Cluster analysis of genes for nitrogen fixation from several diazotrophs

Hierarchical clustering and similarity coefficients of pairwise alignments of the published nucleotide sequences of 27nifH genes suggest thatnif genes are as ancient as the archaebacteria and clostridia. The positions ofnifHl ofMethanococcus thermolithotrophicus, nifH3 ofClostridium pasteurianum, nifH3 ofAzotobacter vinelandii andnifH ofFrankia suggest that a variety of lateral transfers may have occurred during evolution ofnifH gene. The genes for type 3 nitrogenase ofA. vinelandii may have diverged early from methanogens and clostridia. A high similarity coefficient with the derived amino acid sequence of type 3 nitrogenase suggests the presence of a functionally similar enzyme inC. pasteurianum. The type 2 nitrogenase genenifH2 of azotobacters seems to have originated recently from the genenifHl for conventional type I nitrogenase. RhizobialnifH genes comprise two closely related but discrete clusters that are in consonance with the plasmid or chromosomal location ofnif genes. The chromosomal and plasmid locatednifH of rhizobia seem to have evolved independently but contemporaneously.     

Identification of a regulatory nifA type gene and physical mapping of cloned new nif regions of Azospirillum brasilense

Summary Three new Tn5-mutagenized nif genes of Azospirillum brasilense were characterized. The sizes of the restriction fragments and the restriction maps of the cloned nif DNA regions showed that these nif genes are distinct from those reported earlier, e.g. nifHDK, nifE, nifUS, fixABC. The Nif27 mutant was identified as a nifA type regulatory gene of A. brasilense (a) by genetic complementation with nifA of Klebsiella pneumoniae, (b) by the absence of nitrogenase iron protein in western protein blots and (c) by its inability to activate expression of a nijH-lacZ fusion. The growth characteristics of the three mutants showed that none of them is defective in general nitrogen regulatory (ntr) genes. Also, no homology was detected between the three nif DNA regions of the mutants, cloned in pMS188, pMS189 and pMS197, and the K. pneumoniae nif, gInA or ntr genes. In addition, the fixABC genes of Bradyrhizobium japonicum did not show any hybridization with the cloned Azospirillum genes. Unlike the situation in enteric bacteria, the nif genes in A. brasilense are scattered and span a region of about 65 kb.     

The Evolutionary History of Nitrogen Fixation, as Assessed by NifD

The evolutionary history of nitrogen fixation has been vigorously debated for almost two decades. Previous phylogenetic analyses of nitrogen fixation genes (nif) have shown support for either evolution by vertical descent or lateral transfer, depending on the specific nif gene examined and the method of analyses used. The debate centers on the placement and monophyly of the cyanobacteria, proteobacteria, and Gram-positive bacteria (actinobacteria and firmicutes). Some analyses place the cyanobacteria and actinobacteria within the proteobacteria, which suggests that the nif genes have been laterally transferred since this topology is incongruent with ribosomal phylogenies, the standard marker for comparison. Other nif analyses resolve and support the monophyly of the cyanobacteria, proteobacteria, and actinobacteria, supporting vertical descent. We have revisited these conflicting scenarios by analyzing nifD from an increased number of cyanobacteria, proteobacteria, and Gram-positive bacteria. Parsimony analyses of amino acid sequences and maximum likelihood analysis of nucleic acid sequences support the monophyly of the cyanobacteria and actinobacteria but not the proteobacteria, lending support for vertical descent. However, distance analysis of nucleic acid sequences placed the actinobacteria within the proteobacteria, supporting lateral transfer. We discuss evidence for both vertical descent and lateral transfer of nitrogen fixation.     

