Structural features of multiple nifH-like sequences and very biased codon usage in nitrogenase genes of Clostridium pasteurianum.

The structural gene (nifH1) encoding the nitrogenase iron protein of Clostridium pasteurianum has been cloned and sequenced. It is located on a 4-kilobase EcoRI fragment (cloned into pBR325) that also contains a portion of nifD and another nifH-like sequence (nifH2). C. pasteurianum nifH1 encodes a polypeptide (273 amino acids) identical to that of the isolated iron protein, indicating that the smaller size of the C. pasteurianum iron protein does not result from posttranslational processing. The 5' flanking region of nifH1 or nifH2 does not contain the nif promoter sequences found in several gram-negative bacteria. Instead, a sequence resembling the Escherichia coli consensus promoter (TTGACA-N17-TATAAT) is present before C. pasteurianum nifH2, and a TATAAT sequence is present before C pasteurianum nifH1. Codon usage in nifH1, nifH2, and nifD (partial) is very biased. A preference for A or U in the third position of the codons is seen. nifH2 could encode a protein of 272 amino acid residues, which differs from the iron protein (nifH1 product) in 23 amino acid residues (8%). Another nifH-like sequence (nifH3) is located on a nonadjacent EcoRI fragment and has been partially sequenced. C. pasteurianum nifH2 and nifH3 may encode proteins having several amino acids that are conserved in other proteins but not in C. pasteurianum iron protein, suggesting a possible role for the multiple nifH-like sequences of C. pasteurianum in the evolution of nifH. Among the nine sequenced iron proteins, only the C. pasteurianum protein lacks a conserved lysine residue which is near the extended C terminus of the other iron proteins. The absence of this positive charge in the C. pasteurianum iron protein might affect the cross-reactivity of the protein in heterologous systems.     

Amplification, cloning, and sequencing of a nifH segment from aquatic microorganisms and natural communities.

By use of the polymerase chain reaction and degenerate oligonucleotide primers for highly conserved regions of nifH, a segment of nifH DNA was amplified from several aquatic microorganisms, including an N2-fixing bacterium closely associated with the marine filamentous cyanobacterium Trichodesmium sp., a heterotrophic isolate from the root/rhizome of the seagrass Ruppia maritima, and the heterocystous freshwater cyanobacterium Anabaena oscillarioides. nifH segments were amplified directly from DNA extracted from the rhizosphere of roots of the seagrass Halodule wrightii. The nifH fragments were then cloned and sequenced. The DNA and deduced amino acid sequences were compared with known sequences, revealing distinct differences between taxonomic groups. This technique was shown to be useful for (i) the detection of N2-fixing microorganisms and (ii) rapidly obtaining the DNA sequence of the nifH gene, which provides information about general taxonomic groups of N2-fixing microorganisms.     

Amplification, cloning, and sequencing of a nifH segment from aquatic microorganisms and natural communities.

By use of the polymerase chain reaction and degenerate oligonucleotide primers for highly conserved regions of nifH, a segment of nifH DNA was amplified from several aquatic microorganisms, including an N2-fixing bacterium closely associated with the marine filamentous cyanobacterium Trichodesmium sp., a heterotrophic isolate from the root/rhizome of the seagrass Ruppia maritima, and the heterocystous freshwater cyanobacterium Anabaena oscillarioides. nifH segments were amplified directly from DNA extracted from the rhizosphere of roots of the seagrass Halodule wrightii. The nifH fragments were then cloned and sequenced. The DNA and deduced amino acid sequences were compared with known sequences, revealing distinct differences between taxonomic groups. This technique was shown to be useful for (i) the detection of N2-fixing microorganisms and (ii) rapidly obtaining the DNA sequence of the nifH gene, which provides information about general taxonomic groups of N2-fixing microorganisms.     

Nucleotide sequence of the gene encoding the nitrogenase iron protein of Thiobacillus ferrooxidans.

