Subunits Of Nitrogenase Research Articles

Immunolocalization and Western Blot Analysis of Nitrogenase in Oscillatoria limosa During a Light‐dark Cycle

AbstractNitrogenase of the non‐heterocystous nitrogen‐fixing cyanobacterium Oscillatoria limosa was subjected to western blot analysis and immunogold electron microscopy using antisera raised against dinitrogenase (MoFe‐protein, Component I) and dinitrogenase reductase (Fe‐protein, Component II). O. limosa was grown diazotrophically under an alternating light‐dark cycle (16–8h light‐dark). Although nitrogenase activity (acetylene reduction) was found predominantly during the dark phase, being absent during most of the light period, immunogold electron microscopy revealed label of both subunits of nitrogenase in samples taken throughout the light‐dark cycle. It was also shown that the nitrogenase label was distributed homogeneously in the cell and that it was present in every cell of every trichome whether fixing nitrogen or not. On average, 34 (± 6) gold particles μm−2 thin section were detected. Nitrate‐grown cells did not contain nitrogenase label. Western blot analysis of the Fe‐protein in samples taken during the light phase, revealed a single band with an apparent molecular weight of 37 kDa. At the end of the light period, and during the dark phase when high nitrogenase activities were observed, an additional band of 36 kDa was found. The anti‐MoFe‐protein antiserum revealed a single band of 56 kDa which was present throughout the light‐dark cycle. Nitrate‐grown cells were not recognized by either antiserum. It is concluded that nitrogenase enzyme is present in O. limosa throughout the light‐dark cycle but that the Fe‐protein is modified (inactive form) during the light period when nitrogenase activity is absent.

Immunocytochemical localization of nitrogenase in bacteria symbiotically associated with Azolla spp

In situ immunogold labeling and transmission electron microscopy were used to detect nitrogenase in bacteria (bactobionts) symbiotically associated with leaf cavities of Azolla caroliniana and Azolla filiculoides. In A. caroliniana, the Fe protein of the nitrogenase complex was detected in a subset of the distinct bactobiont types present in leaf cavities of all ages. Similar results were obtained for the bactobionts of A. filiculoides with antisera against both the Fe and MoFe subunits of nitrogenase.

Nucleotide sequence and mutational analysis of the structural genes (anfHDGK) for the second alternative nitrogenase from Azotobacter vinelandii.

The nucleotide sequence of a region of the Azotobacter vinelandii genome exhibiting sequence similarity to nifH has been determined. The order of open reading frames within this 6.1-kilobase-pair region was found to be anfH (alternative nitrogen fixation, nifH-like gene), anfD (nifD-like gene), anfG (potentially encoding a protein similar to the product of vnfG from Azotobacter chroococcum), anfK (nifK-like gene), followed by two additional open reading frames. The 5'-flanking region of anfH contains a nif promoter similar to that found in the A. vinelandii nifHDK gene cluster. The presumed products of anfH, anfD, and anfK are similar in predicted Mr and pI to the previously described subunits of nitrogenase 3. Deletion plus insertion mutations introduced into the anfHDGK region of wild-type strain A. vinelandii CA resulted in mutant strains that were unable to grow in Mo-deficient, N-free medium but grew in the presence of 1 microM Na2MoO4 or V2O5. Introduction of the same mutations into the nifHDK deletion strain CA11 resulted in strains that grew under diazotrophic conditions only in the presence of vanadium. The lack of nitrogenase 3 subunits in these mutant strains was demonstrated through two-dimensional gel analysis of protein extracts from cells derepressed for nitrogenase under Mo and V deficiency. These results indicate that anfH, anfD, and anfK encode structural proteins for nitrogenase 3.

Tn5-induced mutants of Azotobacter vinelandii affected in nitrogen fixation under Mo-deficient and Mo-sufficient conditions.