Nitrogen fixation in strains ofAzotobacter chroococcum bearing deletions of a cluster of genes coding for nitrogenase

Strains of the obligately aerobic nitrogen fixing organismAzotobacter chroococcum were constructed which contained defined chromosomal deletions in which the nitrogenase structural genenifHDK cluster (nifH for the polypeptide of the Fe-protein component of nitrogenase andnifD andnifK for the alpha and beta subunits respectively of the MoFe-protein component of the enzyme) was replaced by a kanamycin resistance gene. N₂ fixation was nevertheless observed in deletion strains though only in a molybdenum-deficient medium or in spontaneously arising tungstate-resistant derivatives. In comparison with the parent strain growing in molybdenum-sufficient medium, diazotrophic growth was slow and the nitrogenase activity in vivo was characterised by disproportionately low rates of C₂H₂-reduction compared to H₂-evolution and relative insensitivity of H₂-evolution to inhibition by C₂H₂. The findings show reiteration of functional structural genes for nitrogenase inA. chroococcum consistent with our previous observation of twonifH genes in this organism and detection in this work of a secondnifK-like sequence in the genomes of both parent and deletion strains whenA. chroococcum nifK DNA was used as a probe.     

Indigenous plasmids and cultural characteristics of rhizobia nodulating chickpeas (Cicer arietinum L.)

We examined 27 strains of chickpea rhizobia from different geographic origins for indigenous plasmids, location and organization of nitrogen fixation (nif) genes, and cultural properties currently used to separate fast- and slow-growing groups of rhizobia. By using an in-well lysis and electrophoresis procedure one to three plasmids of molecular weights ranging from 35 to higher than 380 Mdal were demonstrated in each of 19 strains, whereas no plasmids were detected in the eight remaining strains. Nitrogenase structural genes homologous to Rhizobium meliloti nifHD, were not detected in plasmids of 26 out of the 27 strains tested. Hybridization of EcoRI digested total DNA from these 26 strains to the nif probe from R. meliloti indicated that the organization of nifHD genes was highly conserved in chickpea rhizobia. The only exception was strain IC-72 M which harboured a plasmid of 140 Mdal with homology to the R. meliloti nif DNA and exhibited also a unique organization of nifHD genes. The chickpea rhizobia strains showed a wide variation of growth rates (generation times ranged from 4.0 to 14.5 h) in yeast extract-mannitol medium but appear to be relatively homogeneous in terms of acid production in this medium and acid reaction in litmus milk. Although strains with fast and slow growth rates were identified, DNA/DNA hybridization experiments using a nifHD-specific probe, and the cultural properties examined so far do not support the separation of chickpea rhizobia into two distinct groups of the classical fast- and slow-growing types of rhizobia.     

携带Paenibacillus polymyxa WLY78固氮基因簇(nif)的重组大肠杆菌Escherichia coli78-7转录组分析

[目的]来自Paenibacillus polymyxa WLY78的固氮基因簇（nifBHDKEfNXhesAnifV）可以转化入Escherichia coli中表达并使重组大肠杆菌合成有固氮活性的固氮酶。本文拟通过对重组大肠杆菌E.coli 78-7的转录组分析以提高其固氮能力。[方法]对固氮条件（无氧无NH₄⁺）和非固氮条件（空气和100 mmol/L NH₄⁺）培养的重组大肠杆菌E.coli 78-7进行转录组分析。[结果]nif基因在两种培养条件下显著表达,说明在重组大肠杆菌中可规避原菌中氧气和NH₄⁺对nif基因的负调控。对于固氮过程必需的非nif基因,如参与钼、硫、铁元素转运的mod、cys和feoAB,这些基因在两种培养条件下表达水平有差异。而参与铁硫簇合成的suf和isc基因簇在两条件下表达水平差异巨大。此外,参与氮代谢的基因在固氮条件下显著上调。[结论]重组大肠杆菌中与固氮相关的非nif基因在该菌的固氮过程中具有较大影响,本文对在异源宿主中调高固氮酶活性研究具有重要意义。     

Conserved plasmid/chromosome sequences in fast- and slow-growing rhizobia that nodulate the same plant

