Nitrogenase Maturation Research Articles

Characterization of a Nitrogenase Iron Protein Substituted with a Synthetic [Fe4Se4] Cluster

AbstractThe Fe protein of nitrogenase plays multiple roles in substrate reduction and cluster maturation via its redox‐active [Fe4S4] cluster. Here we report the synthesis and characterization of a water‐soluble [Fe4Se4] cluster that is used to substitute the [Fe4S4] cluster of the Azotobacter vinelandii Fe protein (AvNifH). Biochemical, EPR and XAS/EXAFS analyses demonstrate the ability of the [Fe4Se4] cluster to adopt the super‐reduced, all‐ferrous state upon its incorporation into AvNifH. Moreover, these studies reveal that the [Fe4Se4] cluster in AvNifH already assumes a partial all‐ferrous state ([Fe4Se4]0) in the presence of dithionite, where its [Fe4S4] counterpart in AvNifH exists solely in the reduced state ([Fe4S4]1+). Such a discrepancy in the redox properties of the AvNifH‐associated [Fe4Se4] and [Fe4S4] clusters can be used to distinguish the differential redox requirements for the substrate reduction and cluster maturation of nitrogenase, pointing to the utility of chalcogen‐substituted FeS clusters in future mechanistic studies of nitrogenase catalysis and assembly.

Characterization of a Nitrogenase Iron Protein Substituted with a Synthetic [Fe4 Se4 ] Cluster.

The Fe protein of nitrogenase plays multiple roles in substrate reduction and cluster maturation via its redox-active [Fe4 S4 ] cluster. Here we report the synthesis and characterization of a water-soluble [Fe4 Se4 ] cluster that is used to substitute the [Fe4 S4 ] cluster of the Azotobacter vinelandii Fe protein (AvNifH). Biochemical, EPR and XAS/EXAFS analyses demonstrate the ability of the [Fe4 Se4 ] cluster to adopt the super-reduced, all-ferrous state upon its incorporation into AvNifH. Moreover, these studies reveal that the [Fe4 Se4 ] cluster in AvNifH already assumes a partial all-ferrous state ([Fe4 Se4 ]0 ) in the presence of dithionite, where its [Fe4 S4 ] counterpart in AvNifH exists solely in the reduced state ([Fe4 S4 ]1+ ). Such a discrepancy in the redox properties of the AvNifH-associated [Fe4 Se4 ] and [Fe4 S4 ] clusters can be used to distinguish the differential redox requirements for the substrate reduction and cluster maturation of nitrogenase, pointing to the utility of chalcogen-substituted FeS clusters in future mechanistic studies of nitrogenase catalysis and assembly.

AnfO controls fidelity of nitrogenase FeFe protein maturation by preventing misincorporation of FeV-cofactor.

Azotobacter vinelandii produces three genetically distinct, but structurally and mechanistically similar nitrogenase isozymes designated as Mo‐dependent, V‐dependent, or Fe‐only based on the heterometal contained within their associated active site cofactors. These catalytic cofactors, which provide the site for N2 binding and reduction, are, respectively, designated as FeMo‐cofactor, FeV‐cofactor, and FeFe‐cofactor. Fe‐only nitrogenase is a poor catalyst for N2 fixation, when compared to the Mo‐dependent and V‐dependent nitrogenases and is only produced when neither Mo nor V is available. Under conditions favoring the production of Fe‐only nitrogenase a gene product designated AnfO preserves the fidelity of Fe‐only nitrogenase by preventing the misincorporation of FeV‐cofactor, which results in the accumulation of a hybrid enzyme that cannot reduce N2. These results are interpreted to indicate that AnfO controls the fidelity of Fe‐only nitrogenase maturation during the physiological transition from conditions that favor V‐dependent nitrogenase utilization to Fe‐only nitrogenase utilization to support diazotrophic growth.

Identification of Genes Involved in Fe-S Cluster Biosynthesis of Nitrogenase in Paenibacillus polymyxa WLY78.

