nifH Gene Product Research Articles

Endophytic bacteria from paddy with double 1-aminocyclopropane-1-carboxylic acid deaminase and nitrogenase activity

Plant growth promoting bacteria with dual activity, 1-aminocyclopropane-1-carboxylic-acid deaminase (ACCD) and nitrogenase, is more effective in supporting plant growth under stress condition. Previously, we were obtained several endophytic bacterial strains that exhibited dual activity, one of which was Raoultella terrigena PCM8. This study aimed to characterize the ACCD and nitrogenase genes of PCM8 strain. The acdS gene was obtained from the results of Whole Genomic Sequencing analyis, while the nifH gene was obtained by PCR. The characterization of both of the genes was carried out by means of in-silico analysis. WGS annotation analysis, showed that the acdS gene of PCM8 was located at the locus 19090 of genomic DNA and contains 978 nucleotides. In silico analysis of both acdS and nifH gene products showed that the ACCD enzyme of PCM8 had 325 amino acids, with molecular weight of 34.95 kDa, while nitrogenase as represented by nifH subunit product consist of 96 amino acids with molecular weight of 93.98 kDa, respectively. The ACCD had pI value of 5.06, and catalytic residues of Lys51, Ser78, Tyr287, and Thr288, while nifH gene product had the pI value of 11.77. The results suggested that R. terrigena PCM8 potentially produce double activity of ACCD and nitrogenase and therefore it can be a good candidate as plant growth promoting under stress condition.
 Keywords: acdS gene, 1-aminocyclopropane-1-carboxylic acid deaminase, endophytic bacteria, nifH gene, nitrogenase Raoultella terrigena

Diversity of free-Living nitrogen fixing Streptomyces in soils of the badlands of South Dakota

Biological Nitrogen Fixation is critical for ecosystem productivity. Select members of Bacteria and Archaea express a nitrogenase enzyme complex that reduces atmospheric nitrogen to ammonia. Several nitrogen fixing bacteria form symbiotic associations with plants, but free-living diazotrophs also contribute a substantial amount of nitrogen to ecosystems. The aim of this study was to isolate and characterize free-living diazotrophs in arid lands of South Dakota Badlands. Samples were obtained from sod tables and the surrounding base in spring and fall. Diazotrophs were isolated on solid nitrogen free medium (NFM) under hypoxic conditions, and their16S rRNA and nifH genes sequenced. nifH was also amplified directly from soil DNA extracts. The 16S rRNA gene data indicated a diversity of putative free-living diazotrophs across 4 phyla (Actinomycetes, Proteobacteria, Bacteroidetes, and Firmicutes), but ∼50% of these clustered with Streptomyces. These Streptomyces isolates grew in liquid NFM in an ammonia-depleted environment. Only 5 of these yielded a nifH gene product using the PolF/PolR primer set. Four of these aligned with nifH of the cyanobacteria Scytonema and Nostoc, and the other one aligned with nifH of Bradyrhizobium. Six selected Streptomyces isolates, three of which were nifH positive by PCR, all indicated 15N2 incorporation, providing strong support of nitrogen fixation. All nifH amplicons from soil DNA extract resembled Cyanobacteria. This is the first known report of diazotrophic Streptomyces, other than the thermophilic, autotrophic S. thermoautotrophicus. nifH genes of these Streptomyces were related to those from Cyanobacteria. It is possible that the cyanobacteria-like nifH amplicons obtained from soil DNA were associated with Streptomyces.

Purification of a NifEN Protein Complex That Contains Bound Molybdenum and a FeMo-Co Precursor from an Azotobacter vinelandii ΔnifHDK Strain

The NifEN protein complex serves as a molecular scaffold where some of the steps for the assembly of the iron-molybdenum cofactor (FeMo-co) of nitrogenase take place. A His-tagged version of the NifEN complex has been previously purified and shown to carry two identical [4Fe-4S] clusters of unknown function and a [Fe-S]-containing FeMo-co precursor. We have improved the purification of the his-NifEN protein from a DeltanifHDK strain of Azotobacter vinelandii and have found that the amounts of iron and molybdenum within NifEN were significantly higher than those reported previously. In an in vitro FeMo-co synthesis system with purified components, the NifEN protein served as a source of both molybdenum and a [Fe-S]-containing FeMo-co precursor, showing significant FeMo-co synthesis activity in the absence of externally added molybdate. Thus, the NifEN scaffold protein, purified from DeltanifHDK background, contained the Nif-Bco-derived Fe-S cluster and molybdenum, although these FeMo-co constituents were present at different levels within the protein complex.

