fixation. There is a symbiotically regulated NifA-dependent promoter upstream of fixA (PnifA1), as well as an additional basal constitutive promoter driving background expression of nifA (PnifA2). These were confirmed by 5'-end mapping of transcription start sites using differential RNA-seq. Complementation of polar fixAB and fixX mutants (Fix- strains) confirmed expression of nifA from PnifA1 in symbiosis. Electron microscopy combined with single-cell Raman microspectroscopy characterization of fixAB mutants revealed previously unknown heterogeneity in bacteroid morphology within a single nodule. Two morphotypes of mutant fixAB bacteroids were observed. One was larger than wild-type bacteroids and contained high levels of polyhydroxy-3-butyrate, a complex energy/reductant storage product. A second bacteroid phenotype was morphologically and compositionally different and resembled wild-type infection thread cells. From these two characteristic fixAB mutant bacteroid morphotypes, inferences can be drawn on the metabolism of wild-type nitrogen-fixing bacteroids.[Formula: see text] Copyright © 2021 The Author(s). This is an open access article distributed under the CC BY 4.0 International license.

Resource Allocation During the Transition to Diazotrophy in Klebsiella oxytoca

Free-living nitrogen-fixing bacteria can improve growth yields of some non-leguminous plants and, if enhanced through bioengineering approaches, have the potential to address major nutrient imbalances in global crop production by supplementing inorganic nitrogen fertilisers. However, nitrogen fixation is a highly resource-costly adaptation and is de-repressed only in environments in which sources of reduced nitrogen are scarce. Here we investigate nitrogen fixation (nif) gene expression and nitrogen starvation response signaling in the model diazotroph Klebsiella oxytoca (Ko) M5a1 during ammonium depletion and the transition to growth on atmospheric N2. Exploratory RNA-sequencing revealed that over 50% of genes were differentially expressed under diazotrophic conditions, among which the nif genes are among the most highly expressed and highly upregulated. Isotopically labelled QconCAT standards were designed for multiplexed, absolute quantification of Nif and nitrogen-stress proteins via multiple reaction monitoring mass spectrometry (MRM-MS). Time-resolved Nif protein concentrations were indicative of bifurcation in the accumulation rates of nitrogenase subunits (NifHDK) and accessory proteins. We estimate that the nitrogenase may account for more than 40% of cell protein during diazotrophic growth and occupy approximately half the active ribosome complement. The concentrations of free amino acids in nitrogen-starved cells were insufficient to support the observed rates of Nif protein expression. Total Nif protein accumulation was reduced 10-fold when the NifK protein was truncated and nitrogenase catalysis lost (nifK1–1203), implying that reinvestment of de novo fixed nitrogen is essential for further nif expression and a complete diazotrophy transition. Several amino acids accumulated in non-fixing ΔnifLA and nifK1–1203 mutants, while the rest remained highly stable despite prolonged N starvation. Monitoring post-translational uridylylation of the PII-type signaling proteins GlnB and GlnK revealed distinct nitrogen regulatory roles in Ko M5a1. GlnK uridylylation was persistent throughout the diazotrophy transition while a ΔglnK mutant exhibited significantly reduced Nif expression and nitrogen fixation activity. Altogether, these findings highlight quantitatively the scale of resource allocation required to enable the nitrogen fixation adaptation to take place once underlying signaling processes are fulfilled. Our work also provides an omics-level framework with which to model nitrogen fixation in free-living diazotrophs and inform rational engineering strategies.