NifS and NifU (encoded by nifS and nifU) are generally dedicated to biogenesis of the nitrogenase Fe–S cluster in diazotrophs. However, nifS and nifU are not found in N2-fixing Paenibacillus strains, and the mechanisms involved in Fe–S cluster biosynthesis of nitrogenase is not clear. Here, we found that the genome of Paenibacillus polymyxa WLY78 contains the complete sufCDSUB operon, a partial sufC2D2B2 operon, a nifS-like gene, two nifU-like genes (nfuA-like and yutI), and two iscS genes. Deletion and complementation studies showed that the sufC, sufD, and sufB genes of the sufCDSUB operon, and nifS-like and yutI genes were involved in the Fe–S cluster biosynthesis of nitrogenase. Heterologous complementation studies demonstrated that the nifS-like gene of P. polymyxa WLY78 is interchangeable with Klebsiella oxytoca nifS, but P. polymyxa WLY78 SufCDB cannot be functionally replaced by K. oxytoca NifU. In addition, K. oxytoca nifU and Escherichia coli nfuA are able to complement the P. polymyxa WLY78 yutI mutant. Our findings thus indicate that the NifS-like and SufCDB proteins are the specific sulfur donor and the molecular scaffold, respectively, for the Fe–S cluster formation of nitrogenase in P. polymyxa WLY78. YutI can be an Fe–S cluster carrier involved in nitrogenase maturation in P. polymyxa WLY78.

In silico determination of nitrogen metabolism in microbes from extreme conditions using metagenomics.

The acid ponds of the Danakil Depression in northern Ethiopia are polyextreme environments that exceed the normal physicochemical limits of pH, salinity, ion content, and temperature. We tested for the occurrence of DNA-based life in this environment using Metagenomic Shotgun DNA sequencing approaches. The obtained sequences were examined by the bioinformatic tools MetaSpades, DIAMOND and MEGAN 6-CE, and we were able to bin more than 90% of the metagenomics contigs of Dallol and Black Water to the Bacteria domain, and to the Proteobacteria phylum. Predictions of gene function based on SEED disclosed the presence of different nutrient cycles in the acid ponds. For this study, we focused on partial or completely sequenced genes involved in nitrogen metabolism. The KEGG nitrogen metabolism pathway mapping results for both acid ponds showed that all the predicted genes are involved directly or indirectly in the assimilation of ammonia and no dissimilation or nitrification process was identified. Furthermore, the deduced nitrogen fixation in the two acid ponds based on SEED classification indicated the presence of different sets of nitrogen fixing (nif) genes for biosynthesis and maturation of nitrogenase. Based on the in silico analysis, the predicted proteins involved in nitrogen fixation, especially the cysteine desulfurase and [4Fe-4S] ferredoxin, from both acid ponds are unique with less than 80% sequence similarity to the next closest protein sequence. Considering the extremity of the environmental conditions of the two acid ponds in the Danakil depression, this metagenomics dataset can add to the study of unique gene functions in nitrogen metabolism that enable thriving biocommunities in hypersaline and highly acidic conditions.

Biosynthesis of the nitrogenase active-site cofactor precursor NifB-co in Saccharomyces cerevisiae

The radical S-adenosylmethionine (SAM) enzyme NifB occupies a central and essential position in nitrogenase biogenesis. NifB catalyzes the formation of an [8Fe-9S-C] cluster, called NifB-co, which constitutes the core of the active-site cofactors for all 3 nitrogenase types. Here, we produce functional NifB in aerobically cultured Saccharomyces cerevisiae Combinatorial pathway design was employed to construct 62 strains in which transcription units driving different expression levels of mitochondria-targeted nif genes (nifUSXB and fdxN) were integrated into the chromosome. Two combinatorial libraries totaling 0.7 Mb were constructed: An expression library of 6 partial clusters, including nifUSX and fdxN, and a library consisting of 28 different nifB genes mined from the Structure-Function Linkage Database and expressed at different levels according to a factorial design. We show that coexpression in yeast of the nitrogenase maturation proteins NifU, NifS, and FdxN from Azotobacter vinelandii with NifB from the archaea Methanocaldococcus infernus or Methanothermobacter thermautotrophicus yields NifB proteins equipped with [Fe-S] clusters that, as purified, support in vitro formation of NifB-co. Proof of in vivo NifB-co formation was additionally obtained. NifX as purified from aerobically cultured S. cerevisiae coexpressing M. thermautotrophicus NifB with A. vinelandii NifU, NifS, and FdxN, and engineered yeast SAM synthase supported FeMo-co synthesis, indicative of NifX carrying in vivo-formed NifB-co. This study defines the minimal genetic determinants for the formation of the key precursor in the nitrogenase cofactor biosynthetic pathway in a eukaryotic organism.

