Inferring the Evolutionary History of Mo-Dependent Nitrogen Fixation from Phylogenetic Studies of nifK and nifDK

The ability to fix nitrogen is widely, but sporadically distributed among the Bacteria and Archaea suggesting either a vertically inherited, ancient function with widespread loss across genera or an adaptive feature transferred laterally between co-inhabitants of nitrogen-poor environments. As previous phylogenetic studies of nifH and nifD have not completely resolved the evolutionary history of nitrogenase, sixty nifD, nifK, and combined nifDK genes were analyzed using Bayesian, maximum likelihood, and parsimony algorithms to determine whether the individual and combined datasets could provide additional information. The results show congruence between the 16S and nifDK phylogenies at the phyla level and generally support vertical descent with loss. However, statistically significant differences between tree topographies suggest a complex evolutionary history with the underlying pattern of vertical descent obscured by recurring lateral transfer events and different patterns of evolution between the genes. Results support inheritance from the Last Common ancestor or an ancient lateral transfer of the nif genes between Bacteria and Archaea, ongoing gene transfer between cohabitants of similar biogeographic regions, acquisition of nitrogen-fixing capability via symbiosis islands, possible xenologous displacement of one gene in the operon, and possible retention of ancestral genes in heterocystous cyanobacteria. Analyses support the monophyly of the Cyanobacteria, αβγ-Proteobacteria, and Actinobacteria (Frankia) and provide strong support for the placement of Frankia nif genes at the base of combined the Cyanobacteria/Proteobacteria clades.     

Hybridization of DNA from methanogenic bacteria with nitrogenase structural genes (nifHDK)

Summary Using the Southern hybridization technique, homologies were examined between restricted DNA of four methanogenic bacteria (Methanobacterium ivanovi, Methanobacterium thermoautotrophicum, Methanococcus voltae, Methanosarcina barkeri) and the nif (nitrogen fixation) genes of Klebsiella pneumoniae and Anabaena strain 7120. With K. pneumoniae probes, no hybridization was observed with nifA, nifNE, and nifJ but positive results were obtained with the nifHDK genes coding for nitrogenase. Homology was detected, in the four strains, with K. pneumoniae and Anabaena nifH probes. In M. voltae and M. ivanovi, the homology found with nifH was estimated to be about 70% and a weaker hybridization was observed also with nifD and nifK. In M. voltae, the sequence homologous to nifH was found on a 3.0 kbp HindIII fragment and sequences homologous to nifD and nifK on a 3.8 kbp HindIII fragment. The 3.0 kbp fragment was cloned and the region homologous to nifH was localized more precisely. When this fragment was used as a probe against other DNAs, it behaved as a K. pneumoniae and Anabaena nifH probe. The results suggest that the structural genes for nitrogenase may be present in archaebacteria and raise interesting questions regarding their evolution.     

Insertion and deletion mutations within the nif region of Rhizobium japonicum

Insertion and deletion mutants were used to characterize a genomic region of Rhizobium japonicum where the nitrogenase structural genes are located on two separate operons nifDK and nifH. In addition to previously described nifD:: Tn5 and nifK:: Tn5 mutations we have now generated, by localized mutagenesis, further Tn5 insertion mutations in the vicinity of nifDK as well as within and adjacent to nifH. The nifD:: Tn5, nifK:: Tn5, and nifH:: Tn5 mutant strains were of the Nod⁺ Fix^- phenotype whereas all other mutants were symbiotically fully effective (Nod⁺ Fix⁺). The nifH:: Tn5 mutation was helpful in the identification of the nifH gene product (the dinitrogenase reductase) by two-dimensional gel electrophoresis: due to its polar effect this insertion specifically abolished the synthesis of that protein under microaerobic culture conditions. The ultrastructure of soybean root nodules infected with either the nif ⁺ wild-type or with the nif ^- (but otherwise isogenic) mutant strains was analyzed by electron microscopy. All contained fully developed bacteroids, but the nitrogen non-fixing mutants showed massive accumulation of PHB.Of Tn5-containing strains, kanamycin sensitive derivatives were obtained which contained deletions. Several classes of deletion mutants were found which, as judged by their physical DNA structure and their phenotypes, allowed the following most important conclusions: (i) deletions lacking both the nifDK and nifH regions indicate linkage between the two operons whereby at least 15 kb of DNA separate them; (ii) one deletion ending upstream from nifH, and lacking only nifDK, indicates that the nifDK operon is located on the 5-flanking side of the nifH operon; (iii) all deletion mutants are Nod⁺ indicating that there are no essential nodulation gnes located between and adjacent to nifDK and nifH.     