Artificial activation of nif gene expression in nodule bacteria Ex Planta

Background. Rhizobia are the most effective nitrogen-fixing organisms that can fix nitrogen only in symbiosis with leguminous plants. The general transcriptional activator of nitrogen fixation genes in diazotrophic bacteria is NifA. In this work, the possibility of modifying the regulation of nitrogen fixation in the nodule bacteria Mesorhizobium, Ensifer and Rhizobium was studied by introducing an additional copy of the nifA gene into the bacterial genomes during the regulation of induced bacterial promoters.&#x0D; Materials and methods. A series of expression genetic constructs with NifA genes of nodule bacteria strains under the control of an inducible promoter Pm were created. The resulting constructs were transformed into strains of nodule bacteria. The obtained recombinant strains were investigated for the appearance of their nitrogen-fixing activity in the free-living state.&#x0D; Results. It was shown that the expression of nifA in recombinant cells of all three genera of bacteria leads to the appearance of insignificant nitrogenase activity. At the same time, the level of nitrogenase activity does not have a correlation with the level of expression of the introduced nifA gene, which, most likely, is a consequence of the multilevel regulation of nitrogen fixation.&#x0D; Conclusion. The possibility of artificial activation of nitrogenase activity in nodule bacteria in the free-living state by introducing the NifA regulatory protein gene into bacteria was shown.

α-Lys424 Participates in Insertion of FeMoco to MoFe Protein and Maintains Nitrogenase Activity in Klebsiella oxytoca M5al.

Our previous investigation of substrates reduction catalyzed by nitrogenase suggested that α-Ile423 of MoFe protein possibly functions as an electron transfer gate to Mo site of active center-“FeMoco”. Amino acid residue α-Lys424 connects directly to α-Ile423, and they are located in the same α-helix (α423-431). In the present study, function of α-Lys424 was investigated by replacing it with Arg (alkaline, like Lys), Gln (neutral), Glu (acidic), and Ala (neutral) through site-directed mutagenesis and homologous recombination. The mutants were, respectively, termed 424R, 424Q, 424E, and 424A. Studies of diazotrophic cell growth, cytological, and enzymatic properties indicated that none of the substitutions altered the secondary structure of MoFe protein, or normal expression of nifA, nifL, and nifD. Substitution of alkaline amino acid (i.e., 424R) maintained acetylene (C2H2) and proton (H+) reduction activities at normal levels similar to that of wild-type (WT), because its FeMoco content did not reduce. In contrast, substitution of acidic or neutral amino acid (i.e., 424Q, 424E, 424A) impaired the catalytic activity of nitrogenase to varying degrees. Combination of MoFe protein structural simulation and the results of a series of experiments, the function of α-Lys424 in ensuring insertion of FeMoco to MoFe protein was further confirmed, and the contribution of α-Lys424 in maintaining low potential of the microenvironment causing efficient catalytic activity of nitrogenase was demonstrated.

Positive and negative regulation of transferred nif genes mediated by indigenous GlnR in Gram-positive Paenibacillus polymyxa.

Ammonia is a major signal that regulates nitrogen fixation in most diazotrophs. Regulation of nitrogen fixation by ammonia in the Gram-negative diazotrophs is well-characterized. In these bacteria, this regulation occurs mainly at the level of nif (nitrogen fixation) gene transcription, which requires a nif-specific activator, NifA. Although Gram-positive and diazotrophic Paenibacilli have been extensively used as a bacterial fertilizer in agriculture, how nitrogen fixation is regulated in response to nitrogen availability in these bacteria remains unclear. An indigenous GlnR and GlnR/TnrA-binding sites in the promoter region of the nif cluster are conserved in these strains, indicating the role of GlnR as a regulator of nitrogen fixation. In this study, we for the first time reveal that GlnR of Paenibacillus polymyxa WLY78 is essentially required for nif gene transcription under nitrogen limitation, whereas both GlnR and glutamine synthetase (GS) encoded by glnA within glnRA operon are required for repressing nif expression under excess nitrogen. Dimerization of GlnR is necessary for binding of GlnR to DNA. GlnR in P. polymyxa WLY78 exists in a mixture of dimers and monomers. The C-terminal region of GlnR monomer is an autoinhibitory domain that prevents GlnR from binding DNA. Two GlnR-biding sites flank the -35/-10 regions of the nif promoter of the nif operon (nifBHDKENXhesAnifV). The GlnR-binding site Ⅰ (located upstream of -35/-10 regions of the nif promoter) is specially required for activating nif transcription, while GlnR-binding siteⅡ (located downstream of -35/-10 regions of the nif promoter) is for repressing nif expression. Under nitrogen limitation, GlnR dimer binds to GlnR-binding siteⅠ in a weak and transient association way and then activates nif transcription. During excess nitrogen, glutamine binds to and feedback inhibits GS by forming the complex FBI-GS. The FBI-GS interacts with the C-terminal domain of GlnR and stabilizes the binding affinity of GlnR to GlnR-binding site Ⅱ and thus represses nif transcription.