The DNA sequence was determined for the cloned Thiobacillus ferrooxidans nifH and part of the nifD genes. A putative T. ferrooxidans nifH promoter was identified whose sequences showed perfect consensus with those of the Klebsiella pneumoniae nif promoter. Two putative consensus upstream activator sequences were also identified. The amino acid sequence was deduced from the DNA sequence. In a comparison of nifH DNA sequences from T. ferrooxidans and eight other nitrogen-fixing microbes, a Rhizobium sp. isolated from Parasponia andersonii showed the greatest homology (74%) and Clostridium pasteurianum (nifH 1) showed the least homology (54%). In a comparison of the amino acid sequences of the Fe proteins, the Rhizobium sp. and Rhizobium japonicum showed the greatest homology (both 86%) and C. pasteurianum (nifH 1 gene product) demonstrated the least homology (56%) to the T. ferrooxidans Fe protein.     

Cloning of nifHD from Nostoc commune UTEX 584 and of a flanking region homologous to part of the Azotobacter vinelandii nifU gene.

The heterocystous cyanobacterium Nostoc commune UTEX 584 contains two nifH-like sequences (nifH1 and nifH2) in addition to nifHD. A region of DNA 1 kilobase upstream from the 5' end of nifH showed considerable sequence similarity to part of the published nifU sequences of Azotobacter vinelandii and Klebsiella pneumoniae.     

Nucleotide sequence of the gene coding for the nitrogenase iron protein from Klebsiella pneumoniae

We report the complete DNA sequence of the Klebsiella pneumoniae nifH gene, the gene which codes for component 2 (Fe protein or nitrogenase reductase) of the nitrogenase enzyme complex. The amino acid sequence of the K. pneumoniae nitrogenase Fe protein is deduced from the DNA sequence. The K. pneumoniae Fe protein contains 292 amino acids, has a Mr = 31,753, and contains 9 cysteine residues. We compare the amino acid sequence of the K. pneumoniae protein with available amino acid sequence data on nitrogenase Fe proteins from two other species, Clostridium pasteurianum and Azotobacter vinelandii. The C. pasteurianum Fe protein, for which the complete sequence is known, shows 67% homology with the K. pneumoniae Fe protein. Extensive regions of strong conservation (90-95%) are found, while other regions show relatively poor conservation (30-35%). It is suggested that these strongly conserved regions are of special importance to the function of this enzyme, and the findings are discussed in the light of evolutionary theories on the origin of nif genes.     

Cloning and nucleotide sequence of a frxC-ORF469 gene cluster of Synechocystis PCC6803: conservation with liverwort chloroplast frxC-ORF465 and nif operon.

A gene, frxC, which is unique to the chloroplast genome of the liverwort Marchantia polymorpha, has sequence similarity to nifH, the product of which is an iron protein of a nitrogenase. Although frxC is expressed to produce a protein in liverwort chloroplasts, its function is not known. Using a probe of liverwort chloroplast DNA, a 10.1-kb region containing a gene cluster consisting of open reading frames (ORF278-frxC-ORF469-ORF248) was isolated from the cyanobacterium Synechocystis PCC6803. In this region, frxC and ORF469 showed sequence similarities to liverwort chloroplast frxC (83%) and immediately downstream ORF465 (74%), respectively. Synechocystis frxC showed 31% amino acid sequence identity with nifH1 from Clostridium pasteurianum. Additionally, Synechocystis ORF469 showed a sequence similarity (19% identity) to C. pasteurianum nifK product, which is the beta subunit of a molybdenum-iron protein of a nitrogenase complex. Conservation of the gene arrangement between liverwort and Synechocystis suggests that the liverwort chloroplast frxC-ORF465 cluster may have evolved from an ancestor common to Synechocystis, and that these two genes may have been transferred to the nuclear genome in tobacco and rice during evolution.     

Role of molybdenum in dinitrogen fixation by Clostridium pasteurianum.

The role of Mo in the activity and synthesis of the nitrogenase components of Clostridium pasteurianum has been studied by observing the competition of Mo with its structural analogue W. Clostridial cells when fixing N2 appeared strictly dependent upon the available Mo, showing maximal N2-fixing activity at molybdate concentrations in the media of 10 muM. Cells grown in media with 3 times 10(-6) muM Mo, although showing good growth, had only 15% as much N2-fixing activity. In the presence of W the synthesis of both nitrogenase components, molybdoferredoxin and azoferredoxin, was affected. Attempts to produce nitrogenase in W-grown cells by addition of high molybdenum to the media in the presence of inhibitors of protein synthesis showed that Mo incorporation into a possible inactive preformed apoenzyme did not occur. Unlike other molybdoenzyme-containing cells, in which W either is incorporated in place of Mo to yield inactive protein or initiates the production of apoprotein, C. pasteurianum forms neither a tungsten substituted molybdoferredoxin nor an apoprotein. It is concluded that in C. pasteurianum molybdenum is an essential requirement for both the biosynthesis and activity of its nitrogenase.     

