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PubMed1989

The roles of the nifW, nifZ and nifM genes of Klebsiella pneumoniae in nitrogenase biosynthesis.

Active Fe protein of nitrogenase was synthesised in a non-nitrogen fixing organism when Escherichia coli was transformed with a plasmid encoding only two nif-specific genes, nifH and nifM of Klebsiella pneumoniae. Hence proteins NifH and NifM are sufficient to produce active Fe protein in E. coli. K. pneumoniae strains carrying chromosomal nifW- and nifZ- mutations were constructed and shown to be significant C2H2-reducing activity and to grow on N-free plates. Nevertheless, derepressing cultures of the mutant strains had reduced levels of MoFe protein activity, and consequently significantly lower levels of nitrogenase activity, than the nif+ parent strain. NifW and NifZ therefore appear to be involved in the formation or accumulation of active MoFe protein, but are not essential for nitrogen fixation in K. pneumoniae under the conditions tested.

Enzyme Repression↗

PubMed1994

Nodulating ability of Rhizobium tropici is conditioned by a plasmid-encoded citrate synthase.

Rhizobium species elicit the formation of nitrogen-fixing root nodules through a complex interaction between bacteria and plants. Various bacterial genes involved in the nodulation and nitrogen-fixation processes have been described and most have been localized on the symbiotic plasmids (pSym). We have found a gene encoding citrate synthase on the pSym plasmid of Rhizobium tropici, a species that forms nitrogen-fixing nodules on the roots of beans (Phaseolus vulgaris) and trees (Leucaena spp.). Citrate synthase is a key metabolic enzyme that incorporates carbon into the tricarboxylic acid cycle by catalysing the condensation of acetyl-CoA and oxaloacetic acid to form citrate. R. tropici pcsA (the plasmid citrate synthase gene) is closely related to the corresponding genes of Proteobacteria. pcsA inactivation by a Tn5-mob insertion causes the bacteria to form fewer nodules (30-50% of the original strain) and to have a decreased citrate synthase activity in minimal medium with sucrose. A clone carrying the pcsA gene complemented all the phenotypic alterations of the pcsA mutant, and conferred Rhizobium leguminosarum bv. phaseoli (which naturally lacks a plasmid citrate synthase gene) a higher nodulation and growth capacity in correlation with a higher citrate synthase activity. We have also found that pcsA gene expression is sensitive to iron availability, suggesting a possible role of pcsA in iron uptake.

Agrobacterium tumefaciens↗

PubMed1987

The nifA gene of Rhizobium meliloti is oxygen regulated.

Experiments using plasmid-borne gene fusions and direct RNA measurements have revealed that expression from the nifA gene is induced in Rhizobium meliloti when the external oxygen concentration is reduced to microaerobic levels. Induction occurs in the absence of alfalfa and in the presence of fixed nitrogen and does not require ntrC. The production of functional nifA gene product (NifA) can be demonstrated by its ability to activate the nitrogenase promoter P1. Aerobic induction of nifA can also occur during nitrogen starvation at low pH, but in this case induction is dependent on ntrC and does not lead to P1 activation. The data indicate that reduced oxygen tension is potentially a major trigger for symbiotic activation of nitrogen fixation in Rhizobium species.

Energy Metabolism↗

PubMed1978

Nitrogen-fixing communities in an intertidal ecosystem.

Nitrogen gixation (acetylene reduction) associated with various communities in the intertidal sand and mud flat was measured in situ. Areas which were colonized by algae, plants, and animals generally had significantly higher activities than areas which were visibly uncolonized. The highest activities were measured at sites colonized by a mixed bloom of Oscillatoria-Euglena-photosynthetic bacteria. These areas occupied only about 1% of the surface of the sediments, yet contributed nearly 50% of the biologically fixed nitrogen. Enteromorpha communities also exhibited relatively high activities. Sites were grouped according to the statistical significance of the mean of their activities: group I: uncolonized sand, mud and gravel flats, Eelgrass (Zostera) communities, and snail (amphibola) beds, with an estimated contribution of 1 g N ha-1 day-1; group II: Cockel (Chione) beds, 3 g N ha-1 day-1; group III: Salicornia, Ulva, Juncus, and Enteromorpha, 10 g N ha-1 day-1; and group IV: Oscillatoria-Euglena-photosynthetic bacteria association, 200 g N ha-1 day-1. Enteromorpha and Oscillatoria-Euglena-photosynthetic bacteria communities showed significantly lower rates of nitrogen fixation in the dark than in the light.

