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PubMed2000

Mtsym6, a gene conditioning Sinorhizobium strain-specific nitrogen fixation in Medicago truncatula.

The availability of a wide range of independent lines for the annual medic Medicago truncatula led us to search for natural variants in the symbiotic association with Sinorhizobium meliloti. Two homozygous lines, Jemalong 6 and DZA315.16, originating from an Australian cultivar and a natural Algerian population, respectively, were inoculated with two wild-type strains of S. meliloti, RCR2011 and A145. Both plant lines formed nitrogen-fixing (effective) nodules with the RCR2011 strain. However, the A145 strain revealed a nitrogen fixation polymorphism, establishing an effective symbiosis (Nod(+)Fix(+)) with DZA315.16, whereas only small, white, non-nitrogen fixing nodules (Nod(+)Fix(-)) were elicited on Jemalong 6. Cytological studies demonstrated that these non-fixing nodules are encircled by an endodermis at late stages of development, with no visible meristem, and contain hypertrophied and autofluorescent infection threads, suggesting the induction of plant defense reactions. The non-fixing phenotype is independent of growth conditions and determined by a single recessive allele (Mtsym6), which is located on linkage group 8.

Chromosome Mapping↗

PubMed1993

Symbiotic nitrogen fixation by a nifA deletion mutant of Rhizobium meliloti: the role of an unusual ntrC allele.

In the N2-fixing alfalfa symbiont Rhizobium meliloti, the three sigma 54 (NTRA)-dependent positively acting regulatory proteins NIFA, NTRC, and DCTD are required for activation of promoters involved in N2 fixation (pnifHDKE and pfixABCX), nitrogen assimilation (pglnII), and C4-dicarboxylate transport (pdctA), respectively. Here, we describe an allele of ntrC which results in the constitutive activation of the above NTRC-, NIFA-, and DCTD-regulated promoters. The expression and activation of wild-type NTRC occur in response to nitrogen availability, whereas in cells carrying the ntrC283 allele, the NTRC283 protein appears constitutively active and is constitutively expressed. The ntrC283 allele was shown to carry a single mutation resulting in the replacement of an Asp by a Tyr residue in the helix-turn-helix motif of ntrC283. Introduction of the ntrC283 allele into a nifA deletion mutant restores the N2-fixation ability to 70 to 80% of the wild-type level. Thus, the nifA gene is dispensable for symbiotic N2 fixation.

Alleles↗

PubMed1982

Symbiotic nitrogen fixation: molecular cloning of Rhizobium genes involved in exopolysaccharide synthesis and effective nodulation.

A transposon (Tn5)-induced mutant (strain ANU437) of Rhizobium trifolii was isolated in which no water-soluble exopolysaccharide (EPS) could be detected. This mutant was also incapable of forming nitrogen-fixing root nodules on clover plants. Molecular cloning has demonstrated that the Tn5 transposon was responsible for both of these mutant phenotypes and that there is a direct correlation between EPS synthesis in this bacterial strain and its ability to carry out symbiotic nitrogen fixation. In the mutant ANU437, Tn5 was located in a 9.4-kb EcoRI fragment that was cloned into the amplifiable plasmid pBR322. The recombinant plasmid was used as a hybridization probe to isolate the corresponding wild-type DNA sequence of R. trifolii from a lambda Charon 28 genomic clone bank. This DNA sequence was subcloned into the broad host range conjugative plasmid RP4 and introduced into the Escherichia coli strain RR1. It was then transferred to the mutant ANU437 by conjugation. The acquisition of the wild-type DNA sequence by the mutant ANU437 resulted in the restoration of its ability to synthesize normal levels of EPS and to form nitrogen-fixing nodules on white and subterranean clovers.

Cloning, Molecular↗

PubMed1977

Regulation of nitrogen fixation in Rhizobium spp. Isolation of mutants of Rhizobium trifolii which induce nitrogenase activity.