Apart from the ability to nodulate legumes, fast-and slow-growing rhizobia have few bacteriological traits in common. Given that there is only one pathway to nodulation, DNA sequences conserved in fast- and slow-growing organisms that nodulate the same host should be strongly enriched in infectivity genes. We tested this hypothesis with seven fast-growing and five slow-growing strains that produced responses varying from fully effective nodulation through various ineffective associations to non-nodulation on four different hosts (Lotus pedunculatus, Lupinus nanus, Macroptilium atropurpureum, and Vigna unguiculata). When restriction enzyme digested total DNA from 10 of the strains was separately hybridized with nick-translated plasmid DNA isolated from 4 fast-growing strains, variable but significant homologies were found with all 10 strains. Part of this homology was shown to be associated with the nifKDH genes for nitrogenase and part with putative nodulation genes carried on pC2, a cosmid clone containing a 37 kbp region of the large sym plasmid present in the fast-growing broad-host range Rhizobium sp. strain NGR234. Analysis of the extent of homology between the plasmids of 3 fastgrowing strains (NGR234, TAL 996 and UMKL 19) able to effectively nodulate Vigna unguiculata showed them to have homologous DNA fragments totalling 47 kbp. This core homology represents less than 12% of the total coding capacity of the sym plasmid present in each of these strains.Abbreviations Sym symbiotic sequences/plasmids - nod genes required for nodulation - nod putative nod genes - nif genes required for the synthesis of the enzyme nitrogenase     

Phylogeny of nitrogenase sequences inFrankia and other nitrogen-fixing microorganisms

Summary The complete nucleotide sequence of a nitrogenase (nifH) gene was determined from a second strain (HRN18a) ofFrankia, an aerobic soil bacterium. The open reading frame is 870 bp long and encodes a polypeptide of 290 amino acids. The amino acid and nucleotide sequences were compared with 21 other published sequences. The twoFrankia strains were 96% similar at the amino acid level and 93% similar at the nucleotide level. A number of methods were used to infer phylogenies of these nitrogen fixers, based onnifH amino acid and nucleotide sequences. The results obtained do not agree completely with other phylogenies for these bacteria and thus make probable occurrences of lateral transfer of thenif genes. The time of divergence of the twoFrankia strains could be estimated at about 100 million years. The vanadium-dependent (Type 2) nitrogenase present inAzotobacter spp. appears to be a recent derivation from the conventional molybdenum-dependent (Type 1) enzyme, whereas the iron-dependent (Type 3) alternative nitrogenase would have a much older origin.     

The effects of gibberellins and mepiquat chloride on nitrogenase activity in <Emphasis Type="Italic">Bradyrhizobium japonicum</Emphasis>

Application of plant growth regulators (PGRs) to soybean plants is known to induce changes in nitrogenase activity in root nodules, and this led us to hypothesize that PGRs would affect nitrogenase activity in free-living rhizobia cultures. Little is known about the molecular basis of the effects of PGRs on nitrogenase activity in free-living rhizobia cultures. Therefore, a comparative study was conducted on the effects of gibberellins (GA₃) and mepiquat chloride (PIX), which regulate plant growth, on the nitrogenase activity of the nitrogen-fixing bacterium Bradyrhizobium japonicum. Fix and nif gene regulation and protein expression in free-living cultures of B. japonicum were investigated using real-time PCR and two-dimensional electrophoresis after treatment with GA₃ or PIX. GA₃ treatment decreased nitrogenase activity and the relative expression of nifA, nifH, and fixA genes, but these effects were reversed by PIX treatment. As expected, several proteins involved in nitrogenase synthesis were down-regulated in the GA₃-treated group. Conversely, several proteins involved in nitrogenase synthesis were up-regulated in the PIX-treated group, including bifunctional ornithine acetyltransferase/N-acetylglutamate synthase, transaldolase, ubiquinol-cytochrome C reductase iron-sulfur subunit, electron transfer flavoprotein subunit beta, and acyl-CoA dehydrogenase. Two-pot experiments were conducted to evaluate the effects of GA₃ and PIX on nodulation and nitrogenase activity in Rhizobium-treated legumes. Interestingly, GA₃ treatment increased nodulation and depressed nitrogenase activity, but PIX treatment decreased nodulation and enhanced nitrogenase activity. Our data show that the nif and fix genes, as well as several proteins involved in nitrogenase synthesis, are up-regulated by PIX and down-regulated by GA₃, respectively, in B. japonicum.     