Acetylene↗

PubMed2005

Genome-directed isolation of the key nitrogen fixer Leptospirillum ferrodiazotrophum sp. nov. from an acidophilic microbial community.

Analysis of assembled random shotgun sequence data from a low-diversity, subsurface acid mine drainage (AMD) biofilm revealed a single nif operon. This was found on a genome fragment belonging to a member of Leptospirillum group III, a lineage in the Nitrospirae phylum with no cultivated representatives. Based on the prediction that this organism is solely responsible for nitrogen fixation in the community, we pursued a selective isolation strategy to obtain the organism in pure culture. An AMD biofilm sample naturally abundant in Leptospirillum group III cells was homogenized, filtered, and serially diluted into a nitrogen-free liquid medium. The resulting culture in the terminal dilution grew autotrophically to a maximum cell density of approximately 10(6) cells/ml, oxidizing ferrous iron as the sole energy source. 16S rRNA-internal transcribed spacer region clone library analysis confirmed that the isolate is a member of Leptospirillum group III and that the culture is axenic. We propose the name Leptospirillum ferrodiazotrophum sp. nov. for this iron-oxidizing, free-living diazotroph. This study highlights how environmental sequence data can provide insights for culturing previously uncultured microorganisms.

Bacteria↗

PubMed2006

Halomonas maura is a physiologically versatile bacterium of both ecological and biotechnological interest.

Halomonas maura is a bacterium of great metabolic versatility. We summarise in this work some of the properties that make it a very interesting microorganism both from an ecological and biotechnological point of view. It plays an active role in the nitrogen cycle, is capable of anaerobic respiration in the presence of nitrate and has recently been identified as a diazotrophic bacterium. Of equal interest is mauran, the exopolysaccharide produced by H. maura, which contributes to the formation of biofilms and thus affords the bacterium advantages in the colonisation of its saline niches. Mauran is highly viscous, shows thixotropic and pseudoplastic behaviour, has the capacity to capture heavy metals and exerts a certain immunomodulator effect in medicine. All these attributes have prompted us to make further investigations into its molecular characteristics. To date we have described 15 open reading frames (ORF's) related to exopolysaccharide production, nitrogen fixation and nitrate reductase activity among others.

Biotechnology↗

PubMed1971

Poly- -hydroxybutyrate biosynthesis and the regulation of glucose metabolism in Azotobacter beijerinckii.

Azotobacter beijerinckii possesses the enzymes of both the Entner-Doudoroff and the oxidative pentose phosphate cycle pathways of glucose catabolism and both pathways are subject to feedback inhibition by products of glucose oxidation. The allosteric glucose 6-phosphate dehydrogenase utilizes both NADP(+) and NAD(+) as electron acceptors and is inhibited by ATP, ADP, NADH and NADPH. 6-Phosphogluconate dehydrogenase (NADP-specific) is unaffected by adenosine nucleotides but is strongly inhibited by NADH and NADPH. The formation of pyruvate and glyceraldehyde 3-phosphate from 6-phosphogluconate by the action of the Entner-Doudoroff enzymes is inhibited by ATP, citrate, isocitrate and cis-aconitate. Glyceraldehyde 3-phosphate dehydrogenase is unaffected by adenosine and nicotinamide nucleotides but the enzyme is non-specific with respect to NADP and NAD. Citrate synthase is strongly inhibited by NADH and the inhibition is reversed by the addition of AMP. Isocitrate dehydrogenase, a highly active NADP-specific enzyme, is inhibited by NADPH, NADH, ATP and by high concentrations of NADP(+). These findings are discussed in relation to the massive synthesis of poly-beta-hydroxybutyrate that occurs under certain nutritional conditions. We propose that synthesis of this reserve material, to the extent of 70% of the dry weight of the organism, serves as an electron and carbon ;sink' when conditions prevail that would otherwise inhibit nitrogen fixation and growth.