Cloning and sequence analysis ofAlcaligenes faecalis nifHDK gene cluster

Total DNA ofAlcaligenes faeculis was probed with both the nifH and nifHD sequences fromK. pneumoniae.One positive band of about 4. 6 kb was discovered. This nifH homologous fragment was cloned into the vector pBluescript SK(+) to construct the recombinant plasmid pBZ1. The inserted fragment in pBZl was analyzed by physical mapping and was further subcloned for sequencing. It was found that thisA. faecalis nifHDK homology possessed a typical Sigma(54)-dependent promoter region with upstream activator sequence (UAS) and A-T rich region. The nifH and nifD ORFs were 888 and 1 476 bp long respectively. The GC contents of these two genes were about 61.6% and 60.0%. The intergenic regions of nifH-nifD and nifn-nifK were 101 and 105 bp respectively. There were separate SD sequences upstream of all the three genes. The deduced amino acid sequences of the nifH gene product (the Feprotein) and the nifD gene product (the Mo-Fe-protein) were also highly homologous to other nitrogen-fixing bacteria, especially in those conserved motif. TheA. faeculis sequence has the highest similarity to that ofA. uinelandii.

In Vitro Synthesis of the Iron-Molybdenum Cofactor and Maturation of the nif-encoded Apodinitrogenase: EFFECT OF SUBSTITUTION OF VNFH FOR NIFH

NIFH (the nifH gene product) has several functions in the nitrogenase enzyme system. In addition to reducing dinitrogenase during nitrogenase turnover, NIFH functions in the biosynthesis of the iron-molybdenum cofactor (FeMo-co), and in the processing of alpha2beta2 apodinitrogenase 1 (a catalytically inactive form of dinitrogenase 1 that lacks the FeMo-co) to the FeMo-co-activatable alpha2beta2gamma2 form. The molybdenum-independent nitrogenase 2 (vnf-encoded) has a distinct dinitrogenase reductase protein, VNFH. We investigated the ability of VNFH to function in the in vitro biosynthesis of FeMo-co and in the maturation of apodinitrogenase 1. VNFH can replace NIFH in both the biosynthesis of FeMo-co and in the maturation of apodinitrogenase 1. These results suggest that the dinitrogenase reductase proteins do not specify the heterometal incorporated into the cofactors of the respective nitrogenase enzymes. The specificity for the incorporation of molybdenum into FeMo-co was also examined using the in vitro FeMo-co synthesis assay system.

Induction of complex intracytoplasmic membranes related to nitrogen fixation in Azoarcus sp. BH72

We report the discovery of novel subcellular structures related to bacterial nitrogen fixation in the strictly respiratory diazotrophic bacterium Azoarcus sp. BH72, which was isolated as an endophyte from Kallar grass. Nitrogenase is derepressed under microaerobic conditions at O2 concentrations in the micromolar range. With increasing O2 deprivation, bacteria can develop into a hyperinduced state, which is characterized by high specific rates of respiration and efficient nitrogen fixation at approximately 30 nM O2. Ultrastructural analysis of cells in the course of hyperinduction revealed that complex intracytoplasmic membrane systems are formed, which consist of stacks of membranes and which are absent under standard nitrogen-fixing conditions. The iron protein of nitrogenase was highly enriched on these membranes, as evidenced by immunohistochemical studies. Membrane deficiency in NifH/K- mutants, a deletion mutant in the nifK gene and the character of NH+4-grown cells suggested, in concert with the membrane localization of nitrogenase, that these structures are specialized membranes related to nitrogen fixation. We propose the term 'diazosomes' for them. Development of intracytoplasmic membranes coincides with the appearance of a high-molecular-mass form of the iron protein of nitrogenase, which was detectable in membrane fractions. Mutational analysis, and determination of the N-terminal amino acid sequence indicate that the nifH gene product is covalently modified by a mechanism probably different from adenosine diphosphoribosylation. Development of diazosomes in nitrogen-fixing cells can be induced in pure cultures and in co-culture with a fungus isolated from the rhizosphere of Kallar grass.