Quantitative proteomics of psychotrophic diazotroph in response to nitrogen deficiency and cold stress

Effective protocols and novel biomarkers are the need of this hour to screen potential cold adapted diazotrophs for sustainable mountain agricultural plans. LC-MS/MS based gel less quantitative proteomics was employed to investigate the metabolic response of Himalayan cold adapted diazotroph Pseudomonas palleroniana N26 (JN055435) for nitrogen deficiency and cold stress. More than 5000 proteins were identified, and 125 of them showed significant difference with a 2-fold or greater change (p < .05) between normal and stress conditions, including 29 up-regulated proteins and 35 down-regulated proteins. Expression of nifA, nifL, nifH, nifB, nifD, and nifK during N2 fixing conditions reveals that nitrogenase system was successfully activated. Further, 8% of the upregulated proteins showed similarity with uncharacterized proteins of several nitrogen fixing genera which suggests their in-depth investigation. Additionally, as per earlier studies, cowN was differentially expressed under nitrogen fixing conditions; thereby, confirming its potential to be a potent biomarker for monitoring the nitrogen fixation in cold niches. Biological significanceUnderstanding of nitrogenase expression and regulation is essential to employ potential diazotrophs under diverse ecological niches to achieve agricultural as well as environmental sustainability. The molecular mechanisms of cold adapted diazotrophy are still unaddressed. In this scenario, present study, besides characterizing diazotrophic proteins, is helpful in identifying the protein(s) or a biomarker viz. CowN to facilitate the monitoring of nitrogen fixation in cold niches. To the best of our knowledge, this is the first gel-less quantitative free-living diazotrophic proteome study using label free mass spectrometry having high mass accuracy in both MS and MS/MS scans. It enriches the diazotrophic proteome database and will complement the other “omics” technologies for improved crop protection and sustainability strategies.

MsmiR156 affects global gene expression and promotes root regenerative capacity and nitrogen fixation activity in alfalfa.

MicroRNA156 (miR156) regulates a network of downstream genes to affect plant growth and development. We previously generated alfalfa (Medicago sativa) plants that overexpress homologous miR156 (MsmiR156OE), and identified three of its SPL target genes. These plants exhibited increased vegetative yield, delayed flowering and longer roots. In this study, we aimed to elucidate the effect of miR156 on the root system, including effect on nodulation and nitrogen fixation. We found that MsmiR156 overexpression increases root regeneration capacity in alfalfa, but with little effect on root biomass at the early stages of root development. MsmiR156 also promotes nitrogen fixation activity by upregulating expression of nitrogenase-related genes FixK, NifA and RpoH in roots inoculated with Sinorrhizobium meliloti. Furthermore, we conducted transcriptomics analysis of MsmiR156OE alfalfa roots and identified differentially expressed genes belonging to 132 different functional categories, including plant cell wall organization, peptidyl-hypusine synthesis, and response to water stress. Expression analysis also revealed miR156 effects on genes involved in nodulation, root development and phytohormone biosynthesis. The present findings suggest that miR156 regulates root development and nitrogen fixation activity. Taken together, these findings highlight the important role that miR156 may play as a tool in the biotechnological improvement of alfalfa, and potentially other crops.

Formation of Nitrogenase NifDK Tetramers in the Mitochondria of Saccharomyces cerevisiae.