Isolation and gene quantification of heterotrophic N2-fixing bacterioplankton in the Baltic Sea

Cyanobacteria are regarded as the main N(2)-fixing organisms in marine waters. However, recent clone libraries from various oceans show a wide distribution of the dinitrogenase reductase gene (nifH) originating from heterotrophic bacterioplankton. We isolated heterotrophic N(2)-fixing bacteria from Baltic Sea bacterioplankton using low-nitrogen plates and semi-solid diazotroph medium (SSDM) tubes. Isolates were analysed for the nitrogenase (nifH) gene and active N(2) fixation by nested polymerase chain reaction (PCR) and acetylene reduction respectively. A primer-probe set targeting the nifH gene from a gamma-proteobacterial isolate, 97% 16S rDNA similarity to Pseudomonas stutzeri, was designed for measuring in situ dynamics using quantitative real-time PCR. This nifH gene sequence was detected at two of 11 stations in a Baltic Proper transect at abundances of 3 x 10(4) and 0.8 x 10(3) copies per litre seawater respectively. Oxygen requirements of isolates were examined by cultivation in SSDM tubes where oxygen gradients were determined with microelectrodes. Growth, and thereby N(2) fixation, was observed as horizontal bands formed at oxygen levels of 0-6% air saturation. The apparent microaerophilic or facultative anaerobic nature of the isolates explains why the SSDM approach is the most appropriate isolation method. Our study illustrates how combined isolation, functional analyses and in situ quantification yielded insights into the oxygen requirements of heterotrophic N(2)-fixing bacterioplankton isolates, which were confirmed to be present in situ.     

Detection and quantification of the nifH gene in shoot and root of cucumber plants

A real-time polymerase chain reaction (PCR) method was applied to quantify the nifH gene pool in cucumber shoot and root and to evaluate how nitrogen (N) supply and plant age affect the nifH gene pool. In shoots, the relative abundance of the nifH gene was affected neither by different stages of plant growth nor by N supply. In roots, higher numbers of diazotrophic bacteria were found compared with that in the shoot. The nifH gene pool in roots significantly increased with plant age, and unexpectedly, the pool size was positively correlated with N supply. The relative abundance of nifH gene copy numbers in roots was also positively correlated (r = 0.96) with total N uptake of the plant. The data suggest that real-time PCR-based nifH gene quantification in combination with N-content analysis can be used as an efficient way to perform further studies to evaluate the direct contribution of the N2-fixing plant-colonizing plant growth promoting bacteria to plant N nutrition.     

Design and functional expression in Escherichia coli of a synthetic gene encoding Clostridium pasteurianum 2[4Fe-4S] ferredoxin.

A gene encoding the exact sequence of Clostridium pasteurianum 2[4Fe-4S] ferredoxin and containing 11 unique restriction endonuclease cleavage sites has been synthesized and cloned in Escherichia coli. The synthetic gene is efficiently expressed in E. coli and its product has been purified and characterized. The N-terminal sequence is identical to that of the protein isolated from C. pasteurianum and the recombinant ferredoxin contains the exact amount of [4Fe-4S] clusters (2 per monomer) expected for homogeneous holoferredoxin. It displays reduction potential and kinetic parameters as electron donor to C. pasteurianum hydrogenase I identical to those determined for the native ferredoxin. All of these properties demonstrate that the 2[4Fe-4S] ferredoxin expressed in E. coli is identical to the parent clostridial protein.     