Mutants of Azotobacter vinelandii affected in N2 fixation in the presence of 1 microM Na2MoO4 (conventional system), 50 nM V2O5, or under Mo deficiency (alternative system) have been isolated after Tn5 mutagenesis with the suicide plasmid pSUP1011. These mutants can be grouped into at least four broad phenotypic classes. Mutants in the first class are Nif- under Mo sufficiency but Nif+ under Mo deficiency or in the presence of V2O5. A nifk mutant and a mutant apparently affected in regulation of the conventional system belong to this class. Mutants in the second class are Nif- under all conditions. An FeMo-cofactor-negative mutant (NifB-) belongs to this class, implying an involvement of nifB in both the conventional and the alternative N2 fixation systems. The third mutant class consists of mutants incapable of N2-dependent growth under Mo deficiency. Most of the mutants in this class are also affected in N2 fixation in the presence of 1 microM Na2MoO4, with acetylene reduction rates ranging from 28 to 51% of the rates of the wild type. Strains constructed by genetic transfer of the Kanr marker of mutants from this class into nifHDK or nifK deletion mutants showed N2-dependent growth only in the presence of V2O5, suggesting that growth in the presence of V2O5 and growth under Mo deficiency are independent phenomena. The only mutant in the fourth class shows wild-type nitrogenase activity under Mo sufficiency, but only 10% of the acetylene reduction activity of the wild type in the presence of 50 nM V2O5. The acetylene reduction rates of whole cells of this mutant are identical in Mo-deficient medium and in medium containing V2O5. The conventional nitrogenase subunits are expressed in this mutant even under Mo deficiency or in the presence of V2O5; however, the NH4+- and Mo-repressible proteins normally seen under these conditions could not be detected on two-dimensional gels. The Tn5 insertion carried by this mutant makes N2 fixation dependent solely on the conventional system and consequently abolishes the vanadium effect.

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.

Genetic Engineering and Nitrogen Fixation

Nitrogen is extremely important in agriculture because it is a constituent of proteins, nucleic acids and other essential molecules in all organisms. Most of this nitrogen is derived from reduced or oxidized forms of N in the soil by growing plants, because plants and animals are unable to utilize N2, which is abundant in the atmosphere. Under most cropping conditions N is limiting for growth and is provided in fertilizers, usually at rates of between 50 and 300 kg of N per ha per year (Anonymous, 1979). The only other sources available to plants are from decomposing organic matter, soil reserves, biological nitrogen fixation, the little that is deposited in rainfall and from other sources such as automobile exhausts. Biological nitrogen fixation, the enzymic conversion of N2 gas to ammonia, is much the most important source of fixed nitrogen entering those soils which receive less than about 5 kg N per ha per year from fertilizers. The reduction of N2 is catalysed by the nitrogenase system, which is very similar in composition and function in all prokaryotes which produce it Indeed, subunits of nitrogenase obtained from different nitrogen-fixing species can often be mixed to produce a functional system (Emerich and Burris, 1978). In addition, DNA coding for the structural proteins is so highly conserved in sequence that this coding has been used in hybridization experiments to demonstrate the presence of these genes in all nitrogen-fixing species of prokaryotes tested (Mazur, Rice and Haselkorn, 1980; Ruvkun and Ausubel, 1980). Nitrogenase is found only in prokaryotic micro-organisms and thus eukaryotes, such as plants!» can benefit from N2 fixation only jf they interact with N2-fixing species of micro-organism or obtain the fixed N after the death of the organisms. Nitrogenase functions only under anaerobic conditions because it is irreversibly inactivated by oxygen. The fixation ofN2 requires large amounts of energy, about 30 moles of ATP per mole N2 reduced (Hill, 1976; Schubert and Wolk, 1982), and thus can act as a major drain for energy produced by N2-fixing micro-organisnls. The requirement for an anaerobic environment and large amounts of energy presents problems to the micro-organisms that fix N2 and to the geneticists who wish to extend the range of N2..fixing organisms. Many micro..organisms fix N2 anaerobically and thus avoid the oxygen problem. However, energy production from organic compounds is usually much more efficient when they are metabolized by oxidative phosphorylation. Thus, in general, nitrogen fixation under aerobic or microaerobic conditions should be more efficient, unless too much energy is lost in protecting the enzyme from oxygen or replacing oxygen-damaged proteins. An important consequence of the large energy cost for biological nitrogen fixation is that the activity of nitrogenase needs to be regulated very carefully to ensure that only the required amount of fixed N is produced. We discuss the regulation of N2 fixation in Klebsiella pneumoniae in some detail in this chapter because a full understanding of how nitrogenase is regulated will be necessary if the transfer of N 2 fixation genes (nij') into other species, or even plants, is to be beneficial to the recipient organism. The preceding remarks about the energy requirement and oxygen stability of nitrogenase point to two of the most important problems that will be faced in transferring nijgenes to new hosts. In this review we will discuss other potential problems and show how our knowledge of the genetics of nitrogen fixation might be exploited in future.