Transferring the prokaryotic enzyme nitrogenase into a eukaryotic host with the final aim of developing N2 fixing cereal crops would revolutionize agricultural systems worldwide. Targeting it to mitochondria has potential advantages because of the organelle’s high O2 consumption and the presence of bacterial-type iron–sulfur cluster biosynthetic machinery. In this study, we constructed 96 strains of Saccharomyces cerevisiae in which transcriptional units comprising nine Azotobacter vinelandii nif genes (nifHDKUSMBEN) were integrated into the genome. Two combinatorial libraries of nif gene clusters were constructed: a library of mitochondrial leading sequences consisting of 24 clusters within four subsets of nif gene expression strength, and an expression library of 72 clusters with fixed mitochondrial leading sequences and nif expression levels assigned according to factorial design. In total, 29 promoters and 18 terminators were combined to adjust nif gene expression levels. Expression and mitochondrial targeting was confirmed at the protein level as immunoblot analysis showed that Nif proteins could be efficiently accumulated in mitochondria. NifDK tetramer formation, an essential step of nitrogenase assembly, was experimentally proven both in cell-free extracts and in purified NifDK preparations. This work represents a first step toward obtaining functional nitrogenase in the mitochondria of a eukaryotic cell.

The effects of gibberellins and mepiquat chloride on nitrogenase activity in Bradyrhizobium japonicum

Application of plant growth regulators (PGRs) to soybean plants is known to induce changes in nitrogenase activity in root nodules, and this led us to hypothesize that PGRs would affect nitrogenase activity in free-living rhizobia cultures. Little is known about the molecular basis of the effects of PGRs on nitrogenase activity in free-living rhizobia cultures. Therefore, a comparative study was conducted on the effects of gibberellins (GA3) and mepiquat chloride (PIX), which regulate plant growth, on the nitrogenase activity of the nitrogen-fixing bacterium Bradyrhizobium japonicum. Fix and nif gene regulation and protein expression in free-living cultures of B. japonicum were investigated using real-time PCR and two-dimensional electrophoresis after treatment with GA3 or PIX. GA3 treatment decreased nitrogenase activity and the relative expression of nifA, nifH, and fixA genes, but these effects were reversed by PIX treatment. As expected, several proteins involved in nitrogenase synthesis were down-regulated in the GA3-treated group. Conversely, several proteins involved in nitrogenase synthesis were up-regulated in the PIX-treated group, including bifunctional ornithine acetyltransferase/N-acetylglutamate synthase, transaldolase, ubiquinol-cytochrome C reductase iron-sulfur subunit, electron transfer flavoprotein subunit beta, and acyl-CoA dehydrogenase. Two-pot experiments were conducted to evaluate the effects of GA3 and PIX on nodulation and nitrogenase activity in Rhizobium-treated legumes. Interestingly, GA3 treatment increased nodulation and depressed nitrogenase activity, but PIX treatment decreased nodulation and enhanced nitrogenase activity. Our data show that the nif and fix genes, as well as several proteins involved in nitrogenase synthesis, are up-regulated by PIX and down-regulated by GA3, respectively, in B. japonicum.

The Sulfur Carrier Protein TusA Has a Pleiotropic Role in Escherichia coli That Also Affects Molybdenum Cofactor Biosynthesis*

The Escherichia coli L-cysteine desulfurase IscS mobilizes sulfur from L-cysteine for the synthesis of several biomolecules such as iron-sulfur (FeS) clusters, molybdopterin, thiamin, lipoic acid, biotin, and the thiolation of tRNAs. The sulfur transfer from IscS to various biomolecules is mediated by different interaction partners (e.g. TusA for thiomodification of tRNAs, IscU for FeS cluster biogenesis, and ThiI for thiamine biosynthesis/tRNA thiolation), which bind at different sites of IscS. Transcriptomic and proteomic studies of a ΔtusA strain showed that the expression of genes of the moaABCDE operon coding for proteins involved in molybdenum cofactor biosynthesis is increased under aerobic and anaerobic conditions. Additionally, under anaerobic conditions the expression of genes encoding hydrogenase 3 and several molybdoenzymes such as nitrate reductase were also increased. On the contrary, the activity of all molydoenzymes analyzed was significantly reduced in the ΔtusA mutant. Characterization of the ΔtusA strain under aerobic conditions showed an overall low molybdopterin content and an accumulation of cyclic pyranopterin monophosphate. Under anaerobic conditions the activity of nitrate reductase was reduced by only 50%, showing that TusA is not essential for molybdenum cofactor biosynthesis. We present a model in which we propose that the direction of sulfur transfer for each sulfur-containing biomolecule is regulated by the availability of the interaction partner of IscS. We propose that in the absence of TusA, more IscS is available for FeS cluster biosynthesis and that the overproduction of FeS clusters leads to a modified expression of several genes.

