Genome-wide transcriptome profiling of nitrogen fixation in Paenibacillus sp. WLY78
© Shi et al. 2016
Received: 29 July 2015
Accepted: 23 February 2016
Published: 1 March 2016
Diazotrophic (nitrogen-fixing) Gram-positive and endospore-formed Paenibacillus spp. have potential uses as a bacterial fertilizer in agriculture. The transcriptional analysis of nitrogen fixation in Paenibacillus is lacking, although regulation mechanisms of nitrogen fixation have been well studied in Gram-negative diazotrophs.
Here we report a global transcriptional profiling analysis of nitrogen fixation in Paenibacillus sp. WLY78 cultured under N2-fixing condition (without O2 and NH4 +) and non-N2-fixing condition (air and 100 mM NH4 +). The nif (nitrogen fixation) gene operon composed of 9 genes (nifBHDKENXhesAnifV) in this bacterium was significantly up-regulated in N2-fixing condition compared to non-N2-fixing condition, indicating that nif gene transcription is strictly controlled by NH4 + and O2. qRT-PCR confirmed that these nif genes were differently expressed. Non-nif genes specifically required in nitrogen fixation, such as mod, feoAB and cys encoding transporters of Mo, Fe and S atoms, were coordinately transcribed with nif genes in N2-fixing condition. The transcript abundance of suf operon specific for synthesis of Fe-S cluster was up-regulated in N2-fixing condition, suggesting that Sul system, which takes place of nifS and nifU, plays important role in the synthesis of nitrogenase. We discover potential specific electron transporters which might provide electron from Fe protein to MoFe protein of nitrogenase. The glnR whose predicted protein might mediate nif transcription regulation by NH4 + is significantly up-regulated in N2-fixing condition. The transcription levels of nitrogen metabolism and anaerobic respiration were also analyzed.
The nif gene operon (nifBHDKENXhesAnifV) in Paenibacillus sp. WLY78 is significantly up-regulated in N2-fixing condition compared to non-N2-fixing condition. Non-nif genes specifically required in nitrogen fixation were also significantly up-regulated in N2-fixing condition. Fur and Fnr which are involved in anaerobic regulation and GlnR which might mediate nif gene transcription regulation by NH4 + were significantly up-regulated in N2-fixing condition. This study provides valuable insights into nitrogen fixation process and regulation in Gram-positive firmicutes.
KeywordsPaenibacillus sp. WLY78 nif gene Transcription Up-regulation Nitrogen metabolism
Biological nitrogen fixation, the conversion of atmospheric N2 to NH3, plays an important role in the global nitrogen cycle and in world agriculture . The ability to fix nitrogen is widely, but sporadically distributed among Archaea and Bacteria which includes these families: Proteobacteria, Firmicutes, Cyanobacteria, Actinobacteria and Chlorobi [2–5]. The nif gene number and organization vary greatly among diazotrophs [6–13]. For example, twenty nif genes, nifJHDKTYENXUSVWZMFLABQ, organized in several transcriptional units, are clustered in a single 23-kb region in the chromosome of Klebsiella oxytoca . Genetic and biochemical studies on the two model diazotrophs (K. oxytoca and Azotobacter vinelandii) revealed that 16 nif gene (nifH, D, K, Y, T, E, N, X, U, S, V, Z, W, M, B, Q) products are probably essential for efficient biosynthesis of nitrogenase [3, 14]. In addition to those genes specifically required for the biosynthesis and activity of nitrogenase, the non-nif genes encoding transporters for Mo, Fe and S play important roles in nitrogen fixation. Almost all of the nif genes from Gram-negative diazotrophs possess a σ54-dependent promoter which requires a form of RNA polymerase holoenzyme containing a unique sigma factor, σN (σ54) encoded by the rpoN gene. Transcription of nif genes in these diazotrophs is stringently regulated in response to environmental oxygen and ammonium. In K. oxytoca, nif genes are subject to two levels of regulation, one global and the other nif specific. The nif-specific regulation is mediated by the NifA (nifA gene product) which is a transcriptional activator required for the expression of all K. oxytoca nif operons, except its own . The global level of nif regulation in K. oxytoca is mediated by the global nitrogen regulator NtrC.