The Molybdenum Storage Protein: A soluble ATP hydrolysis-dependent molybdate pump.

A continuous FeMo cofactor supply for nitrogenase maturation is ensured in Azotobacter vinelandii by developing a cage-like molybdenum storage protein (MoSto) capable to store ca. 120 molybdate molecules ( ) as discrete polyoxometalate (POM) clusters. To gain mechanistic insight into this process, MoSto was characterized by Mo and ATP/ADP content, structural, and kinetic analysis. We defined three functionally relevant states specified by the presence of both ATP/ADP and POM clusters (MoStofunct ), of only ATP/ADP (MoStobasal ) and of neither ATP/ADP nor POM clusters (MoStozero ), respectively. POM clusters are only produced when ATP is hydrolyzed to ADP and phosphate. Vmax was ca. 13 μmolphosphate ·min-1 ·mg-1 and Km for molybdate and ATP/Mg2+ in the low micromolar range. ATP hydrolysis presumably proceeds at subunit α, inferred from a highly occupied α-ATP/Mg2+ and a weaker occupied β-ATP/no Mg2+ -binding site found in the MoStofunct structure. Several findings indicate that POM cluster storage is separated into a rapid ATP hydrolysis-dependent molybdate transport across the protein cage wall and a slow molybdate assembly induced by combined auto-catalytic and protein-driven processes. The cage interior, the location of the POM cluster depot, is locked in all three states and thus not rapidly accessible for molybdate from the outside. Based on Vmax , the entire Mo storage process should be completed in less than 10 s but requires, according to the molybdate content analysis, ca. 15 min. Long-time incubation of MoStobasal with nonphysiological high molybdate amounts implicates an equilibrium in and outside the cage and POM cluster self-formation without ATP hydrolysis. DATABASES: The crystal structures MoSto in the MoSto-F6, MoSto-F7, MoStobasal , MoStozero , and MoSto-F1vitro states were deposited to PDB under the accession numbers PDB 6GU5, 6GUJ, 6GWB, 6GWV, and 6GX4.

Biological iron-sulfur storage in a thioferrate-protein nanoparticle

Iron–sulfur clusters are ubiquitous in biology and function in electron transfer and catalysis. They are assembled from iron and cysteine sulfur on protein scaffolds. Iron is typically stored as iron oxyhydroxide, ferrihydrite, encapsulated in 12 nm shells of ferritin, which buffers cellular iron availability. Here we have characterized IssA, a protein that stores iron and sulfur as thioferrate, an inorganic anionic polymer previously unknown in biology. IssA forms nanoparticles reaching 300 nm in diameter and is the largest natural metalloprotein complex known. It is a member of a widely distributed protein family that includes nitrogenase maturation factors, NifB and NifX. IssA nanoparticles are visible by electron microscopy as electron-dense bodies in the cytoplasm. Purified nanoparticles appear to be generated from 20 nm units containing ∼6,400 Fe atoms and ∼170 IssA monomers. In support of roles in both iron–sulfur storage and cluster biosynthesis, IssA reconstitutes the [4Fe-4S] cluster in ferredoxin in vitro.

A Sterile α-Motif Domain in NafY Targets Apo-NifDK for Iron-Molybdenum Cofactor Delivery via a Tethered Domain

NafY participates in the final steps of nitrogenase maturation, having a dual role as iron-molybdenum cofactor (FeMo-co) carrier and as chaperone to the FeMo-co-deficient apo-NifDK (apo-dinitrogenase). NafY contains an N-terminal domain of unknown function (n-NafY) and a C-terminal domain (core-NafY) necessary for FeMo-co binding. We show here that n-NafY and core-NafY have very weak interactions in intact NafY. The NMR structure of n-NafY reveals that it belongs to the sterile α-motif (SAM) family of domains, which are frequently involved in protein-protein interactions. The presence of a SAM domain in NafY was unexpected and could not be inferred from its amino acid sequence. Although SAM domains are very commonly found in eukaryotic proteins, they have rarely been identified in prokaryotes. The n-NafY SAM domain binds apo-NifDK. As opposed to full-length NafY, n-NafY impaired FeMo-co insertion when present in molar excess relative to FeMo-co and apo-NifDK. The implications of these observations are discussed to offer a plausible mechanism of FeMo-co insertion. NafY domain structure, molecular tumbling, and interdomain motion, as well as NafY interaction with apo-NifDK are consistent with the function of NafY in FeMo-co delivery to apo-NifDK.