Purification and characterization of a FeMo cofactor-deficient MoFe protein.

Previous studies have shown that the nifH gene product is required for FeMo cofactor biosynthesis and insertion and that a delta nifH strain of Azotobacter vinelandii designated DJ54 accumulates a FeMo cofactor-deficient MoFe protein that is distinct from the FeMo cofactor-deficient protein synthesis by Nif B-, N-, or E- strains [Tal, S., Chun, T., Gavini, N., & Burgess, B. K. (1991) J. Biol. Chem. 266, 10654-10657]. Here we report the purification and activation of the MoFe protein from DJ54. The purified protein is an alpha 2 beta 2 tetramer that is indistinguishable from the wild-type MoFe protein by the criteria of SDS-polyacrylamide gel electrophoresis, native gel electrophoresis, and two-dimensional gel electrophoresis. It binds normally to its redox partner, the Fe protein, by the criterion of chemical cross-linking. It does not contain FeMo cofactor and does not catalyze significant C2H2 reduction or reduction-independent MgATP hydrolysis. It can, however, be activated with FeMo cofactor following the addition of the Fe protein and MgATP when an additional required component(s) is supplied by cell-free extracts from a delta nifD strain of A. vinelandii. The purified DJ54 MoFe protein does contain P-clusters by the criteria of metal analysis, CD spectroscopy, cluster extrusion, and electrochemical reduction of the POX state. In the presence of dithionite it exhibits an axial S = 1/2 EPR signal that integrates to 0.1-0.3 spin per alpha 2 beta 2 tetramer.(ABSTRACT TRUNCATED AT 250 WORDS)

Nucleotide Sequence and Characterization of Rhizobium meliloti Nodulation Competitiveness Genes nfe

Rhizobium meliloti large plasmid pRmeGR4b carries the nodulation competitiveness locus nfe responsible for the nodulation efficiency and competitive ability of strain GR4 on alfalfa roots. We report here the nucleotide sequence and characterization of a 3345 base-pair DNA section of the nfe region. Sequence analysis revealed four open reading frames (ORFs), two of them with rightward polarity, termed nfe1 and nfe2, are preceded by functional nif consensus sequences and NifA-binding motifs. An additional, NifA-independent, transcriptional start site for nfe1 was also found. Two other ORFs with leftward polarity, designated ORFA and ORFB, were indentified upstream from nfe1 and nfe2 but no nif consensus sequences were found. However, expression of ORFA might be indirectly coupled to the NifA-NtrA regulatory network. The gene products of nfe1 and nfe2 were identified using in vitro transcription/translation and bacteriophage T7 RNA polymerase/promoter system, respectively. A high degree of homology between the amino terminal domain of Nfe1 and the nifH gene product was found. In addition, nfe1 shows homology with the upstream non-coding DNA region of the fixABCX operon. Furthermore, the putative ORFB encoded protein contains a helix-turn-helix motif that resembles the DNA-binding consensus sequence proposed for many prokaryotic regulatory proteins.

The delta nifB (or delta nifE) FeMo cofactor-deficient MoFe protein is different from the delta nifH protein