In contrast to these Gram-negative diazotrophs, Paenibacillus sp. WLY78, a Gram-positive bacterium, possesses a minimal and compact nif gene cluster consisting of 9 genes (nifBnifHnifDnifKnifEnifNnifXhesAnifV) . The 9 nif genes are organized as an operon and possess a σ70-dependent promoter located in front of nifB gene. The genome of Paenibacillus sp. WLY78 does not have nifA . The nitrogease activity of Paenibacillus sp. WLY78 was inhibited by high concentration of NH4 + and O2 . These data suggest that regulation mechanisms of nitrogen fixation differ greatly between Gram-positive Paenibacillus and Gram-negative K. oxytoca and A. vinelandii [17, 18].
Here we performed genome-wide transcription profiling analysis of Paenibacillus sp. WLY78 cultured under N2-fixing (without O2 and NH4 +) and non-N2-fixing (air and 100 mM NH4 +) conditions. Our results revealed that the nif genes and non-nif genes specifically required for nitrogen fixation in Paenibacillus were coordinately expressed in N2-fixing condition compared to non-N2-fixing condition. The transcription levels of nitrogen metabolism and anaerobic respiration were also analyzed. Our study provides valuable insights into nitrogen fixation process and regulation of Gram-positive Paenibacillus.
Genome-wide transcription analysis of Paenibacillus sp. WLY78
Transcriptional analysis of the nitrogen fixation genes
Relative expression level (Reads Per Kilobase per Million, RPKM) in Paenibacillus sp. WLY78
Gene numbers/Paenibacillus sp. WLY78
Non-N2-fixation (100 mM NH4 + and Air)
N2-fixation (without NH4 + and O2)
Transcriptional analysis of molybdate transporters
Molybdenum is essential in bacteria for the activity of a limited number of microbial enzymes , including nitrogenase  and nitrate reductase . As molybdate is present in the environment in only trace amounts, bacteria employ an energy-dependent high-affinity molybdate transporter to accumulate it. Molybdate is transported mainly by the high-affinity ModABC system. It was reported that the CysPTWA (SulT) sulfate-thiosulfate permease, which transports sulfate, also can transport molybdenum with lower affinity that requires high molybdate concentrations .
Transcriptional analyis of sulfate transporters
Sulfur is an essential element for microorganisms, especially for diazotrophs whose nitrogenase contains iron-sulfur clusters [3–5, 23]. Sulfur can be obtained from varied compounds, sulfate (SO4 2−) and thiosulfate (S2O3 2−) being the preferred sulfur sources for the majority of organisms. Sulfate and thiosulfate are taken up by membrane transporters called sulfate permeases. Bacterial sulfate permeases belong to the SulT (Sbp/CysPTWA), SulP, CysP/(PiT) and CysZ families . It is reported that sulfate is structurally related to the oxyanions molybdate, and it can also be transported by the ModABC molybdate transport system .
Sulfate permeases in Paenibacillus sp. WLY78 include the SulT (SbpCysTWA), SulP and CysP/(PiT). The SulT (sulfate-thiosulfate) permease of Paenibacillus sp. WLY78 was constituted by sbp, cysT, cysW and cysA gene products. The sbp and cysTW form an operon, while cysA is located in another chromosomal region. The Paenibacillus CysP sulfate permease, which is similar to CysP of B. subtilis, belongs to the PiT family of phosphate transporters, and may also transport sulfate. As shown in Fig. 3b and Additional file 1: Table S3, sbpcysTWA (encoding the SulT), sulP (encoding SulP), and cysP (encoding CysP/(PiT)) in Paenibacillus sp. WLY78 were up-regulated from 2.5-fold to 32.9-fold. We also find that Sul1, which is a putative sulfate permease, was up-regulated in N2-fixing condition. In addition, the cysK1, cysK3, cysK5 and cysK7 encoding cysteine synthase were up-regulated, and cysH, cysS, cysC, cysE, cysG, cysI and cysJ genes involved in sulfur metabolism were also up-regulated in N2-fixing condition compared to non-N2-xondition.
Transcriptional analyis of Fe transporter
Iron (Fe) is an essential element for almost all organisms and is required in cofactors for many enzymes, including nitrogenase. At neutral pH, iron is often biologically unavailable because of the poor solubility of ferric iron . Many bacteria excrete ferric chelators, known as siderophores, to take up ferric iron (Fe3+). Usually, bacteria take up ferric complexes (e.g. ferri-siderophores, haem and haem–protein complexes, ferric–transferrin/lactoferrin complexes, and ferric–citrate) . At acidic pH or under anaerobic condition, Fe is the soluble Fe2+ form (ferrous iron). The major route for bacterial-ferrous-iron uptake would appear to be, in many cases, via Feo (Ferrous iron transport) . Enterobacterial Feo systems are composed of three proteins: FeoA, FeoB and FeoC. FeoB is responsible for ferrous iron transport, but the functions of FeoA and FeoC remain unclear. The feoABC genes constitute an operon. However, the feoA and feoC genes are not always present alongside feoB in some bacteria [26, 27].