Full structure building and docking of NifS from extremophile Acidithiobacillus ferrooxidans

The gene iscS-2 from extremophile Acidithiobacillus ferrooxidans may play a crucial role in nitrogenase maturation. To investigate the protein encoded by this gene, a reliable integral three-dimensional molecular structure was built. The obtained structure was further used to search binding sites, carry out the flexible docking with cofactor pyridoxal 5′-phosphate(PLP) and substrate cysteine, and identify its key residues. The docking results of PLP reveal that the residues of Lys203, His100, Thr73, Ser200, His202, Asp177 and Gln180 have large interaction energies and/or hydrogen bonds fixation with PLP. The docking results of cysteine show that the amino group in cysteine is very near His100, Lys203 and PLP, and the interaction energies for cysteine with them are very big. These identified residues are in line with the experimental facts of NifS from other sources. Moreover, the four residues of Asn152, Val179, Ala102 and Met148 in the PLP docking and the two residues of Lys208 and Ala102 in the cysteine docking also have large interaction energies, which are fitly conserved in NifS from all kinds of sources but have not been identified before. According to these results, this gene encodes NifS protein, and the substrate cysteine can be effectively recruited into the active site. Furthermore, all of the above detected key residues are directly responsible for the binding and/or catalysis of PLP and cysteine.

The iron–sulphur protein Ind1 is required for effective complex I assembly

NADH:ubiquinone oxidoreductase (complex I) of the mitochondrial inner membrane is a multi-subunit protein complex containing eight iron-sulphur (Fe-S) clusters. Little is known about the assembly of complex I and its Fe-S clusters. Here, we report the identification of a mitochondrial protein with a nucleotide-binding domain, named Ind1, that is required specifically for the effective assembly of complex I. Deletion of the IND1 open reading frame in the yeast Yarrowia lipolytica carrying an internal alternative NADH dehydrogenase resulted in slower growth and strongly decreased complex I activity, whereas the activities of other mitochondrial Fe-S enzymes, including aconitase and succinate dehydrogenase, were not affected. Two-dimensional gel electrophoresis, in vitro activity tests and electron paramagnetic resonance signals of Fe-S clusters showed that only a minor fraction (approximately 20%) of complex I was assembled in the ind1 deletion mutant. Using in vivo and in vitro approaches, we found that Ind1 can bind a [4Fe-4S] cluster that was readily transferred to an acceptor Fe-S protein. Our data suggest that Ind1 facilitates the assembly of Fe-S cofactors and subunits of complex I.

A Proposed Role for the Azotobacter vinelandii NfuA Protein as an Intermediate Iron-Sulfur Cluster Carrier

Iron-sulfur clusters ([Fe-S] clusters) are assembled on molecular scaffolds and subsequently used for maturation of proteins that require [Fe-S] clusters for their functions. Previous studies have shown that Azotobacter vinelandii produces at least two [Fe-S] cluster assembly scaffolds: NifU, required for the maturation of nitrogenase, and IscU, required for the general maturation of other [Fe-S] proteins. A. vinelandii also encodes a protein designated NfuA, which shares amino acid sequence similarity with the C-terminal region of NifU. The activity of aconitase, a [4Fe-4S] cluster-containing enzyme, is markedly diminished in a strain containing an inactivated nfuA gene. This inactivation also results in a null-growth phenotype when the strain is cultivated under elevated oxygen concentrations. NifU has a limited ability to serve the function of NfuA, as its expression at high levels corrects the defect of the nfuA-disrupted strain. Spectroscopic and analytical studies indicate that one [4Fe-4S] cluster can be assembled in vitro within a dimeric form of NfuA. The resultant [4Fe-4S] cluster-loaded form of NfuA is competent for rapid in vitro activation of apo-aconitase. Based on these results a model is proposed where NfuA could represent a class of intermediate [Fe-S] cluster carriers involved in [Fe-S] protein maturation.