Analysis of codon usage in genes for nitrogen fixation from phylogenetically diverse diazotrophs

Summary A cluster analysis based on codon usage in genes for biological nitrogen fixation (nif genes) grouped diazotrophs into three distinct classes: anaerobes, cyanobacteria, and aerobes. In thenif genes ofKlebsiella pneumoniae there was no evidence for selection pressure in favor of highly translatable codons. However, in the nitrogen regulatory operonglnAntrBntrC of enteric bacteria the stoichiometrically high level of glutamine synthetase may be facilitated by the presence of efficiently translatable codons inglnA. Thenif genes of the cyanobacteriumAnabaena showed codon selection in favor of translational efficiency. Computation of codon adaptation indices for expression in heterologous systems indicated that the reading frames most suitable for expression ofnif genes inEscherichia coli, Bacillus subtilis, andSaccharomyces cerevisiae were present in azotobacters, clostridia, and cyanobacteria, respectively. In codon-usage-based cluster analysis, type 3 nitrogenase genes ofAzotobacter vinelandii grouped along with type 1 and type 2 genes. This is in contrast to the nucleotide sequence-based multiple alignment in which type 3 nitrogenase genes ofA. vinelandii have been reported to cluster with entirely unrelated diazotrophs such as methanogens and clostridia. This may be indicative of lateral transfer ofnif genes among widely divergent taxons. The chromosomal- and plasmid-locatednif genes of rhizobia also cluster separately in nucleotide sequence-based analysis but showed similar codon usage. These analyses suggested that the phylogeny ofnif genes drawn on the basis of nucleotide sequence homology was not masked by the taxon-specific pressure on codon usage.     

Identification of nifE-, nifN- and nifS-like genes in Bradyrhizobium japonicum

Summary The 17 kb region between the Bradyrhizobium japonicum nitrogenase genes (nifDK and nifH) was investigated for the presence of further nif or fix genes by site-directed insertion or deletion/replacement mutagenesis and interspecies hybridization. Mutant strains were tested for their ability to reduce acetylene in free-living, microaerobic culture (Nif phenotype) and in soybean root nodules (Fix phenotype). The presence of a gene, previously identified by hybridization with the Klebsiella pneumoniae nifB gene, was proved by isolation of a nifB insertion mutant which was completely Nif^- and Fix^-. Three other regions were found to be homologous to the K. pneumoniae genes nifE, nifN, and nifS, NifE and nifN insertion mutants were completely Nif^-/Fix^- whereas nifS mutants were leaky with 30% residual Fix activity. Taken together, the data show that the B. japonicum genome harbours a cluster of closely adjacent genes which are directly concerned with nitrogenase function.     

Regulation of symbiotic nitrogen fixation in root nodules of alfalfa (Medicago sativa) infected with Rhizobium meliloti

Symbiotic nitrogen fixation of Rhizobium meliloti bacteroids in Medicago sativa root nodules was suppressed by several inorganic nitrogen sources. Amino acids like glutamine, glutamic acid and aspartic acid, which can serve as sole nitrogen sources for the unnodulated plant did not influence nitrogenase activity of effective nodules, even at high concentrations.Ammonia and nitrate suppressed symbiotic nitrogen fixation in vivo only at concentrations much higher than those needed for suppression of nitrogenase activity in free living nitrogen fixing bacteria. The kinetics of suppression were slow compared with that of free living nitrogen fixing bacteria. On the other hand, nitrite, which acts as a direct inhibitor of nitrogenase, suppressed very quickly and at low concentrations. Glutamic acid and glutamine enhanced the effect of ammonia dramatically, while the suppression by nitrate was enhanced only slightly.     