Our study reveals that there are 48 Fe transporter genes in the genome of Paenibacillus sp. WLY78, indicating this bacterium is very rich in the Fe transporter (Fig. 3c and Additional file 1: Table S4). Except for three genes yfmD (Fe3+siderophore ABC-transporter permease), yfmE (Fe3+dicitrate ABC-transporter permease) and ftpA (ABC-type Fe3+ transport system) being weakly down-regulated, the other 45 genes were up-regulated from 1- to 205-fold in N2-fixing condition compared to non-N2-fixing condition. Notably, of the 48 Fe transporter genes, 41 genes belong to Fe3+ transport systems including Fe3+ siderophores transport systems and Fe3+ hydroxamate transport systems, and 6 genes were involved in Fe2+ uptake and regulation and 1 gene (ftn) encodes iron storage. The highest expressed Fe transporter in N2-fixing condition was fhuD7 encoding Fe3+ transporter. Feo system of Paenibacillus sp. WLY78 is composed of FeoA and FeoB and responsible for uptake of Fe2+. feoAB were up-regulated 54- and 12-fold, respectively, in N2-fixing condition. fit gene encoding iron storage protein was up-regulated 16-fold. Notably, Paenibacillus contains 2 fur genes, which were up-regulated 43- and 11-fold, respectively. The fur gene codes for the transcriptional activator Fur (Ferric uptake regulator), which controls its own synthesis as well as the transcription of genes involved in the iron homeostasis [28, 29]. It also participates in the regulation of other cellular functions such as oxidative stress, glycolysis, TCA cycle, respiration, 2, 3-dihydroxybenzoate biosynthesis [28, 29]. In addition to fur genes, Paenibacillus has 2 genes perC3 and perR1 encoding Fe2+/Zn2+ uptake regulation proteins and they were significantly up-regulated 44- and 108-fold, respectively, in N2-fixing condition compared to non-N2-fixing condition. These data indicate that both Fe2+ and Fe3+ uptake play important role in nitrogen fixation of Paenibacillus sp. WLY78. Especially, Fe3+ transporters may play a major role in nitrogen fixation of Paenibacillus sp. WLY78, in accordance with that bacterium is grown in neutral pH where Fe is in the form of insoluble Fe3+.
Transcriptional analysis of iron-sulfur cluster assembly system
Nitrogenase is a complex [Fe-S] enzyme and the [Fe-S] clusters of nitrogenase play a critical function in electron transfer and in the reduction of substrates driven by the free energy liberated from Mg-ATP hydrolysis [3–5]. NifUS (nifU and nifS gene products), which mobilizes Fe and S for the assembly of small Fe/S fragments, were generally thought to be specialized for the assembly of the Fe4-S4 cluster of NifH. NifU and NifS are also involved in the assembly of the P-cluster and the FeMo-co of the NifDK component of nitrogenase . nifSU are widely distributed in diazotrophs, such as K. oxytoca and A. vinelandii. In addition to nifSU, isc (iscR, iscU, iscS, iscA, hscB, hscA, fdx, and iscX)system also contributes the assembly of Fe-S cluster in A. vinelandii .
Transcriptional analysis of electron transporters for nitrogenase
Nitrogen fixation is carried out by the enzyme nitrogenase, which transfers electrons originating from low-potential electron carriers, such as flavodoxin or ferredoxin molecules, to molecular N2 . In K. oxytoca, the physiological electron flow to nitrogenase involves specifically the products of the nifF and nifJ genes . The nifF gene product, a flavodoxin, mediates electron transfer from the nifJ gene product, a pyruvate: flavodoxin oxidoreductase, to the Fe protein of nitrogenase .
