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PubMed1981

Biological nitrogen fixation: primary structure of the Klebsiella pneumoniae nifH and nifD genes.

A DNA fragment carrying the Klebsiella pneumoniae nifK, D, and H genes was isolated from the nif- strain UNF841 (Tn5::nifK) by molecular cloning into the Escherichia coli plasmid pBR325. The nucleotide sequences of both the nifH gene, which encodes the Fe protein of the nitrogenase enzyme complex, and 622 nucleotides of the nifD gene, which encodes the alpha-subunit of the Mo-Fe protein, were determined by direct DNA sequencing by both the chemical and chain termination methods. A comparison of the primary structure of the Klebsiella nifH gene and its product with that recently determined for the blue-green alga Anabaena demonstrates that the gene sequences are more divergent than the protein sequence data would suggest. This implies that despite the strong, presumably functional, constraints that act at the protein structure level, the nucleotide sequence of the gene and its mRNA are only restrained by the coding requirements, allowing substantial drift in codon usage.

Amino Acid Sequence↗

PubMed1978

Fine-structure mapping and complementation analysis of nif (nitrogen fixation) genes in Klebsiella pneumoniae.

Four hundred and eighty-nine independent Nif- strains containing 260 point, 130 millimicron-induced, and 99 deletion mutations in nif in the Klebsiella pneumoniae chromosome were isolated. Three hundred and ninety insertion and point mutations were mapped with millimicron-induced deletions carried on 44 plasmids derived from pTM4010, a recombinant R factor containing the his-nif region of K. pneumoniae. The 99 chromosomal deletions in the nif region were mapped with 69 derivatives of pTM4010 carrying insertion and point mutations in nif. Complementation analysis between 84 derivatives of pTM4010 carrying nif mutations and Rec- derivatives of the 390 Nif- mutants identified 14 genes. The nif mutations were ordered into 49 deletion groups with a gene order of his...nifQBALFMVSNEKDHJ. Complementation analysis of millimicron-induced, amber, frameshift, and deletion mutations indicates there are five polycistronic and two monocistronic operons: nifQ nifB, nifA nifL, nifF, nifM nifV nifS, nifN nifE, nifK nifD nifH, and nifJ. Transcription is from right to left in all polycistronic operons.

Chromosome Mapping↗

PubMed1979

Effect of cyclic guanosine 3',5'-monophosphate on nitrogen fixation in Rhizobium japonicum.

The addition of exogenous cyclic guanosine 3',5'-monophosphate (cGMP) at a concentration of 0.1 mM to a free-living culture of Rhizobium japonicum 3I1b110 was found to completely inhibit the expression of nitrogenase activity and markedly inhibit the expression of hydrogenase and nitrate reductase activities. The effect was specific for cGMP. Experiments on the in vivo incorporation of radioactive methionine and subsequent analysis of the labeled proteins on polyacrylamide gels showed that the biosynthesis of nitrogenase polypeptides was inhibited. It appears that the time of addition of cGMP is important since the effect was only seen during the early stages of nif gene expression. The intracellular level of cGMP was found to respond to physiological changes in the cell, and there was a fall in cGMP concentrations when nitrogenase was induced. Microaerophilic-aerobic shift experiments showed that intracellular levels increased from 0.25 pmol/mg of cell protein under microaerophilic conditions to 2.6 pmol/mg of cell protein under aerobic conditions, suggesting that the cellular pool size of cGMP may be under redox control.

Cyclic GMP↗

PubMed2000

Isolation and characterization of nifDK::kanamycin and nitrogen fixation proficient Azotobacter vinelandii strain, and its implication on the status of multiple chromosomes in Azotobacter.

Several lines of experimental analyses on the ploidy status of Azotobacter vinelandii genome lead to the conclusion that it contains more than 40 copies of its chromosome and therefore it is a polyploid organism. The genetic evidence argues against the existence of polyploidy in these cells since the segregation pattern of genetic markers under lack of selection pressure mimic that of haploids. However, when A. vinelandii was made Nif- by inserting a kanamycin resistance marker gene in the nifDK sequence and the cells were selected for kanamycin resistance and Nif+ phenotype, we were able to score colonies that are both kanamycin resistant and Nif+. Therefore, when the cells were subjected to forced double selection of the same locus, they behaved as if they carried at least two chromosomes, one carrying the kanamycin resistance marker in the nifDK genes and the other carrying the intact nifDK genes. These analyses suggested that at least a diploidy status can be induced in these cells under selection pressure.

Azotobacter vinelandii↗

PubMed1985

Expression of nitrogen fixation genes in foreign hosts. Assembly of nitrogenase Fe protein in Escherichia coli and in yeast.