We have examined three strains of Azotobacter vinelandii, which contain defined deletions within the nifH, nifB, or nifE genes. All three strains accumulate inactive FeMo cofactor-deficient forms of the MoFe protein of nitrogenase. These forms can be activated in vitro by addition of isolated FeMo cofactor in N-methylformamide. Although the phenotypes of these strains are superficially the same, our characterizations demonstrate that the FeMo cofactor-deficient MoFe protein synthesized by the delta nifH strain is quite different from that synthesized by either the delta nifB or delta nifE strains. These differences include the following: 1) the activation of the delta nifH protein requires MgATP, whereas the activation of the delta nifB and delta nifE proteins does not; 2) the delta nifH extracts can be activated with FeMo cofactor to wild-type levels of activity, whereas delta nifB and delta nifE extracts cannot; 3) the delta nifH protein is markedly less heat stable than the delta nifB and delta nifE proteins; and 4) the migration of the delta nifH protein on native gels is very different when compared with delta nifB and delta nifE, which look like each other. These data can be explained if the nifB and nifE gene products are only involved in FeMo cofactor biosynthesis, whereas the nifH gene product is involved in both the initial synthesis of FeMo cofactor and in the insertion of preformed FeMo cofactor into the MoFe protein. A model is presented that suggests that the FeMo cofactor-deficient MoFe protein synthesized by the delta nifH strain is the one that normally participates in MoFe protein assembly in wild-type cells.

Genes required for formation of the apoMoFe protein of Klebsiella pneumoniae nitrogenase in Escherichia coli.

A binary plasmid system was used to produce nitrogenase components in Escherichia coli and subsequently to define a minimum set of nitrogen fixation (nif) genes required for the production of the iron-molybdenum cofactor (FeMoco) reactivatable apomolybdenum-iron (apoMoFe) protein of nitrogenase. The active MoFe protein is an alpha 2 beta 2 tetramer containing two FeMoco clusters and 4 Fe4S4 P centers (for review see, Orme-Johnson, W.H. (1985) Annu. Rev. Biophys. Biophys. Chem. 14, 419-459). The plasmid pVL15, carrying a tac-promoted nifA activator gene, was coharbored in E. coli with the plasmid pGH1 which contained nifHDKTYENXUSVWZMF' derived from the chromosome of the nitrogen fixing bacterium Klebsiella pneumoniae. The apoMoFe protein produced in E. coli by pGH1 + VL15 was identical to the apoprotein in derepressed cells of the nifB- mutant of K. pneumoniae (UN106) in its electrophoretic properties on nondenaturing polyacrylamide gels as well as in its ability to be activated by FeMoco. The constituent peptides migrated identically to those from purified MoFe protein during electrophoresis on denaturing gels. The concentrations of apoMoFe protein produced in nif-transformed strains of E. coli were greater than 50% of the levels of MoFe protein observed in derepressed wild-type K. pneumoniae. Systematic deletion of individual nif genes carried by pGH1 has established the requirements for the maximal production of the FeMoco-reactivatable apoMoFe protein to be the following gene products, NifHDKTYUSWZM+A. It appears that several of the genes (nifT, Y, U, W, and Z) are only required for maximal production of the apoMoFe protein, while others (nifH, D, K, and S) are absolutely required for synthesis of this protein in E. coli. One curious result is that the nifH gene product, the peptide of the Fe protein, but not active Fe protein itself, is required for formation of the apoMoFe protein. This suggests the possibility of a ternary complex of the NifH, D, and K peptides as the substrate for the processing to form the apoMoFe protein. We also find that nifM, the gene which processes the nifH protein into Fe protein (Howard, K.S., McLean, P.A., Hansen, F. B., Lemley, P.V., Kobla, K.S. & Orme-Johnson, W.H. (1986) J. Biol. Chem. 261, 772-778) can, under certain circumstances, partially replace other processing genes (i.e. nifTYU and/or WZ) although it is not essential for apoMoFe protein formation. It also appears that nifS and nifU, reported to play a role in Fe protein production in Azotobacter vinelandii, play no such role in K. pneumoniae, although these genes are involved in apoMoFe formation.(ABSTRACT TRUNCATED AT 400 WORDS)

The nifH gene product is required for the synthesis or stability of the iron-molybdenum cofactor of nitrogenase from Klebsiella pneumoniae.

The MoFe protein of nitrogenase from Klebsiella pneumoniae contains an iron-molybdenum cofactor, FeMoco, the synthesis or processing of which involves the products of at least five genes, nifQ, nifB, nifN, nifE and nifV. We have detected FeMoco activity in extracts of strains which synthesise neither of the MoFe protein subunits, indicating that FeMoco can be synthesised prior to combination with the MoFe protein polypeptides. Expression of the nifH gene (or a large part of it), was essential for FeMoco activity to be observed either in the presence or in the absence of the MoFe protein subunits. The nifH gene product was not involved in the control of the transcription of other nif gene products known to be involved in FeMoco synthesis or processing, nor was it essential for the stability of performed FeMoco before its combination with the MoFe protein polypeptides.