Transcriptional analysis of respiration and energy metabolism
Since nitrogenase is very sensitive to oxygen, nitrogen fixation was carried out in anaerobic or microanaerobic conditions. From this study, it is found that the oxygen and nitrogen limitation induced about 20 cytochrome oxidase genes (qoxABCD, ctaCDEF, cydAB and others) in Paenibacillus sp. WLY78 (Fig. 5b and Additional file 1: Table S7). The cydABCD encoding cytochrome bd oxidase, active under microaerobic condition, were up-regulated from 12.2- to 42.35-fold. ctaCDEF were up-regulated from 1.56- to 88.73-fold. The cyoABCD coding for cytochrome bo oxidase, functional under aerobic condition, were weakly induced in N2-fixing condition. qoxABCD are up-regulated from 1.59- to 7.17-fold. There are 3 ndh genes encoding NADH dehydrogenase being up-regulated from 4.7- to 12.94-fold. hcaD encoding NAD(FAD)-dependent dehydrogenases was the highest up-regulated gene with 116.08-fold in N2-fixing condition compared to non-N2-fixing condition. The data suggest that active consumption of oxygen might provide O2 protection for nitrogenase in Paenibacillus.
Many bacteria are able to grow anaerobically using alternative electron acceptors, including nitrate or fumarate . Here we found that there are two sets of narIJHG genes encoding assimilatory nitrite reductases and they were differently expressed from 0.37- to 4.98-fold in Paenibacillus (Additional file 1: Table S8). All of the nas genes (nasE1B, nasE3D1, nasC and nasD3) encoding NAD(P)H-nitrite-reductases and nasA encoding nitrate transporter were up-regulated in N2-fixing condition (Additional file 1: Table S8). The two component regulatory proteins, ResD and ResE, and an anaerobic gene regulator, FNR, were previously shown to be indispensable for nitrate respiration in B. subtilis [33, 34]. Here we show that in Paenibacillus sp. WLY78, transcript level of fnr gene is up-regulated 6.84-fold, and resD-resE encoding two-component regulatory proteins ResD-ResE were up-regulated to 16.2- and 14.6-fold, respectively.
Transcriptional analysis of nitrogen metabolism
The nif gene operon of Paenibacillus possesses a σ70-dependent promoter instead of a σ54-dependent promoter. GlnR, a global regulator of nitrogen metabolism in Bacillus, exists in Paenibacillus . Our previous studies revealed that there is a GlnR/TnrA-binding site in the nif promoter region of Paenibacillus sp. WLY78 . Here, we find that Paenibacillus sp. WLY78 has 3 glnA genes, one of which is linked with glnR, and the other two (here named glnA1 and glnA2) are separately located in other regions of chromosome. The transcript level of glnRA operon was up-regulated 18.7- to 19.06-fold in N2-fixing condition compared to non-N2-fixing condition (Fig. 5c). The other 2 glnA genes, amtB and gltABD involved in nitrogen metabolism were significantly up-regulated in N2-fixing condition compared to non-N2-fixing condition (Fig. 5c and Additional file 1: Table S9).
Transcriptional analysis of ATPase
The nitrogen fixation process is coupled to the hydrolysis of 16 equivalents of ATP. In Paenibacillus sp. WLY78, except for atpD, other atp genes (e.g. atpC, atpG, atpA, atpH, atpF, atpE, atpB and atpI) encoding ATP synthase subunits were highly expressed under N2-fixing condition compared to non-N2-fixing condition (Fig. 5d and Additional file 1: Table S10).
Transcriptional analysis of the sigma factors
Regulation of gene expression in bacteria occurs primarily at the level of transcription. Although activators and repressors can significantly affect the efficiency of transcription, the specificity of the transcription reaction rests on interactions between RNA polymerase (RNAP) and the promoters . The bacterial RNA polymerase holoenzyme (holo RNAP) is composed of core RNAP (α2β'βσω) and sigma (σ) factor. The σ factor of RNA polymerase recognizes promoter regions and initiates transcription .
It was reported that there are at least 10 known sigma factors in B. subtilis, including σA, σB, σC, σD, σE, σF, σG, σH, σK, and σL . The σA is the housekeeping sigma factor that is responsible for expression of essential genes . Here we found that in Paenibacillus sp. WLY78, sigA gene encoding σA which is an equivalent of E. coli σ70, was up-regulated 10.72-fold in N2-fixing condition compared to non-N2-fixing condition (Fig. 5e and Additional file 1: Table S11). Moreover, sigB responsible for the transcription of genes that can confer stress resistance to the vegetative cell was up-regulated 29.64-fold, consistent with the current culture condition (limited nitrogen and oxygen). sigH specific for σH involved in chemotaxis/autolysin/flagellar gene transcription was the highest expressed gene among those genes specific for σ factors in both N2-fixing and non-N2-fixing conditions. sigL encoding σL, which is an equivalent of σ54 of E. coli and responsible for nitrogen metabolism, was up-regulated in N2-fixing condition. sigF encoding σF specific for early forespore gene expression was highly expressed in non-N2-fixing condition and was down-regulated by N2-fixing condition. Other sig genes were transcribed at low levels in both N2-fixing and non-N2-fixing conditions.