In Klebsiella pneumoniae, the nifH gene encodes the Fe protein (Kp2) polypeptide that is assembled into a homodimer responsible for the reduction of nitrogenase. Escherichia coli or the yeast Saccharomyces cerevisiae, transformed with the K. pneumoniae nifH gene in suitable expression vectors, synthesize the Fe protein polypeptide. This study examines the assembly of the nifH gene product into its characteristic dimeric structure in E. coli and in yeast. Immunoblotting methods, as well as 55Fe2- labeling of K. pneumoniae were employed to detect native nitrogenase components in cell lysates. E. coli and yeast transformants contained a protein similar to native Kp2 in its immunoreactivity, apparent molecular weight, and lability in the presence of oxygen or MgATP. While in E. coli the co-introduction of nifH and nifM resulted in enhanced levels of the nifH product, it appears that the nifH gene product alone is sufficient for the assembly of an Fe protein-like structure in foreign prokaryotic and eukaryotic hosts.

Adenosine Triphosphate↗

PubMed1992

Posttranslational modification of Klebsiella pneumoniae flavodoxin by covalent attachment of coenzyme A, shown by 31P NMR and electrospray mass spectrometry, prevents electron transfer from the nifJ protein to nitrogenase. A possible new regulatory mechanism for biological nitrogen fixation.

A strain of Escherichia coli (71-18) that produces ca. 15% of its soluble cytoplasmic protein as a flavodoxin, the Klebsiella pneumoniae nifF gene product, has been constructed. The flavodoxin was purified using FPLC and resolved into two forms, designated KpFldI and KpFldII, which were shown to have identical N-terminal amino acid sequences (30 residues) in agreement with that predicted by the K. pneumoniae nifF DNA sequence. 31P NMR, electrospray mass spectrometry, UV-visible spectra, and thiol group estimations showed that the single cysteine residue (position 68) of KpFldI is posttranslationally modified in KpFldII by the covalent, mixed disulfide, attachment of coenzyme A. KpFldII was inactive as an electron carrier between the K. pneumoniae nifJ product (a pyruvate-flavodoxin oxidoreductase) and K. pneumoniae nifH product (the Fe-protein of nitrogenase). This novel posttranslational modification of a flavodoxin is discussed in terms of the regulation of nitrogenase activity in vivo in response to the level of dissolved O2 and the carbon status of diazotrophic cultures.

Amino Acid Sequence↗

PubMed1981

Effects of Temperature, Nitrogen Fertilization, and Plant Age on Nitrogen Fixation by Setaria italica Inoculated with Azospirillum brasilense (strain cd).

The association between the nitrogen-fixing bacterium Azospirillum brasilense (strain cd) and the grass Setaria italica was studied under different environmental and soil conditions. Highest acetylene reduction rates in intact plants were observed at the booting stage of Setaria (2350 nmol ethylene produced hour(-1) plant(-1)) at 27 C. Higher temperatures, up to 32 C, enhanced ethylene reduction. Significant increases in shoot dry weight, panicle weight, and length were obtained in inoculated plants fertilized with suboptimal NH(4)NO(3) levels. The increase in nitrogen content of plants inoculated with A. brasilense was shown to be due to N(2) fixation. This was demonstrated by growing plants in washed quartz sand with no combined nitrogen. The bacteria also increased branching and development of roots. It was concluded that inoculation of Setaria with A. brasilense may lead both to increases in plant yield and saving of nitrogen fertilizer.

Journal Article↗

PubMed1987

The symbiotic nitrogen fixation regulatory operon (fixRnifA) of Bradyrhizobium japonicum is expressed aerobically and is subject to a novel, nifA-independent type of activation.

The Bradyrhizobium japonicum N2 fixation regulatory gene, nifA, was sequenced and its transcription start site determined. Between the start of transcription and the nifA gene an open reading frame of 278 codons was found and named fixR. A deletion in fixR which allowed transcription into nifA resulted in a 50% reduced Fix activity. The fixRnifA operon was expressed in soybean root nodules, in cultures grown anaerobically with nitrate as terminal electron acceptor, in microaerobic cultures, and in aerobic cultures. The transcription start site (+1) was preceded by a characteristic nif(-24/-12)-type promoter consensus sequence. Double base-pair exchanges in the -12 but not in the -24 region resulted in a 'promoter-down' phenotype. A promoter-upstream DNA region between -50 and -148 was essential for maximal promoter activity. Expression from the promoter was not dependent on nifA. We conclude that the fixRnifA promoter is positively controlled, and that it requires a newly postulated transcriptional factor in order to become activated.