This communication describes the isolation and characterization of mutants of Rhizobium trifolii which can induce nitrogenase activity in defined liquid medium. Two procedures were used for the isolation of these mutants from R. trifolii strain DT-6: (1) following chemical mutagenesis, slow growing mutants were selected which were unable to utilize NH+4 as sole source of nitrogen; (2) as spontaneous mutants resistant to the glutamate analogue L-methionine-DL-sulfoximine. Mutants (DT-71, DT-125) isolated by these procedures induced nitrogenase activity in the free-living state, whereas the parent strain lacked this property. Induction of nitrogenase activity in these mutants occurred during the late exponential phase of growth when the rate of protein synthesis was decreasing. The addition of NH+4 to a medium containing glutamate as the nitrogen-source resulted in a 50--70% reduction (repression?) of nitrogenase activity; in contrast, the rate of protein synthesis or the rate of respiration was not influenced by exogenous NH+4. Biochemical analysis showed that these mutants (strains DT-71 and DT-125) have defects in both nitrogen and carbon metabolism. The levels of glutamate synthase (both NADP+ -and NAD+ -dependent activities) and glutamate dehydrogenase (NAD+-dependent activity) were markedly lower. In addition, the mutants were found to have no detectable ribitol dehydrogenase or beta-galactosidase activity. These findings are discussed in relation to a mechanism of regulation of symbiotic nitrogen fixation.

Ammonia↗

PubMed1980

Comparison of the iron proteins from the nitrogen fixation complexes of Azotobacter vinelandii, Clostridium pasteurianum, and Klebsiella pneumoniae.

The molecular weights, amino acid compositions, amino- and carboxyl-terminal sequences, and ion-exchange peptide maps of the cysteine-containing tryptic peptides were determined for the iron proteins from the nitrogen fixation complexes of Azotobacter vinelandii (Av2) and Klebsiella pneumoniae (Kp2). Our results are compared to the known amino acid sequence of the iron protein from Clostridium pasteurianum (Cp2) [Tanaka, M., Haniu, M., Yasunobu, K. & Mortenson, L. E. (1977) J. Biol. Chem. 252, 7093-7100]. Previous studies have shown the iron proteins to have similar enzymatic functions and spectroscopic properties. Furthermore, the DNAs coding for the iron protein from many different species cross-hybridize [Ruvkun, G. B. & Ausubel, F. M. (1980) Proc. Natl. Acad. Sci. USA 77, 191-195]. Our results indicate that the protein structures are similar yet have significant differences. The amino-terminal sequences of Av2 and Kp2 are extended compared to the amino-terminal methionine of Cp2 and may indicate a different initiation site in these proteins. The aminoterminal sequences for Av2 and Kp2 are more homologous with each other than either of these are with Cp2. The carboxyl-terminal sequences are extended in Av2(14 residues) and Kp2 ( approximately 30 residues) compared to Cp2. The amino- and carboxyl-terminal sequences establish that either the structural gene sizes are different in the three organisms or extensive posttranslational modification must occur in some species. Because cysteinyl residues are involved at the active site of the iron protein, a sensitive peptide mapping technique was used to compare cysteinyl peptides of the iron protein from the three species. Av2 and Kp2 have a redistribution of cysteinyl residues when compared to Cp2. Three important differences in the cysteine distributions were found, namely, residue 4 is valine and residue 148 is alanine in Cp2, but cysteinyl residues occupy these positions in Av2, whereas residue 231 is cysteine in Cp2 but alanine in Av2. The peptide mapping technique provides a method for the investigation of selective chemical modification of cysteinyl residues.

Amino Acid Sequence↗

PubMed1996

Diversity of Nitrogen Fixation Genes in the Symbiotic Intestinal Microflora of the Termite Reticulitermes speratus.

The diversity of nitrogen-fixing organisms in the symbiotic intestinal microflora of a lower termite, Reticulitermes speratus, was investigated without culturing the resident microorganisms. Fragments of the nifH gene, which encodes the dinitrogenase reductase, were directly amplified from the DNA of the mixed microbial population in the termite gut and were clonally isolated. The phylogenetic analysis of the nifH product amino acid sequences showed that there was a remarkable diversity of nitrogenase genes in the termite gut. A large number of the termite nifH sequences were most closely related to those of a firmicute, Clostridium pasteurianum, with a few being most closely related to either the (gamma) subclass of the proteobacteria or a sequence of Desulfovibrio gigas. Some of the others were distantly related to those of the bacteria and were seemingly derived from the domain Archaea. The phylogenetic positions of these nifH sequences corresponded to those of genera found during a previous determination of rRNA-based phylogeny of the termite intestinal microbial community, of which a majority consisted of new, yet-uncultivated species. The results revealed that we have little knowledge of the organisms responsible for nitrogen fixation in termites.