In this study, a genome-wide transcription analysis of the nitrogen fixation in Paenibacillus sp. WLY78 cultured under N2-fixing and non-N2-fixing conditions was performed. Our results reveal that the transcripts of the nif genes (nifBHDKENXhesAnifV) of Paenibacillus sp. WLY78 are significantly up regulated in N2-fixing condition compared to non-N2-fixing condition, suggesting that nif gene expression in Paenibacillus was strongly regulated by ammonium and oxygen. Our data are consistent with the findings that in many diazotrophs such as K. oxytoca and A. vinelandii, expression of the nif genes is tightly controlled at the transcriptional level in response to the concentration of fixed nitrogen and the oxygen . However, regulation mechanisms vary greatly among different diazotrophs. In the well-studied Gram-negative diazotrophic K. oxytoca, nif genes, which possess a σ54-dependent promoter, are subject to two levels of regulation, one global and the other nif specific. The nif-specific regulation is mediated by the NifA (nif gene product) which is a transcriptional activator required for the expression of all K. oxytoca nif operons, except its own . The global level of nif regulation in K. oxytoca is mediated by the global nitrogen regulator NtrC. The level of phosphorylated NtrC in the NtrB-NtrC two component regulatory system controls expression of glnA-ntrBC operon, nifL-nifA operon and glnK-amtB operon. The GlnD and the GlnB control the activity of the NtrB–NtrC two-component regulatory system .
Although regulation mechanism of nitrogen fixation is well-studied in Gram-negative bacteria, regulation mechanism of nitrogen fixation in Gram-positive Paenibacillus and Bacillus is lacking. There is no nifA in Paenibacillus sp. WLY78, and the nif gene operon of Paenibacillus possesses a σ70-dependent promoter instead of a σ54-dependent promoter. GlnR, a global regulator of nitrogen metabolism in Bacillus, exists in Paenibacillus . Our previous studies revealed that there is a GlnR/TnrA-binding site in the nif promoter region of Paenibacillus sp. WLY78 . Our recent studies reveals that GlnR binds the nif promoter in vitro by EMSA (Electrophoretic mobility shift assay) (not published), suggesting that GlnR might mediate nif gene transcription according to ammonium concentration.
Nitrogenase is a complex [Fe-S] enzyme. A lot of researches demonstrated that nifU and nifS, whose products were involved in the assembly of [Fe-S] clusters, were required for nitrogen fixation [30, 31]. The genome of Paenibacillus sp. WLY78 does not have nifSU, but contains a complete suf (sufCBSUD) operon, a partial suf (sufABC) operon, a partial isc system (iscSR and fdx) and two nifS-like genes. This study reveals that the transcript abundances of sufCBSUD were much higher than the other related genes in N2-fixing condition, indicating that they play important roles in the Fe-S cluster assembly of nitrogenase.
Nitrogen fixation is an energy intensive process and requires a suitable reductant to support electron transport to nitrogenase. Unlike K. oxytoca nif gene cluster containing nifF and nifJ, which provide the electron transport, Paenibacillus nif gene cluster does not have nifF and nifJ. In this study, we found that several genes encoding ferredoxins, such as fer, fldA and COG3411, which might be involved in electron transport in nitrogenase of Paenibacillus sp. WLY78 were highly transcribed in N2-fixing condition.
In summary our results demonstrate that the expression of the nif gene operon of Paenibacillus was highly induced inN2-fixing condition. The non-nif genes specially required for nitrogen fixation, such as transporters of Fe, S and Mo were coordinately transcribed with nif genes in Paenibacillus. This study shows that Sul system was up regulated in N2-fixing condition, suggesting that Sul system, which takes place of nifS and nifU, plays important role in the synthesis of Fe-S cluster in Paenibacillus. We discover potential electron transporters which specifically transfer electrons to nitrogenase in Paenibacillus.