Different organization of nif genes in nonheterocystous and heterocystous cyanobacteria

Summary Labeled probes carrying the Anabaena PCC 7120 nitrogenase (nifK and nifD) and nitrogenase reductase (nifH) genes were hybridized to Southern blots of DNA from diverse N₂-fixing cyanobacteria in order to test a previous observation of different nif gene organization in nonheterocystous and heterocystous strains. The nif probes showed no significant hybridization to DNA from a unicellular cyanobacterium incapable of N₂ fixation. All nonheterocystous cyanobacteria examined (unicellular and filamentous) had a contiguous nifKDH gene cluster whereas all of the heterocystous strains showed separation of nifK from contiguous nifDH genes. These findings suggest that nonheterocystous and heterocystous cyanobacteria have characteristic and fundamentally different nif gene arrangements. The noncontiguous nif gene pattern, as shown with two Het^- mutants, is independent of phenotypic expression of heterocyst differentiation and aerobic N₂-fixation. Thus nif arrangement could be a useful taxonomic marker to distinguish between phenotypically Het^- heterocystous cyanobacteria and phylogenetically unrelated nonheterocystous strains.     

Effect of Naturally Occurring nif Reiterations on Symbiotic Effectiveness in Rhizobium phaseoli

Most naturally occurring strains of Rhizobium phaseoli possess reiteration of the nif genes. Three regions contain nitrogenase structural genes in strain CFN42. Two of these regions (a and b) have copies of nifH, nifD, and nifK, whereas the third region (c) contains only nifH. Strains containing mutations in either nif region a or nif region b had significantly diminished symbiotic effectiveness compared with the wild-type strain on the basis of nodule mass, total nitrogenase activity per plant, nitrogenase specific activity, total nitrogen in the shoot, and percentage of nitrogen. A strain containing mutations in both nif region a and nif region b was totally ineffective. These data indicate that both nif region a and nif region b are needed for full symbiotic effectiveness in R. phaseoli.     

Complementation analysis of Klebsiella pneumoniae mutants defective in nitrogen fixation.

Summary A series of mutants defective in nitrogen fixation (nif) were isolated in Klebsiella pneunoniae strain M5a1. The nif mutations were either located on plasmid pRD1 or on the K. pneumoniae chromosome. A total of 37 plasmid mutants and 28 chromosomal mutants were employed in complementation tests using the acetylene reduction technique. Most mutants could be assigned to one of seven nif cistrons: nifA, nifB, nifD, nifE, nifF, nifH, and nifK.Complementation analysis of two nif deletion mutants confirmed transductional evidence that these strains carry nifB-A-F deletions. One deletion mutant had, in contrast to previous transductional analysis, a functional nifK cistron and presumably is deleted for nifB-A-F-E.Examination of the biochemical phenotype of several mutants suggests that the nifA product has a regulatory function, and nifK, nifD and nifH are most probably the structural genes for nitrogenase.     

Conservation of nif sequences in Frankia

Summary Southern blots of Frankia total DNAs were hybridized with nifHDK probes from Rhizobium meliloti, Klebsiella pneumoniae and Frankia strain Arl3. Differences between strains were noted in the size of the hybridizing restriction fragments. These differences were more pronounced among Elaeagnus-compatible strains than among Alnus- or Casuarina-compatible strains. Gene banks constructed for Frankia strains EUN1f, HRN18a, CeD and ACoN24d were used to isolate nif-hybridizing restriction fragments for subsequent mapping and comparisons. The nifH zone had the highest sequence conservation and the nifH and nifD genes were found to be contiguous. The complete nucleotide sequence of the nifH open reading frame (ORF) from Frankia strain Arl3 is 861 bp in length and encodes a polypeptide of 287 amino acids. Comparisons of these nucleic acid and amino acid sequences with other published nifH sequences suggest that Frankia is most similar to Anabaena and Azotobacter spp. and K. pneunoniae and least similar to the Gram-positive Clostridium pasteurianum and to the archaebacterium Methanococcus voltae.     