The NifA-RpoN Regulon of Mesorhizobium loti Strain R7A and Its Symbiotic Activation by a Novel LacI/GalR-Family Regulator

Mesorhizobium loti is the microsymbiont of Lotus species, including the model legume L. japonicus. M. loti differs from other rhizobia in that it contains two copies of the key nitrogen fixation regulatory gene nifA, nifA1 and nifA2, both of which are located on the symbiosis island ICEMlSymR7A. M. loti R7A also contains two rpoN genes, rpoN1 located on the chromosome outside of ICEMlSymR7A and rpoN2 that is located on ICEMlSymR7A. The aims of the current work were to establish how nifA expression was activated in M. loti and to characterise the NifA-RpoN regulon. The nifA2 and rpoN2 genes were essential for nitrogen fixation whereas nifA1 and rpoN1 were dispensable. Expression of nifA2 was activated, possibly in response to an inositol derivative, by a novel regulator of the LacI/GalR family encoded by the fixV gene located upstream of nifA2. Other than the well-characterized nif/fix genes, most NifA2-regulated genes were not required for nitrogen fixation although they were strongly expressed in nodules. The NifA-regulated nifZ and fixU genes, along with nifQ which was not NifA-regulated, were required in M. loti for a fully effective symbiosis although they are not present in some other rhizobia. The NifA-regulated gene msi158 that encodes a porin was also required for a fully effective symbiosis. Several metabolic genes that lacked NifA-regulated promoters were strongly expressed in nodules in a NifA2-dependent manner but again mutants did not have an overt symbiotic phenotype. In summary, many genes encoded on ICEMlSymR7A were strongly expressed in nodules but not free-living rhizobia, but were not essential for symbiotic nitrogen fixation. It seems likely that some of these genes have functional homologues elsewhere in the genome and that bacteroid metabolism may be sufficiently plastic to adapt to loss of certain enzymatic functions.

Rhodobacter sphaeroides의 nif 유전자의 발현에 대한 NifA와 PrrA의 작용

To find out the growth conditions for the maximum activity of nitrogenase which catalyzes nitrogen fixation in Rhodobacter sphaeroides, the promoter activities of nifA and nifH were analyzed and the results indicated that expression of both nifA and nifH was increased in response to deprivation of both O2 concentration and nitrogen source. The nifA mutant was constructed by deleting the gene to investigate the effect of NifA, the transcriptional regulator, on the nifH and nifA expression in R. sphaeroides. Analysis of expression of nif genes using the nifA::lacZ and nifH::lacZ fusions in the nifA mutant revealed that NifA acts as a positive activator for nifH and an autoregulator in its own expression. The promoter activities of nifA and nifH in the prrA mutant grown under anaerobic and <TEX>${NH_4}^+$</TEX>-free conditions were derepressed, comparing with those of the wild-type grown under the same conditions, indicating that the prrA product acts as a positive regulator in expression of nifA and nifH.