Journal Article↗

PubMed2005

Metagenomic marine nitrogen fixation--feast or famine?

Natural populations of bacteria in different environments can be astonishingly diverse, as was revealed graphically by large-scale sequencing of samples of their so-called metagenomes. Among the sequence datasets from four different samples of marine bacterial metagenomes, we noted that nitrogen fixation (nif) genes were conspicuous by their absence from three of them. However, in one sample, more than one-third of the bacteria appeared to have a complement of these genes. Here, some reasons behind this site-to-site variability and their implications for how molecular methods, involving large-scale sequencing and/or functional metagenomics, can best be used to describe bacterial diversity in natural environments are discussed.

Bacteria↗

PubMed1995

Sequences of nifX, nifW, nifZ, nifB and two ORF in the Frankia nitrogen fixation gene cluster.

The actinomycete Frankia alni fixes N2 in root nodules of several non-leguminous plants. It is one of the few known N2-fixing members of the high-GC Gram+ lineage of prokaryotes. Thus, we have undertaken a study of its nitrogen fixation gene (nif) organization to compare with that of the more extensively characterized proteobacteria. A cosmid (pFN1) containing the nif region of Fa CpI1 was isolated from a cosmid library using the nifHDK genes of Fa CpI1 as a probe. A 4.5-kb BamHI fragment that mapped downstream from the previously characterized nifHDK genes was cloned and sequenced. Based on nt and aa sequence similarities to nif from other N2-fixing bacteria, eight ORF were identified and designated nifX, orf3, orf1, nifW, nifZ, nifB, orf2 and nifU. A region that hybridized to Rhizobium meliloti and Klebsiella pneumoniae nifA did not appear to contain a nifA-like gene. We have revised the map of the Fa nif region to reflect current information.

Actinomycetales↗

PubMed1976

Isolation, growth and nitrogen fixation of a gas vacuole-less mutant of the blue-green alga Anabaena aphanizomenoides.

Mutants with loss of gas vacuoles (GVL-) were isolated from a clonal population of the gas vacuole containing (GVL+) blue-green alga Anabaena aphanizomenoides with a spontaneous mutation frequency of 1.7 x 10(-3) in nitrogen-free media. Growth and nitrogen fixation of the mutant were slow when compared to the GVL+ parent. Both the parent and mutant filaments tolerated 0.02 microgram/ml of streptomycin, and higher concentrations were lethal. The reversion of the mutant towards the parent did not occur.

Cyanobacteria↗

PubMed1976

Control of synbiotic nitrogen fixation in Rhizobia. Regulation of NH4+ assimilation.

This communication is concerned with physiological, biochemical, and genetic studies of the regulation of ammonium (NH4+) assimilation by Rhizobia (root nodule bacteria) that infect leguminous plants. The major conclutions are (i) physiological studies show that Rhizobia are able to assimilate NH4+ for growth only when supplemented with certain organic nitrogen sources (e.g., L-aspartate, L-leucine, L-serine). Addition of as little as 2 mug/ml of L-aspartate supported growth on NH4+ as nitrogen source. In contrast, addition of glutamate in combination with NH4+-blocked NH4+ utilization; (ii) biochemical analysis show that glutamate synthase activity (NADP- and NAD-linked) is always present in cells capable of assimilating NH4+; also cells without glutamate synthase activity were found to be incapable of NH4+ utilization. Glutamate synthase levels were observed to fluctuate markedly depending on the available nitrogen source and on the growth stage of the culture; (iii) mutants were selected in which assimilation of NH4+ is no longer subject to inhibition (repression?) by glutamate. The levels of glutamate synthase activity (NADP-linked) (in the presence of glutamate) show approximately a two-fold increase over the level in the parent strain. The mutants no longer require supplementation with small amounts of organic nitrogen for growth in medium containing inorganic nitrogen (e.g., NH4+ or NO3-); (iv) these findings are discussed in relation to the working model of symbiotic nitrogen fixation recently proposed (O'Gara and Shanmugam (1976), Biochim. Biophys. Acta 437, 313--321).