Biodiversity of denitrifying and dinitrogen-fixing bacteria in an acid forest soil

Isolated soil DNA from an oak-hornbeam forest close to Cologne, Germany, was suitable for PCR amplification of gene segments coding for the 16S rRNA and nitrogenase reductase (NifH), nitrous oxide reductase (NosZ), cytochrome cd(1)-containing nitrite reductase (NirS), and Cu-containing nitrite reductase (NirK) of denitrification. For each gene segment, diverse PCR products were characterized by cloning and sequencing. None of the 16S rRNA gene sequences was identical to any deposited in the data banks, and therefore each of them belonged to a noncharacterized bacterium. In contrast, the analyzed clones of nifH gave only a few different sequences, which occurred many times, indicating a low level of species richness in the N2-fixing bacterial population in this soil. Identical nifH sequences were also detected in PCR amplification products of DNA of a soil approximately 600 km distant from the Cologne area. Whereas biodiversity was high in the case of nosZ, only a few different sequences were obtained with nirK. With respect to nirS, cloning and sequencing of the PCR products revealed that many false gene segments had been amplified with DNA from soil but not from cultured bacteria. With the 16S rRNA gene data, many sequences of uncultured bacteria belonging to the Acidobacterium phylum and actinomycetes showed up in the PCR products when isolated DNA was used as the template, whereas sequences obtained for nifH and for the denitrification genes were closely related to those of the proteobacteria. Although in such an experimental approach one has to cope with the enormous biodiversity in soils and only a few PCR products can be selected at random, the data suggest that denitrification and N2 fixation are not genetic traits of most of the uncultured bacteria.     

Genetic characterization of Cylindrospermopsis raciborskii (cyanobacteria) isolates from diverse geographic origins based on nifH and cpcBA-IGS nucleotide sequence analysis

Isolates of the toxic, N(2)-fixing species Cylindrospermopsis raciborskii from various geographic locations were analyzed with respect to their genetic diversity based on the nifH and cpcBA-IGS genes. Gene sequences clustered according to their geographic origin, with the nifH sequences separating into European, Australian, and American groups and the cpcBA-IGS sequences separating into American and European or Australian groups. PCR primers for both genes were designed to exclusively amplify DNA from Cylindrospermopsis species, and an additional primer set for cpcBA-IGS was designed to specifically amplify the American C. raciborskii strains.     

Diazotrophic community structure and function in two successional stages of biological soil crusts from the Colorado Plateau and Chihuahuan Desert

The objective of this study was to characterize the community structure and activity of N2-fixing microorganisms in mature and poorly developed biological soil crusts from both the Colorado Plateau and Chihuahuan Desert. Nitrogenase activity was approximately 10 and 2.5 times higher in mature crusts than in poorly developed crusts at the Colorado Plateau site and Chihuahuan Desert site, respectively. Analysis of nifH sequences by clone sequencing and the terminal restriction fragment length polymorphism technique indicated that the crust diazotrophic community was 80 to 90% heterocystous cyanobacteria most closely related to Nostoc spp. and that the composition of N2-fixing species did not vary significantly between the poorly developed and mature crusts at either site. In contrast, the abundance of nifH sequences was approximately 7.5 times greater (per microgram of total DNA) in mature crusts than in poorly developed crusts at a given site as measured by quantitative PCR. 16S rRNA gene clone sequencing and microscopic analysis of the cyanobacterial community within both crust types demonstrated a transition from a Microcoleus vaginatus-dominated, poorly developed crust to mature crusts harboring a greater percentage of Nostoc and Scytonema spp. We hypothesize that ecological factors, such as soil instability and water stress, may constrain the growth of N2-fixing microorganisms at our study sites and that the transition to a mature, nitrogen-producing crust initially requires bioengineering of the surface microenvironment by Microcoleus vaginatus.     

Nucleotide sequence of the R.meliloti nitrogenase reductase (nifH) gene.

The nucleotide sequence of the structural gene (nifH) of nitrogenase reductase (Fe protein) from R.meliloti 41 with its flanking ends is reported. The amino acid sequence of nitrogenase reductase was deduced from the DNA sequence. The predicted R.meliloti nitrogenase reductase protein consists of 297 amino acid residues, has a molecular weight of 32,740 daltons and contains 5 cysteine residues. The codon usage in the nifH gene is presented. In the 5' flanking region, sequences resembling to consensus sequences of bacterial control regions were found. Comparison of the R.meliloti nifH nucleotide and amino acid sequences with those from different nitrogen-fixing organisms showed that the amino acid sequences are more conserved than the nucleotide sequences. This structural conservation of nitrogenase reductase may be related to its function and may explain the conservation of the nifH gene during evolution.