Bacterial strains, media and growth conditions
Paenibacillus sp. WLY78 used here was isolated from rhizosphere of bamboo by our laboratory (16). The bacterium was routinely grown in LB or LD medium (per liter contains: 2.5 g NaCl, 5 g yeast and 10 g tryptone) at 30°Cwith shaking. Since nitrogenase is very sensitive to oxygen and nitrogenase activity is inhibited by high concentration of ammonium, nitrogen fixation was carried out in anaerobic or microanaerobic condition and without ammonium or with limited ammonium. For transcriptomic analysis and real-time quantitative RT-PCR, Paenibacillus sp. WLY78 was grown in nitrogen-deficient medium under nitrogen-fixing condition (without O2 and NH4 +) or non-nitrogen-fixing condition (21 % O2 and 100 mM NH4 +). Nitrogen-deficient medium contained (per liter) (per liter) 10.4 g Na2HPO4, 3.4 g KH2PO4, 26 mg CaCl2• 2H2O, 30 mg MgSO4, 0.3 mg MnSO4, 36 mg Ferric citrate, 7.6 mg Na2MoO4 · 2H2O, 10 μg p-aminobenzoic acid, 5 μg biotin, 4 g glucose as carbon source and 2 mM glutamate as nitrogen source.
Isolation of RNA
Paenibacillus sp. WLY78 was grown to OD600 = 0.3–0.4 at different concentration of ammonium and oxygen and then were harvested by centrifugation at 4 °C. Total RNA was isolated using a SV Total RNA Isolation System (Promega) according to the manufacturer’s instructions. The possibility of contamination of genomic DNA was eliminated by digestion with RNase-free DNase I (Takara Bio). The integrity and size distribution of the RNA was verified by agarose gel electrophoresis, and the concentration was determined spectrophotometrically.
Total RNA was isolated from Paenibacillus sp. WLY78 grown in N2-fixing and non-N2-fixing conditions, respectively. cDNA library construction and SOLiD sequencing from the total RNA were completed in Beijing Genomics Institute (Chinese Academy of Sciences) and three technical replicates of each sample were carried out. Raw sequencing reads were mapped against the Paenibacillus sp. WLY78 genome, using the programme BWA as previously described [40, 41]. We use DEGseq for identifying differentially expressed genes from RNA-seq data . Transcript level differences with adjusted P values of <0.001 were considered to be significant.
Quantitative real-time RT-PCR
To confirm the results of SOLiD sequencing, 9 nif genes (nifBHDKENXhesAnifV) were chosen for qRT-PCR analyses that were expressed in Paenibacillus sp. WLY78 from the two transcriptomes. qRT-PCR was performed using the SYBR GreenI(ROX) Kit from TakeRa Company according to the manufacturer’s protocol. Reactions were performed in triplicate. The primers used for qRT-PCR reactions are listed in Additional file 1: Table S12.
Availability of data
The RNA-seq sequencing data of Paenibacillus sp. WLY78 have been deposited in NCBI database under accession number SRP053133.
This work was supported by the National Nature Science Foundation of China (Grant No. 31470189).
Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
- Falkowski PG. Evolution of the nitrogen cycle and its influence on the biological sequestration of CO2 in the ocean. Nature. 1997;387(6630):272–5.View ArticleGoogle Scholar
- Capone DG. Marine nitrogen fixation: what’s the fuss? Curr Opin Microbiol. 2001;4(3):341–8.View ArticlePubMedGoogle Scholar
- Rubio LM, Ludden PW. Biosynthesis of the iron-molybdenum cofactor of nitrogenase. Annu Rev Microbiol. 2008;62:93–111.View ArticlePubMedGoogle Scholar
- Hu Y, Ribbe MW. Biosynthesis of nitrogenase FeMoco. Coordin Chem Rev. 2011;255(9–10):1218–24.View ArticleGoogle Scholar
- Roberts GP, MacNeil T, MacNeil D, Brill WJ. Regulation and characterization of protein products coded by the nif (nitrogen fixation) genes of Klebsiella pneumoniae. J Bacteriol. 1978;136(1):267–79.PubMedPubMed CentralGoogle Scholar