The photoheterotrophic H2 evolution of Rhodobacter sphaeroides is enhanced in the presence of ethanol

Rhodobacter sphaeroides evolves H2 by nitrogenase under photoheterotrophic growth conditions. The maximum accumulation level of H2 increases by up to 60% when the culture medium, in which succinate and ammonium ion are the major carbon and nitrogen sources, is supplemented with ethanol at 0.5% (vol/vol). Such an effect by ethanol is not observed when ammonium ion is omitted from the medium. Ethanol is not used as a carbon source under the conditions examined, and the utilization of succinate and ammonium ion by cell is not affected by ethanol. The nitrogenase activity in the presence of ethanol, which is approximately 60% higher compared with that determined in its absence, is regulated at the level of nif transcription since the similar increase in the β-galactosidase activity of nifH-lacZ fusion was reproducibly observed under the same conditions. Interestingly, nifA transcription also increased similarly in the presence of ethanol. Neither glnB nor glnK interruptions abolished the ethanol-mediated enhancement of H2 evolution. Thus, the presence of ethanol results in the enhancement of nitrogenase expression of R. sphaeroides through the increase of NifA expression in a manner independent of GlnB and GlnK.

R. sphaeroides 에서의 orf282 유전자의 분석과 이들의 기능

The orf282 gene of Rhodobacter sphaeroides is located between the ccoNOQP operon encoding cbb3 terminal oxidase and the fnrL gene encoding an anaerobic activator, FnrL. Its function remains unknown. In an attempt to reveal the function of the orf282 gene, we disrupted the gene by deleting a portion of the orf282 gene and constructed an orf282-knockout mutant. Two FnrL binding sites were found to be located upstream of orf282, and it was demonstrated that orf282 is positively regulated by FnrL. The orf282 gene is not involved in the regulation of spectral complex formation. The cbb3 oxidase activity detected in the orf282 mutant was comparable to that in the wild-type sample, indicating that the orf282 gene is not involved in the regulation of the ccoNOQP operon and the biosynthesis of the cbb3 cytochrome c oxidase. The elevated promoter activity of the nifH and nifA genes, which are the structural genes of nitrogenase and its regulator, respectively, in the orf282 mutant, suggests that the orf282 gene product acts as a negative effector for nifH and nifA expression.

Nitrogen fixation control in Herbaspirillum seropedicae

Herbaspirillum seropedicae is a Gram-negative endophytic diazotroph that associates with important agricultural crops. Several studies have shown that this organism can contribute to plant growth suggesting potential for use as a biofertilizer. Nitrogen fixation in H. seropedicae is highly regulated both at the transcriptional and post-translational levels. Both of these regulatory levels respond to the ammonium availability in the external medium through a cascade of interacting proteins. The transcriptional regulation of the process also responds to oxygen, which is probably directly sensed by the transcriptional regulator NifA. Here, we review current knowledge of the regulation of nitrogen fixation in H. seropedicae. The signal transduction protein GlnK is a key regulator of nitrogen fixation at both the transcriptional and post-translational levels. In vitro analysis indicates that GlnK interacts with NifA and probably modulates its activity, thereby controlling nif expression. GlnK, together with the ammonium channel protein AmtB, also participates in the post-translational regulation of nitrogenase activity by an unidentified mechanism. This regulatory system efficiently controls nitrogen fixation according to prevailing fixed nitrogen and oxygen levels in H. seropedicae.

Functional analysis of the fixL/fixJ and fixK genes in Azospirillum brasilense Sp7

This study demonstrates for the first time that the two-component FixL/FixJ system, activating expression of nitrogen fixation genes in response to low oxygen concentration in rhizobia, also exists in Azospirillum brasilense Sp7. A heme staining assay showed that A. brasilense FixL contains a heme cofactor like that of rhizobial FixL proteins. Azospirillum brasilense FixL and FixJ could activate expression of Sinorhizobium meliloti nifA in S. meliloti fixL − and fixJ − mutants and in Escherichia coli, and that activation was dependent on oxygen concentration, indicating that A. brasilense FixL and FixJ have the same function as S. meliloti FixL and FixJ. Azospirillum brasilense FixL and FixJ could activate expression of A. brasilense fixK in E. coli. Azospirillum brasilense FixL, FixJ, and FixK positively regulated expression of A. brasilense nifA in A. brasilense strains but not in E. coli. The results show that FixL and FixJ can activate the fixK expression, with FixK subsequently activating nifA expression in A. brasilense. Azospirillum brasilense fixJ was involved in aerotaxis and S. meliloti fixJ could restore aerotaxis of the A. brasilense fixJ − mutant. Mutations in fixL and fixJ did not affect nitrogen fixation.