Aconitum↗

PubMed1990

Analysis of three nodD genes in Rhizobium leguminosarum biovar phaseoli; nodD1 is preceded by noIE, a gene whose product is secreted from the cytoplasm.

In a strain of Rhizobium leguminosarum biovar phaseoli, three copies of the regulatory nodulation gene nodD were identified on the Sym plasmid and sequenced. Two were closely linked to each other and the third was near, but not adjacent, to the nodABC genes. Each of these nodD genes could correct the Nod- defect of a nodD mutant strain of R. leguminosarum biovar viciae on peas. A truncated form of nodD2 could also correct this mutant, indicating that the C-terminus of NodD2 is not needed for inducing activity. Upstream of nodD1 and in the same operon is a newly described gene, noIE, whose product appears to be exported into the periplasm. Close to nodD2 is another gene, noIP, with no known counterpart in other rhizobia. Both noIP and noIE-nodD1 are preceded by 'nod-box' sequences and, in the former case, there appear to be two tandemly repeated nod-box sequences. Mutations in each of the nodD genes and in the noIE and noIP genes did not abolish nodulation or nitrogen fixation on beans.

Amino Acid Sequence↗

PubMed1994

Characterization of the genome region encoding an fdxH-type ferredoxin and a new 2[4Fe-4S] ferredoxin from the nonheterocystous, nitrogen-fixing cyanobacterium Plectonema boryanum PCC 73110.

A genomic DNA region with four consecutive open reading frames, including an fdxH-type gene, has been sequenced and initially characterized for the nonheterocystous nitrogen-fixing cyanobacterium Plectonema boryanum PCC 73110. The fdxH gene encodes a [2Fe-2S]-type ferredoxin, 98 amino acids in length, with a deduced molecular mass of 10.9 kDa. Conserved residues include two characteristic lysines at positions 10 and 11, shown recently to be important for interaction with nitrogenase reductase (S. Schmitz, B. Schrautermeier, and H. Böhme, Mol. Gen. Genet. 240:455-460, 1993). The gene is transcribed only under anaerobic nitrogenase-inducing conditions, whereas the Plectonema petF gene, encoding a different (type 1) [2Fe-2S] ferredoxin, is only transcribed in cultures growing with combined nitrogen. The fdxH gene was expressed in Escherichia coli as a holoprotein. The purified protein was able to effectively donate electrons to cyanobacterial nitrogenase, whereas PetF from the same organism was not. The occurrence of FdxH in the nonheterocystous genus Plectonema demonstrates for the first time that FdxH-type ferredoxins are not exclusively expressed within heterocysts, as is true for cyanobacteria differentiating these cells for nitrogen fixation under aerobic growth conditions. Two open reading frames that precede fdxH have high similarity to those found at a corresponding location in Anabaena sp. strain PCC 7120. In the latter organism, they are transcribed only under nitrogen-fixing conditions, but the functions of their gene products remain unclear (D. Borthakur, M. Basche, W. J. Buikema, P. B. Borthakur, and R. Haselkorn, Mol. Gen. Genet. 221:227-234, 1990). An fdxB-type gene encoding a 2[4Fe-4S] ferredoxin not previously identified in cyanobacteria is located immediately downstream of fdxH in P. boryanum.

Amino Acid Sequence↗

PubMed1993

Purification and properties of a bacterial-type ferredoxin from the nitrogen-fixing cyanobacterium Anabaena variabilis ATCC29413.