Structural relationships among rhizobium meliloti symbiotic promoters

Symbiotic nitrogen fixation by Rhizobium meliloti requires the developmentally specific expression of certain bacterial genes. One set of these genes encodes the subunits of nitrogenase, the enzyme responsible for the reduction of atmospheric dinitrogen to ammonia, and another set consists of closely linked genes also essential for nitrogen fixation. Examination of promoter and probable regulatory regions for these gene sets has revealed extensive DNA sequence conservation for more than 160 bp upstream of the respective transcription start points. Three such promoter regions have been identified in the nitrogen fixation ( nif) gene cluster of R. meliloti strain 102F34. Using one of these promoter regions as a hybridization probe, three additional sequences were found in the genome of this strain. The DNA of other R. meliloti strains and Rhizobium species were also examined for homology to the symbiotically regulated promoters of R. meliloti 102F34. DNA sequences homologous to these R. meliloti promoters were found among diverse rhizobia, and in at least some cases were associated with nif genes.

Klebsiella pneumoniae nifA product activates the Rhizobium meliloti nitrogenase promoter.

Bacteria in the genus Rhizobium normally fix nitrogen only when they interact with leguminous plants to produce on the roots a highly differentiated structure, the nodule, within which the bacteria differentiate into nitrogen-fixing bacteroids. By contrast, the enteric bacterium Klebsiella pneumoniae reduces nitrogen in a free-living state in conditions of low oxygen tension and deficiency of fixed nitrogen. In K. pneumoniae, the overall circuitry by which nitrogen-fixation (nif) genes are regulated has been elucidated. In response to ammonia starvation, the product of the glnG gene activates transcription of the nifLA operon; this activation is dependent on the product of glnF (ref. 4). The nifA gene product is in turn required for transcription of all the other nif genes, including the nifHDK operon which codes for the subunits of nitrogenase. In contrast, very little is known about the sequence of events involved in the regulated change in rhizobial nif gene expression associated with bacteroid differentiation. In the work described here, we identify the K. pneumoniae and Rhizobium meliloti nifHDK promoters by mapping the in vivo start points of transcription. By defining and comparing the DNA sequences of these two promoters, we find that they share an unexpected degree of homology. Further, by constructing fusions of each of the two promoters to the lacZ gene from Escherichia coli, we show that both promoters are activated by the product of the K. pneumoniae nifA gene.

Isolation and physical mapping of nitrogen fixation genes from the cyanobacterium Anabaena 7120.

DNA restriction fragments containing nitrogen fixation (nifi genes of the cyanobacterium Anabaena 7120 were isolated as inserts in bacteriophage X, using plasmids containing Klebsiella pneumoniae nif genes as heterologous hybridization probes. Four contiguous E m RI fragments, comprising a 39-kilobase pair segment of the Anabaena chromosome, were physically mapped and those regions homologous to IL pneumoniae nif genes were identified by Southern hybridization and by electron microscope examination of heteroduplex DNA molecules. Regions of Anabaena DNA were found homologous to the following K. pneumoniae nif genes: nif H, the structural gene for nitrogenase reductase; nif D and nif K, the structural genes for the two subunits of nitrogenase; and either nif V or nif S, genes whose products are required for the maturation of nitrogenase. The four Anabaena nif genes so identified are transcribed in the same direction, as is the case in K. pneumoniae. However, the genes are rearranged with respect to the map of K. pneumoniae. In K. pneumoniae, nif H, nif D, and nif K are adjacent and cotranscribed. In Anabaena, nif H and nif D are adjacent but nif K is separated from nif D by 11 kilobase pairs of DNA. In K. pneumoniae, the nif V-nif S genes are to the left of the nif HDK cluster, while in Anabaena, the homologous region is to the right of the nif H gene. Hybridization of labeled subclones of Anabaena DNA to filter-bound RNA prepared from cells induced for nitrogenase indicated that most of the 11 kilobase pairs between nif K and nif D are not transcribed, while fragments containing the identified nif genes are transcribed abundantly. An additional fragment of Anabaena 7120 DNA was isolated, in recombinant X, which is apparently unlinked to the 39-kilobase pair region described above. This fragment contains a region homologous to K. pneumoniae nif H, but not to nif D. The restriction map of the nif H-homologous region of this Anabaena fragment is different from that of the nif H gene described above.