Nitrogenase promoter-lacZ fusion studies of essential nitrogen fixation genes in Bradyrhizobium japonicum I110.

DNA fragments containing either the nifD or nifH promoter and 5' structural gene sequences from Bradyrhizobium japonicum I110 were fused in frame to the lacZ gene. Stable integration of these nif promoter-lacZ fusions by homologous double reciprocal crossover into a symbiotically nonessential region of the B. japonicum chromosome provided an easy assay for the effects of potential nif regulatory mutants. The level of beta-galactosidase activity expressed from these two nif promoter-lacZ fusions was assayed in bacteroids of B. japonicum I110 wild type and Fix mutants generated by transposon Tn5 mutagenesis and identified in the accompanying paper. No nif-positive regulatory mutants were identified from among an array of Fix- mutants in which Tn5 was inserted 9 kilobase pairs upstream of the nifDK operon and within the 18-kilobase-pair region separating the nifDK and nifH operons. This result indicates that there are no genes in these regions involved in the regulation of nitrogenase structural gene expression. Interestingly, the level of beta-galactosidase activity expressed from the nifH promoter was twice that expressed from the nifD promoter, suggesting that the normal cellular level of the nifH gene product in bacteroids is in a 2:1 ratio with the nifD gene product instead of in the 1:1 stoichiometry of the nitrogenase enzyme complex.

The pleiotropic nature of symbiotic regulatory mutants: Bradyrhizobium japonicum nifA gene is involved in control of nif gene expression and formation of determinate symbiosis

In the slow-growing soybean symbiont, Bradyrhizobium japonicum (strain 110), a nifA-like regulatory gene was located immediately upstream of the previously mapped fixA gene. By interspecies hybridization and partial DNA sequencing the gene was found to be homologous to nifA from Klebsiella pneumoniae and Rhizobium meliloti, and to a lesser extent, also to ntrC from K. pneumoniae. The B. japonicum nifA gene product was shown to activate B. japonicum and K. pneumoniae nif promoters (using nif::lacZ translational fusions) both in Escherichia coli and B. japonicum backgrounds. In the heterologous E. coli system activation was shown to be dependent on the ntrA gene product. Site-directed insertion and deletion/replacement mutagenesis revealed that nifA is probably the promoter-distal cistron within an operon. NifA- mutants were Fix- and pleiotropic: (i) they were defective in the synthesis of several proteins including the nifH gene product (nitrogenase Fe protein); the same proteins had been known to be repressed under aerobic growth of B. japonicum but derepressed at low O2 tension; (ii) the mutants had an altered nodulation phenotype inducing numerous, small, widely distributed soybean nodules in which the bacteroids were subject to severe degradation. These results show that nifA not only controls nitrogenase genes but also one or more genes involved in the establishment of a determinate, nitrogen-fixing root nodule symbiosis.

Expression of nitrogen fixation genes in foreign hosts. Assembly of nitrogenase Fe protein in Escherichia coli and in yeast.

In Klebsiella pneumoniae, the nifH gene encodes the Fe protein (Kp2) polypeptide that is assembled into a homodimer responsible for the reduction of nitrogenase. Escherichia coli or the yeast Saccharomyces cerevisiae, transformed with the K. pneumoniae nifH gene in suitable expression vectors, synthesize the Fe protein polypeptide. This study examines the assembly of the nifH gene product into its characteristic dimeric structure in E. coli and in yeast. Immunoblotting methods, as well as 55Fe2- labeling of K. pneumoniae were employed to detect native nitrogenase components in cell lysates. E. coli and yeast transformants contained a protein similar to native Kp2 in its immunoreactivity, apparent molecular weight, and lability in the presence of oxygen or MgATP. While in E. coli the co-introduction of nifH and nifM resulted in enhanced levels of the nifH product, it appears that the nifH gene product alone is sufficient for the assembly of an Fe protein-like structure in foreign prokaryotic and eukaryotic hosts.