In vitro synthesis of the iron–molybdenum cofactor of nitrogenase from iron, sulfur, molybdenum, and homocitrate using purified proteins

Biological nitrogen fixation, the conversion of atmospheric N2 to NH3, is an essential process in the global biogeochemical cycle of nitrogen that supports life on Earth. Most of the biological nitrogen fixation is catalyzed by the molybdenum nitrogenase, which contains at its active site one of the most complex metal cofactors known to date, the iron-molybdenum cofactor (FeMo-co). FeMo-co is composed of 7Fe, 9S, Mo, R-homocitrate, and one unidentified light atom. Here we demonstrate the complete in vitro synthesis of FeMo-co from Fe(2+), S(2-), MoO4(2-), and R-homocitrate using only purified Nif proteins. This synthesis provides direct biochemical support to the current model of FeMo-co biosynthesis. A minimal in vitro system, containing NifB, NifEN, and NifH proteins, together with Fe(2+), S(2-), MoO4(2-), R-homocitrate, S-adenosyl methionine, and Mg-ATP, is sufficient for the synthesis of FeMo-co and the activation of apo-dinitrogenase under anaerobic-reducing conditions. This in vitro system also provides a biochemical approach to further study the function of accessory proteins involved in nitrogenase maturation (as shown here for NifX and NafY). The significance of these findings in the understanding of the complete FeMo-co biosynthetic pathway and in the study of other complex Fe-S cluster biosyntheses is discussed.

Controlled Expression of nif and isc Iron-Sulfur Protein Maturation Components Reveals Target Specificity and Limited Functional Replacement between the Two Systems

The nitrogen-fixing organism Azotobacter vinelandii contains at least two systems that catalyze formation of [Fe-S] clusters. One of these systems is encoded by nif genes, whose products supply [Fe-S] clusters required for maturation of nitrogenase. The other system is encoded by isc genes, whose products are required for maturation of [Fe-S] proteins that participate in general metabolic processes. The two systems are similar in that they include an enzyme for the mobilization of sulfur (NifS or IscS) and an assembly scaffold (NifU or IscU) upon which [Fe-S] clusters are formed. Normal cellular levels of the Nif system, which supplies [Fe-S] clusters for the maturation of nitrogenase, cannot also supply [Fe-S] clusters for the maturation of other cellular [Fe-S] proteins. Conversely, when produced at the normal physiological levels, the Isc system cannot supply [Fe-S] clusters for the maturation of nitrogenase. In the present work we found that such target specificity for IscU can be overcome by elevated production of NifU. We also found that NifU, when expressed at normal levels, is able to partially replace the function of IscU if cells are cultured under low-oxygen-availability conditions. In contrast to the situation with IscU, we could not establish conditions in which the function of IscS could be replaced by NifS. We also found that elevated expression of the Isc components, as a result of deletion of the regulatory iscR gene, improved the capacity for nitrogen-fixing growth of strains deficient in either NifU or NifS.

Genes required for rapid expression of nitrogenase activity in Azotobacter vinelandii

Rnf proteins are proposed to form membrane-protein complexes involved in the reduction of target proteins such as the transcriptional regulator SoxR or the dinitrogenase reductase component of nitrogenase. In this work, we investigate the role of rnf genes in the nitrogen-fixing bacterium Azotobacter vinelandii. We show that A. vinelandii has two clusters of rnf-like genes: rnf1, whose expression is nif-regulated, and rnf2, which is expressed independently of the nitrogen source in the medium. Deletion of each of these gene clusters produces a time delay in nitrogen-fixing capacity and, consequently, in diazotrophic growth. Deltarnf mutations cause two distinguishable effects on the nitrogenase system: (i), slower nifHDK gene expression and (ii), impairment of nitrogenase function. In these mutants, dinitrogenase reductase activity is lowered, whereas dinitrogenase activity remains essentially unaltered. Further analysis indicates that deltarnf mutants accumulate an inactive and iron-deficient form of NifH because they have lower rates of incorporation of [4Fe-4S] into NifH. Deltarnf mutations also cause a noticeable decrease in aconitase activity; however, they do not produce general oxidative stress or modification of Fe metabolism in A. vinelandii. Our results suggest the existence of a redox regulatory mechanism in A. vinelandii that controls the rate of expression and maturation of nitrogenase by the activity of the Rnf protein complexes. rnf1 plays a major and more specific role in this scheme, but the additive effects of mutations in rnf1 and rnf2 indicate the existence of functional complementation between the two homologous systems.