Aspartic Acid↗

PubMed1983

Direct measurements of steady-state kinetics of cyanobacterial n(2) uptake by membrane-leak mass spectrometry and comparisons between nitrogen fixation and acetylene reduction.

A mass spectrometer with a membrane-covered inlet was used to measure nitrogen fixation by following changes in the concentration of dissolved N(2) in a stirred suspension of the cyanobacterium Anabaena variabilis in an open system. The results showed a good fit to Michaelis-Menten kinetics with a K(m) for N(2) of 65 muM at 35 degrees C, corresponding to 0.121 atmosphere of N(2). Corresponding values for the K(m) for acetylene reduction were 385 muM (0.011 atmosphere at 35 degrees C). Comparison of the values of V(max) for N(2) uptake with those for the acetylene reduction assay under similar conditions gave an average value of 3.8 for the conversion factor between N(2) and C(2)H(2) reduction. Reduction of protons to hydrogen was completely inhibited at sufficiently high concentrations of C(2)H(2), but even at saturating N(2) concentrations, 1 mol of H(2) was produced for every mole of N(2) reduced. This explains the finding that the observed C(2)H(2)/N(2) ratio is higher than the value of 3 expected from the requirement for two electrons for acetylene reduction and six for nitrogen reduction. The results correlate well with a mechanism for N(2) reduction involving the equation: N(2) + 8H + 8e --> 2NH(3) + H(2) which gives a conversion factor between C(2)H(2) and N(2) of 4. It is proposed that, in general, 4 is a more appropriate value than 3 for the conversion factor.

Journal Article↗

PubMed2002

The fixM flavoprotein modulates inhibition by AICAR or 5'AMP of respiratory and nitrogen fixation gene expression in Sinorhizobium meliloti.

AICAR, a purine-related metabolite, was recently shown to inhibit respiratory and nifA gene expression in Sino-rhizobium meliloti. Here, we demonstrate that AICAR has essentially no or little effect in a wild-type S. meliloti strain and inhibits respiratory and nitrogen fixation gene expression only in specific mutant backgrounds. We have analyzed in detail a mutant in which addition of AICAR inhibited fixK,fixN,fixT and nifA expression. The corresponding gene,fixM, is located just downstream of fixK1 on pSymA megaplasmid and encodes a flavoprotein oxidoreductase. 5'AMP, a structural analogue of AICAR, mimicked AICAR effect as well as the nucleoside precursors AICAriboside and adenosine. The mode of action of AICAR and 5'AMP in vivo was investigated. We demonstrate that AICAR does not affect FixK transcriptional activity and instead regulates fixK and nifA gene expression. We hypothesize that AICAR and 5'AMP may modulate, possibly indirectly, the activity of the FixLJ two-component regulatory system. The possible physiological roles of AICAR, 5'AMP, and fixM in the context of symbiosis are discussed.

Adenosine Monophosphate↗

PubMed1984

Short-term ammonium inhibition of nitrogen fixation in Azotobacter.

Addition of NH4Cl at low concentrations to Azotobacter chroococcum cells caused an immediate cessation of nitrogenase activity, which was recovered once the added NH+4 was exhausted from the medium. In the presence of inhibitors of ammonium assimilation, such as L-methionine-DL-sulfoximine, L-methionine sulfone or 6-diazo-5-oxo-L-norleucine, externally added NH+4 had no effect on nitrogenase activity and the newly-fixed nitrogen was excreted into the medium as NH+4. It is concluded that, in A. chroococcum, NH+4 must be assimilated to exert its short-term inhibitory effect on nitrogen fixation.

Ammonium Chloride↗

PubMed1998

Evidence for a regulatory link of nitrogen fixation and photosynthesis in Rhodobacter capsulatus via HvrA.