Derepressive effect of NH on hydrogen production by deleting the glnA1 gene in Rhodobacter sphaeroides

Purple non-sulfur (PNS) bacteria produce hydrogen by photofermentation of organic acids in wastewater. However, NH(4)(+) in wastewater may inhibit hydrogen synthesis by repressing the expression and activity of nitrogenase, the enzyme catalyzing hydrogen production in PNS bacteria. In this study, the Rhodobacter sphaeroides 6016 glnA gene encoding glutamine synthetase (GS) was knocked out by homologous recombination, and the effects on hydrogen production and nitrogenase activity were examined. Using 3 mM glutamine as the nitrogen source, hydrogen production (1,245-1,588 mL hydrogen/L culture) and nitrogenase activity were detected in the mutant in the presence of relatively high NH(4)(+) concentrations (15-40 mM), whereas neither was detected in the wild-type strain under the same conditions. Further analysis indicated that high NH(4)(+) concentrations greatly inhibited the expression of nifA and nitrogenase gene in the wild-type strain but not in the glnA1(-) mutant. These observations suggest that GS is essential to NH(4)(+) repression of nitrogenase and that deletion of glnA1 results in the complete derepression of nitrogenase by preventing NH(4)(+) assimilation in vivo, thus relieving the inhibition of nifA and nitrogenase gene expression. Knocking out glnA1 therefore provides an efficient approach to removing the inhibitory effects of ammonium ions in R. sphaeroides and possibly in other hydrogen-producing PNS bacteria.

Effects of ammonium ion, acetate and aerobic conditions on hydrogen production and expression levels of nitrogenase genes in Rhodobacter sphaeroides O.U.001

In the present study, expression levels of nitrogenase encoding nifH and control genes nifA and prrA were examined at different physiological conditions in Rhodobacter sphaeroides O.U.001. In addition to variations in expression levels, changes in hydrogen production and growth were also investigated in response to different concentrations of ammonium source, acetate and aerobic conditions. In the present study, increasing concentration of ammonium chloride was found to be caused decrease in hydrogen production. Glutamate containing medium had the capacity for higher hydrogen production. Hydrogen production was observed even in aerobic conditions. The expression levels of nifH and nifA genes decreased with the increasing concentrations of ammonium chloride. Although the expression of nifA was present in the highest concentrations of NH 4Cl under anaerobic conditions, no expression was observed under aerobic conditions of the same culture conditions. This was likely due to transcriptional level inhibition of nitrogenase in the presence of ammonium ion. Negative correlation was observed between the expression levels of prrA gene and its target, nifA gene.

Post-transcriptional regulation of &amp;lt;italic&amp;gt;NifA&amp;lt;/italic&amp;gt; expression by Hfq and RNase E complex in &amp;lt;italic&amp;gt;Rhizobium leguminosarum&amp;lt;/italic&amp;gt; bv. &amp;lt;italic&amp;gt;viciae&amp;lt;/italic&amp;gt;

NifA is the general transcriptional activator of nitrogen fixation genes in diazotrophic bacteria. In Rhizobium leguminosarum bv. viciae strain 8401/pRL1JI, the NifA gene is part of a gene cluster (fixABCXNifAB). In this study, results showed that in R. leguminosarum bv.viciae 8401/pRL1JI, host factor required (Hfq), and RNase E were involved in the post-transcriptional regulation of NifA expression. It was found that Hfq-dependent RNase E cleavage of NifA mRNA was essential for NifA translation. The cleavage site is located at 32 nucleotides upstream of the NifA translational start codon. A possible explanation based on predicted RNA secondary structure of the NifA 5'-untranslated region was that the cleavage made ribosome-binding sites accessible for translation.