Analysis of phylogeny and codon usage bias and relationship of GC content,amino acid composition with expression of the structural nif genes

Bacteria and archaea have evolved with the ability to fix atmospheric dinitrogen in the form of ammonia, catalyzed by the nitrogenase enzyme complex which comprises three structural genes nifK, nifD and nifH. The nifK and nifD encodes for the beta and alpha subunits, respectively, of component 1, while nifH encodes for component 2 of nitrogenase. Phylogeny based on nifDHK have indicated that Cyanobacteria is closer to Proteobacteria alpha and gamma but not supported by the tree based on 16SrRNA. The evolutionary ancestor for the different trees was also different. The GC1 and GC2% analysis showed more consistency than GC3% which appeared to below for Firmicutes, Cyanobacteria and Euarchaeota while highest in Proteobacteria beta and clearly showed the proportional effect on the codon usage with a few exceptions. Few genes from Firmicutes, Euryarchaeota, Proteobacteria alpha and delta were found under mutational pressure. These nif genes with low and high GC3% from different classes of organisms showed similar expected number of codons. Distribution of the genes and codons, based on codon usage demonstrated opposite pattern for different orientation of mirror plane when compared with each other. Overall our results provide a comprehensive analysis on the evolutionary relationship of the three structural nif genes, nifK, nifD and nifH, respectively, in the context of codon usage bias, GC content relationship and amino acid composition of the encoded proteins and exploration of crucial statistical method for the analysis of positive data with non-constant variance to identify the shape factors of codon adaptation index.     

The structural nif genes of four symbiotic Anabaena azollae show a highly conserved physical arrangement

Cloned DNA from Anabaena sp. PCC 7120 was used to determine the distribution of restriction sites around NifHDK genes of endosymbionts extracted from Azolla species. Although many of the restriction sites of the symbiotic Anabaena nifHDK genes differed from those of the free-living Anabaeba sp. PCC 7120, three apparently identical restriction sites were found in and around the nifD region. The arrangement of A. azollae and Anabaena sp. PCC 7120 nif H,D,K genes appears similar, with nifH and nifD linked and nifK some distance away from nifD. The A. azollae nifHDK genes appear strongly conserved among the Azolla species examined, regardless of the geographical origin of the ferns.     

Genetic analysis of nitrogen fixation in a tropical fast-growing Rhizobium

The Rhizobium strain ORS571, which is associated with the tropical legume Sesbania rostrata, has the property of growing in the free-living state at the expense of ammonia or N₂ as sole nitrogen source. Five mutants, isolated as unable to form colonies on plates under conditions of nitrogen fixation, were studied. All of them, which appear as Fix^- in planta, are nif mutants. With mutant 5740, nitrogenase activity of the crude extract was restored by addition of pure Mo-Fe protein of Klebsiella pneumoniae. A 13-kb BamHI DNA fragment from the wild-type strain, which hybridized with a probe carrying the nifHDK genes of K. pneumoniae, was cloned in vector pRK290 to yield plasmid pRS1. The extent of homology between the probe and the BamHI fragment was estimated at 4 kb and hybridization with K. pneumoniae nifH, nifK, and possibly nifD was detected. The pRS1 plasmid was introduced into the sesbania rhizobium nif mutants. Genetic complementation was observed with strain 5740(pRS1) both in the free-living state and in planta. It thus appears that biochemistry and genetics of nitrogen fixation in this particular Rhizobium strain can be performed with bacteria grown under non-symbiotic conditions.     

Contiguous organization of nitrogenase genes in a heterocystous cyanobacterium

The organization of the three structural nitrogen fixation (nif) genes that encode nitrogenase (nif K and nif D) and nitrogenase reductase (nif H) have been examined in a number of cyanobacteria. Hybridization of Anabaena 7120 nif gene probes to restriction endonuclease-digested genomic DNA has shown (a) that cyanobacteria incapable of N₂ fixation have no regions of DNA with significant homology to the three nif probes, (b) that Pseudanabaena sp., a nonheterocystous cyanobacterium, has a contiguous nif KDH gene cluster, and (c) that in contrast with other heterocystous cyanobacteria, Fischerella sp. has a contiguous nif KDH gene cluster.     

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.     

Paenibacillus piscarius sp. nov., a novel nitrogen-fixing species isolated from the gut of the armored catfish Parotocinclus maculicauda

A Gram-positive, nitrogen-fixing and endospore-forming strain, designated P121^T, was isolated from the gut of the armored catfish (Parotocinclus maculicauda) and identified as a member of the genus Paenibacillus based on the sequences of the 16S rRNA encoding gene, rpoB, gyrB and nifH genes and phenotypic analyses. The most closely related species to strain P121^T were Paenibacillus rhizoplanae DSM 103993^T, Paenibacillus silagei DSM 101953^T and Paenibacillus borealis DSM 13188^T, with similarity values of 98.9, 98.3 and 97.6%, respectively, based on 16S rRNA gene sequences. Genome sequencing revealed a genome size of 7,513,698 bp, DNA G?+?C content of 53.9 mol% and the presence of the structural nitrogenase encoding genes (nifK, nifD and nifH) and of other nif genes necessary for nitrogen fixation. Digital DNA-DNA hybridization (dDDH) experiments and average nucleotide identity (ANI) analyses between strain P121^T and the type strains of the closest species demonstrated that the highest values were below the thresholds of 70% dDDH (42.3% with P. borealis) and 95% ANI (84.28% with P. silagei) for bacterial species delineation, indicating that strain P121^T represents a distinct species. Its major cellular fatty acid was anteiso-C_15:0 (42.4%), and the major isoprenoid quinone was MK-7. Based on physiological, genomic, biochemical and chemotaxonomic characteristics, we propose that strain P121^T represents a novel species for which the name Paenibacillus piscarius sp. nov. is proposed (type strain?=?DSM 25072?=?LFB-Fiocruz 1636).