A Rhodobacter capsulatus reporter strain, carrying a constitutively expressed nifA gene and a nifH-lacZ gene fusion, was used for random transposon Tn5 mutagenesis to search for genes required for the NtrC-independent ammonium repression of NifA activity. A mutation in hvrA, which is known to be involved in low-light activation of the photosynthetic apparatus, released both ammonium and oxygen control of nifH expression in this reporter strain, demonstrating a regulatory link of nitrogen fixation and photosynthesis via HvrA. In addition, a significant increase in bacteriochlorophyll alpha (BChl alpha) content was found in cells under nitrogen-fixing conditions. HvrA was not involved in this up-regulation of BChl alpha. Instead, the presence of active nitrogenase seemed to be sufficient for this process, since no increase in BChl alpha content was observed in different nif mutants.

Bacterial Proteins↗

PubMed1988

Introduction of the Escherichia coli gdhA gene into Rhizobium phaseoli: effect on nitrogen fixation.

Rhizobium phaseoli lacks glutamate dehydrogenase (GDH) and assimilates ammonium by the glutamine synthetase-glutamate synthase pathway. A strain of R. phaseoli harboring the Escherichia coli GDH structural gene (gdhA) was constructed. GDH activity was expressed in R. phaseoli in the free-living state and in symbiosis. Nodules with bacteroids that expressed GDH activity had severe impairment of nitrogen fixation. Also, R. phaseoli cells that lost GDH activity and assimilated ammonium by the glutamine synthetase-glutamate synthase pathway preferentially nodulated Phaseolus vulgaris.

Cloning, Molecular↗

PubMed1980

Complementation analysis and characterization of the nitrogen fixation genes, nifH, nifC and nifJ in Klebsiella pneumoniae.

Complementation tests were performed with various nif mutations for delineating the nif genes. The nif-/nif- heterogenotes were constructed by transferring nif plasmid mutant PRD-nif- from E. coli Jc5466 to various Klebsiella pneumoniae nif- recipients. In addition to those nif genes previously reported elsewhere, a new essential gene for nitrogen fixation, nifC was identified. According to the P1-transduction and three factor reciprocal crosses, nifC was tentatively mapped between nifH and nifJ in the chromsome. The order of nif genes obtained was hisD, nifQ, nifB, nifA, nifL, nifF nifM, nifV, nifS, nifU, nifN, nifE, nifK, nifD, nifH, nifC and nifJ. The examination of the biochemical phenotypes of the nif genes suggests that nifC may be concerned with the synthesis or activation of the iron molybdenum cofactor nitrogenase, nifH, besides coding for the structure of nitrogen reductase may also exert a function on the synthesis of nitrogenase, and nifJ presumably is required to turn on the expression of nifK (D), nifH or nifF.

Chromosome Mapping↗

PubMed1995

Characterization of the cycHJKL genes involved in cytochrome c biogenesis and symbiotic nitrogen fixation in Rhizobium leguminosarum.

Mutants of Rhizobium leguminosarum bv. viciae unable to respire via the cytochrome aa3 pathway were identified by the inability to oxidize N,N'-dimethyl-p-phenylenediamine. Two mutants which were complemented by cosmid pIJ1942 from an R. leguminosarum clone bank were identified. Although pea nodules induced by these mutants contained many bacteroids, no symbiotic nitrogen fixation was detected. Heme staining of cellular proteins revealed that all cytochrome c-type heme proteins were absent. These mutants lacked spectroscopically detectable cytochrome c, but cytochromes aa3 and d were present, the latter at a higher-than-normal level. DNA sequence analysis of complementing plasmids revealed four apparently cotranscribed open reading frames (cycH, cycJ, cycK, and cycL). CycH, CycJ, CycK, and CycL are homologous to Bradyrhizobium japonicum and Rhizobium meliloti proteins thought to be involved in the attachment of heme to cytochrome c apoproteins; CycK and CycL are also homologous to the Rhodobacter capsulatus ccl1 and ccl2 gene products and the Escherichia coli nrfE and nrfF gene products involved in the assembly of c-type cytochromes. The absence of cytochrome c heme proteins in these R. leguminosarum mutants is consistent with the view that the cycHJKL operon could be involved in the attachment of heme to apocytochrome c.

Amino Acid Sequence↗

PubMed1996

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.

Bacterial Proteins↗