- Gavini N, Tungtur S, Pulakat L. Peptidyl-prolyl cis/trans isomerase-independent functional nifH mutant of Azotobacter vinelandii. J Bacteriol. 2006;188(16):6020–5.View ArticlePubMedPubMed CentralGoogle Scholar
- Brigle KE, Weiss MC, Newton WE, Dean DR. Products of the iron-molybdenum cofactor-specific biosynthetic genes, nifE and nifN, are structurally homologous to the products of the nitrogenase molybdenum-iron protein genes, nifD and nifK. J Bacteriol. 1987;169(4):1547–53.PubMedPubMed CentralGoogle Scholar
- Arnold W, Rump A, Klipp W, Priefer UB, Puhler A. Nucleotide sequence of a 24,206-base-pair DNA fragment carrying the entire nitrogen fixation gene cluster of Klebsiella pneumoniae. J Mol Biol. 1988;203(3):715–38.View ArticlePubMedGoogle Scholar
- Jacobson MR, Brigle KE, Bennett LT, Setterquist RA, Wilson MS, Cash VL, et al. Physical and genetic map of the major nif gene cluster from Azotobacter vinelandii. J Bacteriol. 1989;171(2):1017–27.PubMedPubMed CentralGoogle Scholar
- Curatti L, Brown CS, Ludden PW, Rubio LM. Genes required for rapid expression of nitrogenase activity in Azotobacter vinelandii. Proc Natl Acad Sci U S A. 2005;102(18):6291–6.View ArticlePubMedPubMed CentralGoogle Scholar
- Rubio LM, Rangaraj P, Homer MJ, Roberts GP, Ludden PW. Cloning and mutational analysis of the gamma gene from Azotobacter vinelandii defines a new family of proteins capable of metallocluster binding and protein stabilization. J Biol Chem. 2002;277(16):14299–305.View ArticlePubMedGoogle Scholar
- Joerger RD, Bishop PE. Nucleotide sequence and genetic analysis of the nifB-nifQ region from Azotobacter vinelandii. J Bacteriol. 1988;170(4):1475–87.PubMedPubMed CentralGoogle Scholar
- Rodriguez-Quinones F, Bosch R, Imperial J. Expression of the nifBfdxNnifOQ region of Azotobacter vinelandii and its role in nitrogenase activity. J Bacteriol. 1993;175(10):2926–35.PubMedPubMed CentralGoogle Scholar
- Dixon R, Cheng Q, Shen G, Day A, Dowson-Day M. Nif gene transfer and expression in chloroplasts: prospects and problems. Plant Soil. 1997;194(1–2):193–203.View ArticleGoogle Scholar
- Dixon R, Kahn D. Genetic regulation of biological nitrogen fixation. Nat Rev Microbiol. 2004;2(8):621–31.View ArticlePubMedGoogle Scholar
- Wang L, Zhang L, Liu Z, Zhao D, Liu X, Zhang B, et al. A minimal nitrogen fixation gene cluster from Paenibacillus sp. WLY78 enables expression of active nitrogenase in Escherichia coli. PLoS Genet. 2013;9(10):e1003865.View ArticlePubMedPubMed CentralGoogle Scholar
- Nieva-Gomez D, Roberts GP, Klevickis S, Brill WJ. Electron transport to nitrogenase in Klebsiella pneumoniae. Proc Natl Acad Sci U S A. 1980;77(5):2555–8.View ArticlePubMedPubMed CentralGoogle Scholar
- Hill S, Kavanagh EP. Roles of nifF and nifJ gene products in electron transport to nitrogenase in Klebsiella pneumoniae. J Bacteriol. 1980;141(2):470–5.PubMedPubMed CentralGoogle Scholar
- Hamilton TL, Ludwig M, Dixon R, Boyd ES, Dos SP, Setubal JC, et al. Transcriptional profiling of nitrogen fixation in Azotobacter vinelandii. J Bacteriol. 2011;193(17):4477–86.View ArticlePubMedPubMed CentralGoogle Scholar
- Self WT, Grunden AM, Hasona A, Shanmugam KT. Molybdate transport. Res Microbiol. 2001;152(3–4):311–21.View ArticlePubMedGoogle Scholar
- Hernandez JA, George SJ, Rubio LM. Molybdenum trafficking for nitrogen fixation. Biochemistry. 2009;48(41):9711–21.View ArticlePubMedPubMed CentralGoogle Scholar
- Pau RN, Lawson DM. Transport, homeostasis, regulation, and binding of molybdate and tungstate to proteins. Met Ions Biol Syst. 2002;39:31–74.PubMedGoogle Scholar
- Aguilar-Barajas E, Diaz-Perez C, Ramirez-Diaz MI, Riveros-Rosas H, Cervantes C. Bacterial transport of sulfate, molybdate, and related oxyanions. Biometals. 2011;24(4):687–707.View ArticlePubMedGoogle Scholar