Expression of a nitrogen-fixation gene encoding a nitrogenase subunit in yeast

Biological nitrogen fixation is catalyzed by nitrogenase, an enzyme complex exclusive to prokaryotes. We used the yeast Saccharomyces cerevisiae to study the synthesis, and subsequently the assembly, of nitrogenase components in a eukaryote. Here, the Klebsiella pneumoniae nifH gene, encoding the subunit of the Fe protein (Kp2) component of nitrogenase, was expressed in S. cerevisiae from the yeast ADHI promoter. The nifH gene product, detected in yeast by immunoblot analysis with anti-Kp2 antibodies, exhibited the same electrophoretic mobility in SDS-polyacrylamide gels as that of the Kp2 subunit synthesized in K. pneumoniae. Estimates of Kp2 antigen and assays of β-galactosidase activity specified by nifH‘-’lacZ fusions showed that the level of nifH product was similar in anaerobically and aerobically grown yeast, but varied with different transforming plasmids and in various haploid and diploid yeast strains. A cistron located downstream to nifH in a transcript resembling the polycistronic mRNA of the nifHDKY operon in K. pneumoniae is not translated in yeast.

Rhizobium japonicum nitrogenase Fe protein gene (nifH)

A 12.1-kilobase PstI fragment from Rhizobium japonicum, which contains homology to both the Klebsiella pneumoniae and the Rhizobium meliloti nifH genes, was cloned into vector pHE3 . The nifH -homologous region was localized on the restriction enzyme cleavage map by Southern blot hybridization experiments. DNA fragments overlapping the R. japonicum nifH gene were subcloned into plasmid vectors to allow the expression of this region in Escherichia coli minicells. The nifH gene product (the polypeptide of the nitrogenase Fe protein) was thus found to have a molecular weight of 33,000. The complete nucleotide sequence of the nifH gene was established, and the amino acid sequence of its gene product was deduced. The reading frame is 882 nucleotides long, corresponding to 294 amino acids which add up to a polypeptide with a molecular weight of 31,525. There was extensive sequence homology with nifH genes or gene products from other nitrogen-fixing bacteria. The transcription initiation site of the R. japonicum nifH gene was found to lie 153 nucleotides upstream from the coding region and was preceded by a characteristic promoter sequence. A potential terminator region was located 13 nucleotides downstream from the coding region.

Insertion and deletion mutations within the nif region of Rhizobium japonicum

Insertion and deletion mutants were used to characterize a genomic region of Rhizobium japonicum where the nitrogenase structural genes are located on two separate operons nifDK and nifH. In addition to previously described nifD:: Tn5 and nifK:: Tn5 mutations we have now generated, by localized mutagenesis, further Tn5 insertion mutations in the vicinity of nifDK as well as within and adjacent to nifH. The nifD:: Tn5, nifK:: Tn5, and nifH:: Tn5 mutant strains were of the Nod(+) Fix(-) phenotype whereas all other mutants were symbiotically fully effective (Nod(+) Fix(+)). The nifH:: Tn5 mutation was helpful in the identification of the nifH gene product (the dinitrogenase reductase) by two-dimensional gel electrophoresis: due to its polar effect this insertion specifically abolished the synthesis of that protein under microaerobic culture conditions. The ultrastructure of soybean root nodules infected with either the nif (+) wild-type or with the nif (-) (but otherwise isogenic) mutant strains was analyzed by electron microscopy. All contained fully developed bacteroids, but the nitrogen non-fixing mutants showed massive accumulation of PHB.Of Tn5-containing strains, kanamycin sensitive derivatives were obtained which contained deletions. Several classes of deletion mutants were found which, as judged by their physical DNA structure and their phenotypes, allowed the following most important conclusions: (i) deletions lacking both the nifDK and nifH regions indicate linkage between the two operons whereby at least 15 kb of DNA separate them; (ii) one deletion ending upstream from nifH, and lacking only nifDK, indicates that the nifDK operon is located on the 5'-flanking side of the nifH operon; (iii) all deletion mutants are Nod(+) indicating that there are no essential nodulation gnes located between and adjacent to nifDK and nifH.