念珠藻属植物nifH基因的适应性进化分析

念珠藻(Nostoc)固氮过程关键在于固氮酶的催化,而固氮酶复合物中的铁蛋白(NifH)是由高度保守的nifH基因编码的,该基因是进化史上现存最古老的功能基因之一。该研究选取念珠藻属及近缘类群的nifH基因序列共40条,采用最大似然法构建系统发育树;运行PAML4.9软件,对nifH基因编码蛋白进行生物信息学分析,并使用分支模型、位点模型和分支-位点模型检测该基因的选择位点,探讨nifH基因的适应性进化特征。结果表明:(1)最大似然树显示内类群中该研究物种共分为6个分支(A、B、C、D、E和F),其中D和E是2个大的分支,每个大分支中又各包含2个特殊的小分支A、F和B、C,其中F分支包含新疆古尔班通古特沙漠采集到的9株念珠藻,A分支包含F分支及该研究测定序列的4株葛仙米,B分支包含本研究测定序列的4株地皮菜和3株未定种的念珠藻,C分支包含NCBI数据库中下载的5株念珠藻、鱼腥藻序列和本研究测定序列的1株念珠藻。(2)在所分析的3种进化模型中,仅通过分支-位点模型检测出14个统计学上显著的正选择位点,即1F、2S、3S、4T、5A、6F、7F、8I、9S、10C、17I、27Y、29D和31R位点,表明念珠藻属植物的nifH基因发生了适应性变化,分支-位点模型是研究藻类基因适应性进化较好的模型。     

Rhizosphere-Bacterial Community in <Emphasis Type="Italic">Eperua falcata</Emphasis> (Caesalpiniaceae) a Putative Nitrogen-Fixing Tree from French Guiana Rainforest

The rainforest of French Guiana is still largely unaffected by human activity. Various pristine sites like the Paracou Research Station are devoted to study this tropical ecosystem. We used culture-independent techniques, like polymerase chain reaction-temperature gradient gel electrophoresis, and construction of clone libraries of partial 16S rRNA and nifH genes, to analyze the composition of the bacterial community in the rhizosphere of mature trees of Eperua falcata and Dicorynia guianensis, both species within the Caesalpiniaceae family. E. falcata is one of the more abundant pioneer tree species in this ecosystem and so far, no root nodules have ever been found. However, its nitrogen-fixing status is regarded as “uncertain”, whereas D. guianensis is clearly considered a non-nitrogen-fixing plant. The rhizospheres of these mature trees contain specific bacterial communities, including several currently found uncultured microorganisms. In these communities, there are putative nitrogen-fixing bacteria specifically associated to each tree: D. guianensis harbors several Rhizobium spp. and E. falcata members of the genera Burkholderia and Bradyrhizobium. In addition, nifH sequences in the rhizosphere of the latter tree were very diverse. Retrieved sequences were related to bacteria belonging to the α-, β-, and γ-Proteobacteria in the E. falcata rhizoplane, whereas only two sequences related to γ-Proteobacteria were found in D. guianensis. Differences in the bacterial communities and the abundance and diversity of nifH sequences in E. falcata rhizosphere suggest that this tree could obtain nitrogen through a nonnodulating bacterial interaction.     

Host genetic control of symbiosis in soybean (Glycine max L.)

Genes controlling nitrogen-fixing symbioses of legumes with specialized bacteria known as rhizobia are presumably the products of many millions of years of evolution. Different adaptative solutions evolved in response to the challenge of survival in highly divergent complexes of symbionts. Whereas efficiency of nitrogen fixation appears to be controlled by quantitative inheritance, genes controlling nodulation are qualitatively inherited. Genes controlling nodulation include those for non-nodulation, those that restrict certain microsymbionts, and those conditioning hypernodulation, or supernodulation. Some genes are naturally occurring polymorphisms, while others were induced or were the result of spontaneous mutations. The geographic patterns of particular alleles indicate the role of coevolution in determining symbiont specificites and compatibilities. For example, the Rj4 allele occurs with higher frequency (over 50%) among the soybean (G. max) from Southeast Asia. DNA homology studies of strains of Bradyrhizobium that nodulate soybean indicated two groups so distinct as to warrant classification as two species. Strains producing rhizobitoxine-induced chlorosis occur only in Group II, now classified as B. elkanii. Unlike B. japonicum, B. elkanii strains are characterized by (1) the ability to nodulate the rj1 genotype, (2) the formation of nodule-like structures on peanut, (3) a relatively high degree of ex planta nitrogenase activity, (4) distinct extracellular polysaccharide composition, (5) distinct fatty acid composition, (6) distinct antibiotic resistance profiles, and (7) low DNA homology with B. japonicum. Analysis with soybean lines near isogenic for the Rj4 versus rj4 alleles indicated that the Rj4 allele excludes a high proportion of B. elkanii strains and certain strains of B. japonicum such as strain USDA62 and three serogroup 123 strains. These groups, relatively inefficient in nitrogen fixation with soybean, tend to predominate in soybean nodules from many US soils. The Rj4 allele, the most common allelic form in the wild species, has a positive value for the host plants in protecting them from nodulation by rhizobia poorly adapted for symbiosis.     