- Guerinot ML. Microbial iron transport. Annu Rev Microbiol. 1994;48:743–72.View ArticlePubMedGoogle Scholar
- Andrews SC, Robinson AK, Rodrı́guez-Quiñones F. Bacterial iron homeostasis. FEMS Microbiol Rev. 2003;27(2–3):215–37.View ArticlePubMedGoogle Scholar
- Cartron ML, Maddocks S, Gillingham P, Craven CJ, Andrews SC. Feo – transport of ferrous iron into bacteria. Biometals. 2006;19(2):143–57.View ArticlePubMedGoogle Scholar
- McHugh JP, Rodriguez-Quinones F, Abdul-Tehrani H, Svistunenko DA, Poole RK, Cooper CE, et al. Global iron-dependent gene regulation in Escherichia coli. A new mechanism for iron homeostasis. J Biol Chem. 2003;278(32):29478–86.View ArticlePubMedGoogle Scholar
- Baichoo N, Wang T, Ye R, Helmann JD. Global analysis of the Bacillus subtilis Fur regulon and the iron starvation stimulon. Mol Microbiol. 2002;45(6):1613–29.View ArticlePubMedGoogle Scholar
- Hantke K. Iron and metal regulation in bacteria. Curr Opin Microbiol. 2001;4(2):172–7.View ArticlePubMedGoogle Scholar
- Zhao D, Curatti L, Rubio LM. Evidence for nifU and nifS participation in the biosynthesis of the iron-molybdenum cofactor of nitrogenase. J Biol Chem. 2007;282(51):37016–25.View ArticlePubMedGoogle Scholar
- Johnson DC, Dos SP, Dean DR. NifU and NifS are required for the maturation of nitrogenase and cannot replace the function of isc-gene products in Azotobacter vinelandii. Biochem Soc Trans. 2005;33(Pt 1):90–3.View ArticlePubMedGoogle Scholar
- Glaser P, Danchin A, Kunst F, Zuber P, Nakano MM. Identification and isolation of a gene required for nitrate assimilation and anaerobic growth of Bacillus subtilis. J Bacteriol. 1995;177(4):1112–5.PubMedPubMed CentralGoogle Scholar
- Hoffmann T, Troup B, Szabo A, Hungerer C, Jahn D. The anaerobic life of Bacillus subtilis: cloning of the genes encoding the respiratory nitrate reductase system. FEMS Microbiol Lett. 1995;131(2):219–25.View ArticlePubMedGoogle Scholar
- Cruz RH, Boursier L, Moszer I, Kunst F, Danchin A, Glaser P. Anaerobic transcription activation in Bacillus subtilis: identification of distinct FNR-dependent and -independent regulatory mechanisms. EMBO J. 1995;14(23):5984–94.Google Scholar
- Xie JB, Du Z, Bai L, Tian C, Zhang Y, Xie JY, et al. Comparative genomic analysis of N2-fixing and non-N2-fixing Paenibacillus spp.: organization, evolution and expression of the nitrogen fixation genes. PLoS Genet. 2014;10(3):e1004231.View ArticlePubMedPubMed CentralGoogle Scholar
- Dong T, Yu R, Schellhorn H. Antagonistic regulation of motility and transcriptome expression by RpoN and RpoS in Escherichia coli. Mol Microbiol. 2011;79(2):375–86.View ArticlePubMedGoogle Scholar
- Helmann JD, Chamberlin MJ. Structure and function of bacterial sigma factors. Annu Rev Biochem. 1988;57:839–72.View ArticlePubMedGoogle Scholar
- Haldenwang WG. The sigma factors of Bacillus subtilis. Microbiol Rev. 1995;59(1):1–30.PubMedPubMed CentralGoogle Scholar
- Jarmer H, Larsen TS, Krogh A, Saxild HH, Brunak S, Knudsen S. Sigma A recognition sites in the Bacillus subtilis genome. Microbiology. 2001;147(Pt 9):2417–24.View ArticlePubMedGoogle Scholar
- Li H, Durbin R. Fast and accurate short read alignment with Burrows-Wheeler transform. Bioinformatics. 2009;25(14):1754–60.View ArticlePubMedPubMed CentralGoogle Scholar
- Li H, Durbin R. Fast and accurate long-read alignment with Burrows-Wheeler transform. Bioinformatics. 2010;26(5):589–95.View ArticlePubMedPubMed CentralGoogle Scholar
- Wang L, Feng Z, Wang X, Wang X, Zhang X. DEGseq: an R package for identifying differentially expressed genes from RNA-seq data. Bioinformatics. 2010;26(1):136–8.View ArticlePubMedGoogle Scholar