Aerobiosis↗

PubMed1988

Rhizobium meliloti genes required for C4-dicarboxylate transport and symbiotic nitrogen fixation are located on a megaplasmid.

A mutant of Rhizobium meliloti unable to transport C4 dicarboxylates (dct) was isolated after Tn5 mutagenesis. The mutant, 4F6, could not grow on aspartate or the tricarboxylic acid cycle intermediates succinate, fumarate, or malate. It produced symbiotically ineffective nodules on Medicago sativa in which bacteroids appeared normal, but the symbiotic zone was reduced and the plant cells contained numerous starch granules at their peripheries. Cosmids containing the dct region were obtained by selecting those which restored the ability of 4F6 to grow on succinate. The Tn5 insertion in 4F6 was found to be within a 5.9-kilobase (kb) EcoRI fragment common to the complementing cosmids. Site-specific Tn5-mutagenesis revealed dct genes in a segment of DNA about 4 kb in size extending from within the 5.9-kb EcoRI fragment into an adjacent 2.9-kb EcoRI fragment. The 4F6 mutation was found to be in a complementation group in which mutations yielded a Fix- phenotype, whereas other dct mutations in the region resulted in mutants which produced effective nodules in most, although not all, plant tests (partially Fix-). The dct region was found to be located on a megaplasmid known to carry genes required for exopolysaccharide production.

Cosmids↗

PubMed1978

Nitrogen fixation (acetylene reduction) associated with roots of winter wheat and sorghum in Nebraska.

Root segments and root-soil cores (6.5-cm diameter) from fields and nurseries of winter wheat and sorghum were tested for N2 fixation by using the acetylene reduction assay. Wheat samples (approximately 1,200) from 109 sites generally had low or no activity (0 to 3.1 nmol of C2H4 produced per h per g [dry weight] of root segments), even after 24 h of incubation. However, a commercial field of Scout 66, located in western Nebraska, exhibited appreciable activity (290 nmol of C2H4 produced per h per g [dry weight] of root segments). Of 400 sorghum lines and crosses, grain sorghums (i.e., CK-60A, Wheatland A, B517, and NP-16) generally exhibited higher nitrogenase activity than forage sorghums or winter wheats. CK-60A, a male sterile grain sorghum, was sampled at four locations and had the most consistent activity of 24 to 1,100 nmol of C2H4 produced per h per core. The maximum rate extrapolated to 2.5 g of N per hectare per day. Numerous N2-fixing bacterial isolates were obtained from wheat and sorghum roots that exhibited high nitrogenase activity. Most isolates were members of the Enterobacteriacae, i.e., Klebsiella pneumoniae, Enterobacter cloacae, and Erwinia herbicola.

Acetylene↗

PubMed1986

Nitrogenase promoter-lacZ fusion studies of essential nitrogen fixation genes in Bradyrhizobium japonicum I110.

DNA fragments containing either the nifD or nifH promoter and 5' structural gene sequences from Bradyrhizobium japonicum I110 were fused in frame to the lacZ gene. Stable integration of these nif promoter-lacZ fusions by homologous double reciprocal crossover into a symbiotically nonessential region of the B. japonicum chromosome provided an easy assay for the effects of potential nif regulatory mutants. The level of beta-galactosidase activity expressed from these two nif promoter-lacZ fusions was assayed in bacteroids of B. japonicum I110 wild type and Fix mutants generated by transposon Tn5 mutagenesis and identified in the accompanying paper. No nif-positive regulatory mutants were identified from among an array of Fix- mutants in which Tn5 was inserted 9 kilobase pairs upstream of the nifDK operon and within the 18-kilobase-pair region separating the nifDK and nifH operons. This result indicates that there are no genes in these regions involved in the regulation of nitrogenase structural gene expression. Interestingly, the level of beta-galactosidase activity expressed from the nifH promoter was twice that expressed from the nifD promoter, suggesting that the normal cellular level of the nifH gene product in bacteroids is in a 2:1 ratio with the nifD gene product instead of in the 1:1 stoichiometry of the nitrogenase enzyme complex.

Bacterial Proteins↗

PubMed1996

A phosphate transport system is required for symbiotic nitrogen fixation by Rhizobium meliloti.