NifU and NifS are required for the maturation of nitrogenase and cannot replace the function of isc-gene products in Azotobacter vinelandii

In recent years, it has become evident that [Fe-S] proteins, such as hydrogenase, nitrogenase and aconitase, require a complex machinery to assemble and insert their associated [Fe-S] clusters. So far, three different types of [Fe-S] cluster biosynthetic systems have been identified and these have been designated nif, isc and suf. In the present work, we show that the nif-specific [Fe-S] cluster biosynthetic system from Azotobacter vinelandii, which is required for nitrogenase maturation, cannot functionally replace the isc [Fe-S] cluster system used for the maturation of other [Fe-S] proteins, such as aconitase. The results indicate that, in certain cases, [Fe-S] cluster biosynthetic machineries have evolved to perform only specialized functions.

Maturation of Nitrogenase: a Biochemical Puzzle

The nitrogenase enzyme catalyzes the reductive breakage of the very strong triple bond of N2 to generate NH3 in a process known as biological nitrogen fixation. Biological nitrogen fixation is an essential step in the nitrogen cycle in the biosphere, and it is a major contributor to the nitrogen

Interchangeability and Distinct Properties of Bacterial Fe-S Cluster Assembly Systems: Functional Replacement of the isc and suf Operons in Escherichia coli with the nifSU-Like Operon from Helicobacter pylori

The assembly of iron-sulfur (Fe-S) clusters, a key step in the post-translational maturation of Fe-S proteins, is mediated by a complex apparatus. In E. coli, this process involves two independent systems called ISC and SUF encoded by the iscSUA-hscBA-fdx gene cluster and sufABCDSE operon, respectively. Another system, termed NIF (nifSU), is required for the maturation of nitrogenase in nitrogen-fixing bacteria. We have developed a novel genetic system to gain further insight into these multi-component systems, and to determine how ISC, SUF and NIF might differ in their roles in Fe-S assembly. We have constructed an E. coli mutant lacking both the isc and suf operons, and this strain can only survive in the presence of a complementing plasmid. Using the plasmid replacement technique, we examined the isc and suf operons, and identified the genes essential for the function. Additionally, we have found that nifSU-like genes cloned from Helicobacter pylori are functionally exchangeable with the isc and suf operons. Thus, the NIF-like system participates in the maturation of a wide variety of Fe-S proteins. An increased ability of NIF to complement isc and suf loss was seen under anaerobic conditions. This may explain why the NIF system is only found in a limited number of bacterial species, and most other organisms prefer the ISC and/or SUF systems. While the differences between ISC and SUF were small with respect to the complementing activity, the SUF system appears to be more advantageous for bacterial growth in the presence of hydrogen peroxide.

A Third Bacterial System for the Assembly of Iron-Sulfur Clusters with Homologs in Archaea and Plastids

The assembly of iron-sulfur (Fe-S) clusters is mediated by complex machinery. In several proteobacteria, this process involves ISC (Fe-S cluster assembly) machinery composed of at least six components also conserved in mitochondria from lower to higher eukaryotes. In nitrogen-fixing bacteria, another system, termed NIF (nitrogen fixation), is required for the maturation of nitrogenase. Here we report the identification of a third system, designated the SUF machinery, the components of which are encoded in Escherichia coli by an unassigned operon, sufABCDSE. We have analyzed spontaneous pseudorevertants isolated from a mutant strain lacking all the components of the ISC machinery. The suppressor mutations in the revertants have been localized to the regulatory region of the suf operon; overexpression of this operon restores the growth phenotypes and activity of Fe-S proteins in mutant cells lacking ISC. Disruption of the suf operon alone does not cause any major defects, but synthetic lethality was observed when both the isc and suf operons were inactivated. These results indicate that proteins encoded by the suf operon participate in the ISC-independent minor pathway for the assembly of Fe-S clusters. The genes homologous to sufBC are present in a wide range of bacteria, Archaea, and plastids, suggesting that this type of system is almost ubiquitous in nature.

Perturbation of nifT expression in Klebsiella pneumoniae has limited effect on nitrogen fixation.

In the nitrogenase system of Klebsiella pneumoniae, nifT is located between nifDK, the structural genes for dinitrogenase, and nifY, whose product is involved in nitrogenase maturation. It is, therefore, a reasonable hypothesis that the NifT protein might also have a role in the maturation of nitrogenase. However, the phenotypic characterization of nifT and nifT-overexpressing strains for effects on the regulation, maturation, and activity of nitrogenase identified no properties that were distinct from those of the wild type. We conclude that the K. pneumoniae NifT protein is not essential for nitrogen fixation under the conditions examined.