Three soluble ferredoxins were purified to homogeneity from nitrogen-fixing cultures of Anabaena variabilis (ATCC 29413) and characterized. The purified proteins have different absorption spectra, molecular mass, iron content, amino-acid composition and resistance to O2 inactivation. Two were plant-type ferredoxins FdI and FdxH, corresponding to the previously reported ferredoxins II and I (Böhme, H. and Schrautemeier, B. (1987) Biochim. Biophys. Acta 891, 1-7). The third ferredoxin (ferredoxin III) (previously not described in cyanobacteria) was a bacterial-type ferredoxin. Ferredoxin III has a molecular mass of about 6 kDa and contains 3-4 atoms Fe/mol. Native (oxidized) ferredoxin III shows an EPR-signal at g = 2.014 that disappears after reduction by dithionite, characteristic of ferredoxins containing three-iron clusters. Ferredoxin III, like ferredoxin FdxH, is inactivated by oxygen. Ferredoxin III supports higher rates of C2H2 reduction by Rhodobacter capsulatus nitrogenase than FdI and higher rates of H2 evolution by clostridial hydrogenase than FdI and FdxH. Combined nitrogen suppresses the synthesis of both nitrogenase and ferredoxin III. These data suggest a possible role of ferredoxin III (bacterial-type) in nitrogen fixation by A. variabilis.

Amino Acids↗

PubMed1999

Identification of nodulation promoter (nod-box) regions of Rhizobium galegae.

A hybridisation analysis of a genomic clone library of Rhizobium galegae HAMBI 1174 located four EcoRI fragments homologous to the nod-box promoter sequence of Sinorhizobium meliloti in two separate gene regions. Two of the five nod-boxes detected in the R. galegae genome were carried on a single cosmid clone, pRg30, upstream from the nodABCIJ and nodF genes, whereas the other three nod-boxes were carried on a different cosmid clone, pRg10. Hybridisations with various nod gene probes from S. meliloti and Rhizobium leguminosarum species detected a nodD homolog in pRg10. The sequence data obtained from regions adjacent to each nod-box in pRg10 confirmed the presence of a second nodD in the R. galegae genome and, in addition, revealed the presence of nodN, nodU, dctA nifH and nifQ-like genes in pRg10. Thus, by using a promoter-specific nod-box probe we could identify a new region carrying genes involved in nitrogen fixation and host specificity functions.

Base Sequence↗

PubMed2002

Role of GlnK in NifL-mediated regulation of NifA activity in Azotobacter vinelandii.

In several diazotrophic species of Proteobacteria, P(II) signal transduction proteins have been implicated in the regulation of nitrogen fixation in response to NH(4)(+) by several mechanisms. In Azotobacter vinelandii, expression of nifA, encoding the nif-specific activator, is constitutive, and thus, regulation of NifA activity by the flavoprotein NifL appears to be the primary level of nitrogen control. In vitro and genetic evidence suggests that the nitrogen response involves the P(II)-like GlnK protein and GlnD (uridylyltransferase/uridylyl-removing enzyme), which reversibly uridylylates GlnK in response to nitrogen limitation. Here, the roles of GlnK and GlnK-UMP in A. vinelandii were studied to determine whether the Nif (-) phenotype of glnD strains was due to an inability to modify GlnK, an effort previously hampered because glnK is an essential gene in this organism. A glnKY51F mutation, encoding an unuridylylatable form of the protein, was stable only in a strain in which glutamine synthetase activity is not inhibited by NH(4)(+), suggesting that GlnK-UMP is required to signal adenylyltransferase/adenylyl-removing enzyme-mediated deadenylylation. glnKY51F strains were significantly impaired for diazotrophic growth and expression of a nifH-lacZ fusion. NifL interacted with GlnK and GlnKY51F in a yeast two-hybrid system. Together, these data are consistent with those obtained from in vitro experiments (Little et al., EMBO J., 19:6041-6050, 2000) and support a model for regulation of NifA activity in which unmodified GlnK stimulates NifL inhibition and uridylylation of GlnK in response to nitrogen limitation prevents this function. This model is distinct from one proposed for the related bacterium Klebsiella pneumoniae, in which unmodified GlnK relieves NifL inhibition instead of stimulating it.

Adenosine Monophosphate↗

PubMed2003

Benefit to N2-fixing alder of extending growth period at the cost of leaf nitrogen loss without resorption.