The bacterium Rhizobium meliloti forms N2-fixing root nodules on alfalfa plants. The ndvF locus, located on the 1,700-kb pEXO megaplasmid of R. meliloti, is required for nodule invasion and N2 fixation. Here we report that ndvF contains four genes, phoCDET, which encode an ABC-type transport system for the uptake of Pi into the bacteria. The PhoC and PhoD proteins are homologous to the Escherichia coli phosphonate transport proteins PhnC and PhnD. The PhoT and PhoE proteins are homologous to each other and to the E. coli phosphonate transport protein PhnE. We show that the R. meliloti phoD and phoE genes are induced in response to phosphate starvation and that the phoC promoter contains two elements which are similar in sequence to the PHO boxes present in E. coli phosphate-regulated promoters. The R. meliloti ndvF mutants grow poorly at a phosphate concentration of 2 mM, and we hypothesize that their symbiotic phenotype results from their failure to grow during the nodule infection process. Presumably, the PhoCDET transport system is employed by the bacteria in the soil environment, where the concentration of available phosphate is normally 0.1 to 1 microM.

Amino Acid Sequence↗

PubMed1983

Promoter mutations that allow nifA-independent expression of the nitrogen fixation nifHDKY operon.

The nifHDKY operon of Klebsiella pneumoniae encodes for structural polypeptides of nitrogenase and requires the nifA gene product for transcription. Mutations that allow transcription of the nifHDKY operon in absence of the nifA gene product were characterized in plasmids containing the regulatory region of nifHDKY and nifH fused in phase to lacZ. beta-Galactosidase activity served as a measure for nifH expression. Most mutations were located in the nif regulatory region and included insertion sequence 2 (IS2) insertions, a sequence duplication, and a base substitution. In Escherichia coli, beta-galactosidase activity expressed from the mutant plasmids in the absence of nifA was 6-30% of the nifA-activated, parental level. Expression from most mutant plasmids was further increased by nifA. In K. pneumoniae, IS2-containing plasmids expressed low levels of beta-galactosidase and responded poorly, if at all, to activation by nifA, whereas expression from other mutant types was similar to that observed in E. coli. Nucleotide sequence analysis of two mutants indicated that sequences within 41 base pairs upstream to the nifH coding sequence were involved in nif-specific regulation. The results suggest that an inverted repeat element in this region, which could theoretically form a cruciform structure in the DNA, is involved in the transcriptional control of the nifHDKY operon.

Bacterial Proteins↗

PubMed1995

Characterization of a nitrogen-fixation (nif) gene cluster from Anabaena azollae 1a shows that closely related cyanobacteria have highly variable but structured intergenic regions.

The exact identity of cyanobacteria that have been cultured from symbiotic associations with the water fern Azolla spp., whether they are required in the symbiotic process, and their relationship to the symbiotic species, is a matter of some debate. We have characterized a 6 kb region containing the nifB operon and the nifH gene from cyanobacterium Anabaena azollae 1a, a putative symbiont of Azolla caroliniana. Five complete open reading frames have been sequenced. All are very highly conserved when compared with the corresponding regions of Anabaena sp. PCC 7120, with 93% to 97% identity at the nucleotide level and 93% to almost 100% at the amino-acid level. The intergenic regions, however, are not highly conserved (53-89% identity) when compared to the corresponding regions of Anabaena 7120: the A. azollae genome contains both more copies and more types of short tandemly repeated repetitive sequences than Anabaena 7120. The start points of transcription for both the nifB and nifH operons were mapped and found to be the same as those in Anabaena 7120. It was not possible to discern an improved consensus nif promoter sequence, but it was possible to define the likely extent of the promoter to within 40 bases upstream of the transcription start-point.

Amino Acid Sequence↗

PubMed2001

Rhizobia from wild legumes: diversity, taxonomy, ecology, nitrogen fixation and biotechnology.

Wild legumes (herb or tree) are widely distributed in arid regions and actively contribute to soil fertility in these environments. The N2-fixing activity and tolerance to drastic conditions may be higher in wild legumes than in crop legumes. The wild legumes in arid zones harbor diverse and promiscuous rhizobia in their root-nodules. Specificity existed only in few rhizobia from wild legumes, however, the majority of them are with wide host range. Based on phenotypic characteristics and molecular techniques (protein profiles, polysaccharides, plasmids, DNA-DNA hybridization, 16SrRNA, etc.), the root-nodule bacteria that was isolated from wild legumes had been classified into four genera (Rhizobium, Bradyrhizobium, Mesorhizobium and Sinorhizobium). The rhizobia of wild legumes in arid zones, exhibit higher tolerance to the prevailing adverse conditions, e.g. salt stress, elevated temperatures and desiccation. These rhizobia may be used to inoculate wild, as well as, crop legumes, cultivated in reclaimed desert lands. Recent reports indicated that the wild-legume rhizobia formed successful symbioses with some grain legumes. Moreover, intercropping of some N2-fixing tree legumes (e.g. Lablab, Leucaena, Sesbania, etc.) to pasture grasses improved biomass yield and herb quality. In recent years, the rhizobia of wild legumes turn the attention of biotechnologists. These bacteria may have specific traits that can be transferred to other rhizobia through genetic engineering tools or used to produce industrially important compounds. Therefore, these bacteria are very important from both economic and environmental points of view.