     

The nifH, nifM and nifN genes of Azotobacter vinelandii: Characterisation by Tn5 mutagenesis and isolation from pLAFR1 gene banks

Summary Tn5 was introduced into Azotobacter vinelandii on a suicide vector, pGS9. Three Nif^- mutants were found to carry Tn5 in nifH (MV6), in nifN (MV22), and in or near nifM (MV21), from the results of hybridisation experiments. For MV21 and MV22 this was also shown by complementation with the nif genes of Klebsiella pneumoniae on pRD1. MV6 failed to synthesis the nifH, D and K gene products. MV6 and MV22 fixed nitrogen in the absence of supplied molybdenum while mutant MV21 did not, suggesting that the nifM gene product may be required for the alternative nitrogenase system synthesised in azotobacteria under conditions of molybdenum deprivation. Reconstitution experiments with mutant extracts showed that MV22 (nifN ^-) lacked the FeMo cofactor and that MV21 (NifM^-) synthesised inactive Fe protein. These biochemical phenotypes are identical to those of the K. pneumoniae nifN and nifM mutants, respectively, demonstrating that these genes have the same function in both K. pneumoniae and A. vinelandii. Complementation of the A. vinelandii mutants with pLAFR1 gene banks of A. vinelandii or a. chroococcum yielded three cosmids of interest. pLV10 complemented UW91, a nifH mutant, and corrected the defect in MV6 after recombination with the mutant genome. It also carried nifD (but not nifK) and about 18 kb of DNA upstream from nifH. pLV1 from the A. vinelandii gene bank complemented both MV21 and MV22 as did pLC11, isolated from the A. chroococcum gene bank. Both pLV1 and pLC11 carried part of the nif cluster downstream of nifHDK which also includes nifEN and nifMVS on about 22 kb of DNA.     

Comparative Genomic Analysis of N2-Fixing and Non-N2-Fixing Paenibacillus spp.: Organization,Evolution and Expression of the Nitrogen Fixation Genes

We provide here a comparative genome analysis of 31 strains within the genus Paenibacillus including 11 new genomic sequences of N₂-fixing strains. The heterogeneity of the 31 genomes (15 N₂-fixing and 16 non-N₂-fixing Paenibacillus strains) was reflected in the large size of the shell genome, which makes up approximately 65.2% of the genes in pan genome. Large numbers of transposable elements might be related to the heterogeneity. We discovered that a minimal and compact nif cluster comprising nine genes nifB, nifH, nifD, nifK, nifE, nifN, nifX, hesA and nifV encoding Mo-nitrogenase is conserved in the 15 N₂-fixing strains. The nif cluster is under control of a σ⁷⁰-depedent promoter and possesses a GlnR/TnrA-binding site in the promoter. Suf system encoding [Fe–S] cluster is highly conserved in N₂-fixing and non-N₂-fixing strains. Furthermore, we demonstrate that the nif cluster enabled Escherichia coli JM109 to fix nitrogen. Phylogeny of the concatenated NifHDK sequences indicates that Paenibacillus and Frankia are sister groups. Phylogeny of the concatenated 275 single-copy core genes suggests that the ancestral Paenibacillus did not fix nitrogen. The N₂-fixing Paenibacillus strains were generated by acquiring the nif cluster via horizontal gene transfer (HGT) from a source related to Frankia. During the history of evolution, the nif cluster was lost, producing some non-N₂-fixing strains, and vnf encoding V-nitrogenase or anf encoding Fe-nitrogenase was acquired, causing further diversification of some strains. In addition, some N₂-fixing strains have additional nif and nif-like genes which may result from gene duplications. The evolution of nitrogen fixation in Paenibacillus involves a mix of gain, loss, HGT and duplication of nif/anf/vnf genes. This study not only reveals the organization and distribution of nitrogen fixation genes in Paenibacillus, but also provides insight into the complex evolutionary history of nitrogen fixation.