This study examines the adaptive role of not resorbing N in N(2)-fixing deciduous trees in terms of their energy balance. The autumnal growth of N(2)-fixing Alnus firma Sieb. et Zucc. (alder) was compared with that of the non-N(2)-fixing Morus bombycis Koizumi (mulberry), which resorbs leaf N. The freezing resistance of leaves of both species was -2 degrees C. Mulberry seedlings lost their photosynthetic ability in mid-October, although the minimum temperature was still above 0 degrees C. Thereafter, their leaves turned yellow and were gradually shed. In contrast, seedlings of the alder maintained their photosynthetic ability until mid-November, when the minimum temperature fell to the freezing resistance limit. Thereafter, their leaves were shed quickly without an autumn tint. The mulberry resorbed 48.9% of leaf N, whereas the alder resorbed hardly any. These results show that, compared with the mulberry tree, the alder extended its growth period for 1 month in return for losing leaf N without resorption. The amount of energy assimilated by the alder in the extended growth period was about six times that required for compensating for the nitrogen loss, if the compensation is dependent only on the tree's own nitrogen fixation. This surplus energy balance has probably allowed N(2)-fixing deciduous trees to evolve their non-N-resorbing trait.

Adaptation, Physiological↗

PubMed1980

Presence of a cytochrome b-containing ferritin in Azotobacter vinelandii.

It has been demonstrated that a cytochrome b-containing ferritin is present in Azotobacter vinelandii. After DEAE cellulose chromatography and purification fractional precipitation by 50% of the saturated ammonium sulfate of the extract prepared from A. vinelandii cells, a hexagonal crystalline preparation is obtained. The protein contains 4--6% of nonheme iron. The protein molecule is made up of an electron dense iron core with a diameter of 70A and a protein shell with a diameter of 120A. The Fe core can be removed from the shell by the treatment with chelating and reducing agents. Electron micrographs and absorption spectra reveal that the protein shells are very similar before and after the removal of the core. The electrophoretic mobility and immunological properties of the Fe-free protein against the antibody of ferritin are very similar to those of the protein before the removal of the iron. From the above characteristics, it can be inferred that the protein belongs to ferritin. The protein part contains protoheme as prosthetic group and so it belongs to cytochrome b. Hence, the protein prepared from A. vinelandii is a kind of cytochrome b-containing ferritins. The possible role of the ferritin in biological nitrogen fixation is discussed in this paper.

Apoferritins↗

PubMed1983

N(2) Fixation by Azospirillum brasilense and Its Incorporation into Host Setaria italica.

Growth and nitrogen fixation were followed during the life cycle of Setaria italica (foxtail millet) inoculated with Azospirillum brasilense in controlled-environment growth chambers. The plants were fertilized at seeding with a limiting amount of combined nitrogen and maintained with an N-free mineral solution. During maturation of the plants, substantial nitrogenase activity, measured by acetylene reduction, developed in the rhizosphere, with total fixation estimated to be equivalent to 20% of the N in the inoculated plants. The peak of this activity coincided with depletion of soluble nitrogen from the system, which in turn was reflected by a sharp decrease in the nitrate reductase activity of the leaves. A. brasilense was found in association with the root populations at 8 x 10 cells per gram of dry weight. An increase in shoot growth occurred at this time, but no significant increase in total plant nitrogen could be demonstrated. N(2) enrichment experiments confirmed that fixation was occurring, but only about 5% of the nitrogen fixed by A. brasilense was incorporated into the plants within 3 weeks. There was thus no evidence of direct bacterium-to-plant transport of fixed nitrogen, but rather a slow transfer suggesting the gradual death of bacteria and subsequent mineralization of their nitrogen, at least under growth-room conditions.

Journal Article↗

PubMed2002

The soybean GmN6L gene encodes a late nodulin expressed in the infected zone of nitrogen-fixing nodules.