Biotechnology↗

PubMed2001

Nitrogen fixation genes in an endosymbiotic Burkholderia strain.

In this paper we report the identification and characterization of a DNA region containing putative nif genes and belonging to a Burkholderia endosymbiont of the arbuscular mycorrhizal fungus Gigaspora margarita. A genomic library of total DNA extracted from the fungal spores was also representative of the bacterial genome and was used to investigate the prokaryotic genome. Screening of the library with Azospirillum brasilense nifHDK genes as the prokaryotic probes led to the identification of a 6,413-bp region. Analysis revealed three open reading frames encoding putative proteins with a very high degree of sequence similarity with the two subunits (NifD and NifK) of the component I and with component II (NifH) of nitrogenase from different diazotrophs. The three genes were arranged in an operon similar to that shown by most archaeal and bacterial diazotrophs. PCR experiments with primers designed on the Burkholderia nifHDK genes and Southern blot analysis demonstrate that they actually belong to the genome of the G. margarita endosymbiont. They offer, therefore, the first sequence for the nif operon described for Burkholderia. Reverse transcriptase PCR experiments with primers designed on the Burkholderia nifH and nifD genes and performed on total RNA extracted from spores demonstrate that the gene expression was limited to the germination phase. A phylogenetic analysis performed on the available nifK sequences placed the endosymbiotic Burkholderia close to A. brasilense.

Amino Acid Sequence↗

PubMed1989

Oxidation of nitrogenase iron protein by dioxygen without inactivation could contribute to high respiration rates of Azotobacter species and facilitate nitrogen fixation in other aerobic environments.

The kinetics of oxidation of the Fe proteins of nitrogenases from Klebsiella pneumoniae (Kp2) and Azotobacter chroococcum (Ac2) by O2 and H2O2 have been studied by stopped-flow spectrophotometry at 23 degrees C, pH 7.4. With excess O2, one-electron oxidation of Kp2 and Ac2 and their 2 MgATP or 2 MgADP bound forms occurs with rate constants (k) in the range 5.3 x 10(3) M-1.S-1 to 1.6 x 10(5) M-1.S-1. A linear correlation between log k and the mid-point potentials (Em) of these protein species indicates that the higher rates of electron transfer from the Ac2 species are due to the differences in Em of the 4Fe-4S cluster. The reaction of Ac2(MgADP)2 with O2 is sufficiently rapid for it to contribute significantly to the high respiration rate of Azotobacter under N2-fixing conditions and may represent a new respiratory pathway. Excess O2 rapidly inactivates Ac2(MgADP)2 and Kp2(MgADP)2; however, when these protein species are in greater than 4-fold molar excess over the concentration of O2, 4 equivalents of protein are oxidized with no loss of activity. The kinetics of this reaction suggest that H2O2 is an intermediate in the reduction of O2 to 2 H2O by nitrogenase Fe proteins and imply a role for catalase or peroxidase in the mechanism of protection of nitrogenase from O2-induced inactivation.

Adenosine Triphosphate↗

PubMed1988

Ammonia-constitutive nitrogen fixation mutants of Rhodobacter capsulatus.

Mutants of R. capsulatus that express nif genes constitutively with respect to ammonia were studied in order to define better the circuit that regulates nif gene transcription. One mutant class could be complemented in trans by a cosmid clone containing a wild-type gene (nifR5) defined by Tn5 inserts as being no longer than 1.6 kb. The nifR5 gene is unlinked to previously described nif genes. A second mutant class could not be complemented by the wild-type cosmid library. For one mutant in this class, a nifH::lac fusion was used to select further mutants that were Lac-. Only two of these could be complemented in trans to Lac+; the complementing gene was nifR4, which is analogous to the ntrA gene of enterobacteria. Both complemented strains were Nifc. Therefore these mutations do not bypass the need for the nifR4 gene product. A third class of constitutive mutant was found by selecting Nif+ revertants of a Nif- strain deleted for the nifR1 and nifR2 genes. The nifR1 and nifR2 genes are homologues of enterobacterial ntrC and ntrB genes, respectively. Not all of the Nif+ revertants were constitutive; some were regulated normally by ammonia. We suspect that the latter revertants use alternate Ntr systems to activate nif gene transcription, a suggestion consistent with the observation that numerous bands in Southern blots of total DNA of R. capsulatus are identified by Escherichia coli ntr gene probes.

Ammonia↗