Identification of a new class of nitrogen fixation genes in Rhodobacter capsalatus: a putative membrane complex involved in electron transport to nitrogenase

DNA sequence analysis of a 12236 by fragment, which is located upstream of nifE in Rhodobacter capsulatus nif region A, revealed the presence of ten open reading frames. With the exception of fdxC and fdxN, which encode a plant-type and a bacterial-type ferredoxin, the deduced products of these coding regions exhibited no significant homology to known proteins. Analysis of defined insertion and deletion mutants demonstrated that six of these genes were required for nitrogen fixation. Therefore, we propose to call these genes rnfA, rnfB, rnfC, rnfD, rnfE and rnfF (for Rhodobacter nitrogen fixation). Secondary structure predictions suggested that the rnf genes encode four potential membrane proteins and two putative iron-sulphur proteins, which contain cysteine motifs (C-X₂-C-X₂-C-X₃-C-P) typical for [4Fe-4S] proteins. Comparison of the in vivo and in vitro nitrogenase activities of fdxN and rnf mutants suggested that the products encoded by these genes are involved in electron transport to nitrogenase. In addition, these mutants were shown to contain significantly reduced amounts of nitrogenase. The hypothesis that this new class of nitrogen fixation genes encodes components of an electron transfer system to nitrogenase was corroborated by analysing the effect of metronidazole. Both the fdxN and rnf mutants had higher growth yields in the presence of metronidazole than the wild type, suggesting that these mutants contained lower amounts of reduced ferredoxins.     

Visualization of bioluminescence as a marker of gene expression in rhizobium-infected soybean root nodules

The linked structural genes lux A and lux B, encoding bacterial luciferase of a marine bacterium Vibrio harveyi, were fused with the nitrogenase nifD promoter from Bradyrhizobium japonicum and with the P1 promoter of pBR322. Both fusions were integrated into the B. japonicum chromosome by site-specific recombination. Soybean roots infected with the two types of rhizobium transconjugants formed nitrogen-fixing nodules that produced bright blue-green light. Cells containing the P1 promoter/lux AB fusion resulted in continuously expressed bioluminescence in both free-living rhizobium and in nodule bacteriods. However, when under control of the nifD promoter, luciferase activity was found only in introgen-fixing nodules. Light emission from bacteroids allowed us to visualize and to photograph nodules expressing this marker gene fusion in vivo at various levels of resolution, including within single, living plant cells. Localization of host cells containing nitrogen-fixing bacteroids within nodule tissue was accomplished using low-light video microscopy aided by realtime image processing techniques developed specifically to enhance extreme low-level luminescent images.     

Corresponding 16S rRNA gene segments in Rhizobiaceae and Aeromonas yield discordant phylogenies

Previous evidence has indicated that the 16S rRNA genes in certain species of Aeromonas may have a history of lateral transfer and recombination. A comparative analysis of patterns of 16S nucleotide sequence polymorphism among species of Rhizobium and Agrobacterium was conducted to determine if there is similar evidence for chimeric 16S genes in members of the Rhizobiaceae. Results from phylogenetic analyses and comparison of patterns of nucleotide sequence polymorphism in portions of rhizobial 16S genes revealed the same type of segment-dependent polymorphic site partitioning that was previously reported for Aeromonas. These results support the hypothesis that certain 16S segments in rhizobia may have a history of lateral transfer and recombination.Abbreviations 16S rRNA 16S ribosomal ribonucleic acid - 16S the 16S rRNA gene     

Expression of Frankia nif genes in nodules of Alnus glutinosa

Expression of Frankia genes involved in nitrogen fixation was studied in Alnus glutinosa nodules using the in situ hybridization technique. The results show that high level expression of nif genes does not occur immediately upon infection of cortical cells by Frankia. Also, only in the infected cells near the tips of the nodule lobes, nif genes are expressed at high levels. In the majority of infected cells, nif gene expression is rather low.