Previously, we determined the N-terminal amino acid sequences of a number of putative peribacteroid membrane proteins from soybean. Here, we report the cloning of a gene, GmN6L, that encodes one of these proteins. The protein encoded by GmN6L is similar in sequence to MtN6, an early nodulin expressed in Medicago truncatula roots in response to infection by Sinorhizobium meliloti. The GmN6L gene was strongly expressed in mature nodules but not in other plant organs. GmN6L protein was first detected 2 weeks after inoculation with Bradyrhizobium japonicum and was limited to the infected zone of nodules. GmN6L protein was found in symbiosomes isolated from mature soybean nodules, both as a soluble protein and as a peripheral membrane protein bound to the peribacteroid membrane. These data indicate that GmN6L is a late nodulin, which is not involved in the infection process. Homology between GmN6L and FluG, a protein involved in signaling in Aspergillus nidulans, suggests that GmN6L may play a role in communication between the host and microsymbionts during symbiotic nitrogen fixation.

Amino Acid Sequence↗

PubMed1981

Effects of aerobiosis and nitrogen source on the proton motive force in growing Escherichia coli and Klebsiella pneumoniae cells.

The electrochemical gradient of hydrogen ions, or proton motive force (PMF), was measured in growing Escherichia coli and Klebsiella pneumoniae in batch culture. The electrical component of the PMF (delta psi) and the chemical component (delta pH) were calculated from the cellular accumulation of radiolabeled tetraphenylphosphonium, thiocyanate, and benzoate ions. In both species, the PMF was constant during exponential phase and decreased as the cells entered stationary phase. Altering the growth rate with different energy substrates had no effect on the PMF. The delta pH (alkaline inside) varied with the pH of the culture medium, resulting in a constant internal pH. During aerobic growth in media at pH 6 to 7, the delta psi was constant at 160 mV (negative inside). The PMF, therefore, was 255 mV in cells growing at pH 6.3, and decreased progressively to 210 mV in pH 7.1 cultures. K. pneumoniae cells and two E. coli strains (K-12 and ML), including a mutant deficient in the H+-translocating ATPase and a pleiotropically energy-uncoupled mutant with a normal ATPase, had the same PMF during aerobic exponential phase. During anaerobic growth, however, both species had delta psi values equal to 0. Therefore, the PMF in anaerobic cells consisted only of the delta pH component, which was 75 mV or less in cells growing at pH 6.2 or greater. These data thus met the expectation that cells deriving metabolic energy from respiration have a PMF above a threshold value of about 200 mV when the ATPase functions in the direction of H+ influx and ATP synthesis; in fermenting cells, a PMF below a threshold value was expected since the enzyme functions in the direction of H+ extrusion and ATP hydrolysis. K. pneumoniae cells growing anaerobically had no delta psi whether the N source added was N2, NH+4 or one of several amino acids; the delta pH was unaffected. Therefore, any energy cost incurred by the process of nitrogen fixation could not be detected as an alteration of the proton gradient.

Aerobiosis↗

PubMed2002

Newly identified cytochrome c oxidase operon in the nitrogen-fixing cyanobacterium Anabaena sp. strain PCC 7120 specifically induced in heterocysts.

Two operons have been cloned from Anabaena sp. strain PCC 7120 DNA, each of which encodes the three core subunits of distinct mitochondrial-type cytochrome c oxidases. The two operons are only 72 to 85% similar to one another at the nucleotide level in the most conserved subunit. One of these, coxBACII, is induced >20-fold in the middle to late stages of heterocyst differentiation. Analysis of green fluorescent protein reporters indicates that this operon is expressed specifically in proheterocysts and heterocysts. The other operon, coxBACI, is induced only 2.5-fold following nitrogen step-down and is expressed in all cells. Surprisingly, a disruption mutant of coxAII, the gene encoding subunit I of the heterocyst-specific oxidase, grows normally in the absence of combined nitrogen. It is likely that coxBACI and/or two other putative terminal oxidases present in the Anabaena sp. strain PCC 7120 genome are able to compensate for the loss of the heterocyst-specific oxidase in providing ATP for nitrogen fixation and maintaining a low oxygen level in heterocysts.

Anabaena↗