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Results reveal that the regulation of the sigma(sup)54-dependent activator NifA activity involves P(sub)II signal transduction protein-like GlnK protein. Uridylylation of GlnK prevents unmodified GlnK stimulation of flavoprotein NifL inhibitory activities in Azotobacter vinelandii.

Research has been conducted on the type I and type II methanotrophs, methane-oxidizing bacteria. Results indicate that both types of methanotrophs contain nitrogenase involved in nitrogen fixation and that nitrogenase genes from these methanotrophs may be present in different environments.

Results demonstrate a strong homology of nitrogen fixing genes of Azoarcus sp. strain BH72 to other Proteobacteria and cotranscription of a component of the nitrogenase structural genes, the ferredoxin gene. Ferredoxin is not involved in the nitrogen fixation but functions as a part of the electron flux 'switch off' mechanism of nitrogenase activity.

Results demonstrate that arginine 140 is essential for ADP-ribosylation of dinitrogenase reductase so is native conformation of the Fe(sub)4S(sub)4 region of the enzyme. Data also indicate that contrary to previous findings changing lysine 143 to glycine does not affect much the interaction between dinitrogenase reductase and its ADP-tibosyltransferase.

Research demonstrates that rnf operon encodes proteins dedicated to electron transport to nitrogenase in the nitrogen fixing bacterium. Data reveal that cells carrying the operon exhibit increase in nitrogenase activity brought about by the Rnf polypeptide overproduction.

Research was conducted to examine novel nitrogen-fixing microbial populations and diverse habitats that can support nitrogen fixation in the open ocean other than those previously reported. Nitrogenase genes were amplified from oligotrophic surface water picoplankton from the tropical Pacific and Atlantic oceans and from the Caribbean and Sargasso seas. Results demonstrate that there are diverse nitrogen-fixing phylotypes in the oceanic environment in the plankton.

The promoters of the anfA, encoding a transcriptional activator specific for the alternative nitrogenase system of Rhodobacter capsulatus, and of the mopA-modABCD operon were found to be activated by NtrC. Therefore, these genes are also members of the R. capsulatus Ntr regulon. In contrast to the NtrC-activated promoters of glnB, nifA1 and nifA2, the anfA and mopA promoters are also negatively regulated by molybdenum, and a region of dyad symmetry overlapping the transcriptional initiation sites may represent the molybdenum-sensitive element.

A study was conducted to determine the role of the nifB promoter of Azotobacter vinelandii in regulating gene expression of three distinct nitrogenases. Genetic and molecular analyses showed that rpoN mutation eliminated expression in A. vinelandii, suggesting that deltaN is required for gene expression. nifB-lacZ fusions in various mutant strains showed that nifB can be activated by NifA, VnfA or AnfA transcriptional activators.

Characterization of a tungsten-tolerant mutant (CA6) of the diazotroph Azotobacter vinelandii based on the results of growth experiments showed that CA6 requires nitrogenases 1 and 3 for growth in nitrogen (N)-free medium containing Na2MoO4. The wild-type strain CA repressed the transcription of the anfHDGK gene cluster, which encode the nitrogenase enzymes. This suggests that expression of nitrogenases is dependent on the presence of molybdenum.

Two open reading frames (orf), designated anfO and anfR, found downstream of anfK are crucial to nitrogenase 3-dependent diazotrophic growth in Azotobacter vinelandii. When in-frame deletions of these orfs are performed in certain strains, an Anf- phenotype appears. This demonstrates that the products of anfO and anfR should be present for N2 reduction by nitrogenase 3 to occur.

The complete nitrogenase structural genes from the methanogenic archaeon Methanosarcina barkeri 227 are all transcribed in a single NH4+-repressed operon preceded by an archaeal promoter. Structural analysis of the nifHDK2 and its associated genes revealed that the nifK2 gene codes for 456 amino acids having a deduced mass of 50 kDa and has a length of 1,371 bp. This makes it the first complete methanogenic gene set to be sequenced.

Mutation studies of the two genes encoding Azotobacter vinelandii's nitrogenase 2 and 3 delta subunits, vnfG and anfG, indicate the necessity of VafG and AnfG for diazotrophic growth of A. vinelandii in a nitrogenase 2- and 3-dependent manner. The cysteine residue of AnfG is not essential for dinitrogenase 3-dependent AnfG function, while nitrogenase 2-dependent cell growth requires the vnfG gene product.

The use of immunochemical labelling and electron microscopy helps examine the subcellular distribution of cytochrome oxidase, a respiratory protein in the nonheterocystous marine cyanobacterium Trichodesmium thiebautii. The protein is related to thylakoid and cytoplasmic membranes, and this is indicated by the immunochemical labeling of cytochrome oxidase cells with enhanced nitrogenase levels.

The Azotobacter vinelandii genes for nitrogenase-2 are organized in two transcriptional units, vnfHorfFd, which codes for dinitrogenase reductase-2, and vnfDGK, which codes for dinitrogenase-2. The effects of ammonium (NH4+), molybdenum (Mo) and vanadium (V) on the expression of these genes were determined. The results showed that the transcription of both genes were repressed by Mo. However, NH4+ appears to act at both the transcriptional and posttranscriptional levels. Lastly, V is required for the translation of vnfDGK products.

Analysis of the phylogenetic diversity of nitrogenase genes indicate that the genes originate from diverse assemblages and phylogeny as shown by samples collected from nitrogen-poor-, nitrogen-rich hydrothermal vents, and background deep seawater habitats. Data show that nitrogenase genes present in hydrothermal fluids originate from diverse gene assemblages and phylogenetically distinct from deep seawater samples.

Biological nitrogen fixation, a process found only in some prokaryotes, is catalyzed by the nitrogenase enzyme complex. Bacteria containing nitrogenase occupy an indispensable ecological niche, supplying fixed nitrogen to the global nitrogen cycle. Due to this inceptive role in the nitrogen cycle, diazotrophs are present in virtually all ecosystems, with representatives in environments as varied as aerobic soils (e.g., Azotobacter species), the ocean surface layer (Trichodesmium) and specialized nodules in legume roots (Rhizobium). In any ecosystem, diazotrophs must respond to varied environmental conditions to regulate the tremendously taxing nitrogen fixation process. All characterized diazotrophs regulate nitrogenase at the transcriptional level. A smaller set also possesses a fast-acting post-translational regulation system. Although there is little apparent variation in the sequences and structures of nitrogenases, there appear to be almost as many nitrogenase-regulating schemes as there are nitrogen-fixing species. Herein are described the paradigms of nitrogenase function, transcriptional control and post-translational regulation, as well as the variations on these schemes, described in various nitrogen-fixing bacteria. Regulation is described on a molecular basis, focusing on the functional and structural characteristics of the proteins responsible for control of nitrogen fixation. J. Nutr. 130: 1081-1084, 2000. KEY WORDS: * nitrogen fixation * nitrogenase * ADP-ribosylation * dinitrogenase reductase ADP-ribosyltransferase * dinitrogenase reductase-activating glycohydrolase

A study was undertaken that cloned and sequenced the nitrogenase structural nifU and nifHDK genes of the Trichodesmium sp. IMS101 for the purpose of comparing the nitrogenase proteins and nif regulatory region of Trichodesmium to other cyanobacterial diazotrophs. Nif genes are transcribed by a RNA polymerase that employs a sigma factor known as RpoN or sigma54. Results indicated that the multiple nif transcripts presented potential stem-loop structures that terminated at the transcription downstream of nifH and nifD. NifHDK transcription was unaffected by changes in light intensity.