Induction of nitrogen-fixing nodules on clover requires only 32 kilobase pairs of DNA from the Rhizobium trifolii symbiosis plasmid.

Overlapping subclones from the Rhizobium trifolii symbiosis plasmid pRt843a were generated by using in vivo and in vitro methods. Subclones were assayed for symbiotic phenotype by introducing them into a derivative of R. trifolii ANU843 cured of its symbiosis plasmid and testing the transconjugant strains for the ability to induce nitrogen-fixing nodules on clover. One subclone spanning 32 kilobase pairs (kb) of DNA from pRt843a was found to restore nitrogen fixation ability. This subclone included all known nodulation genes of R. trifolii ANU843 and the nitrogenase structural genes nifHDK. In addition, regions homologous to fixABC, nifA, nifB, nifE, and nifN genes of other nitrogen-fixing bacteria were identified in this 32-kb subclone by DNA-DNA hybridization. Transposon mutagenesis of this subclone confirmed that regions containing these nif and fix genes were required for induction of nitrogen-fixing nodules on clover. In addition, a region located 5 kb downstream of the nifK gene was found to be required for induction of nitrogen-fixing nodules. No homology to known nif and fix genes could be detected in this latter region.     

Identification and mapping of nitrogen fixation genes of Rhodobacter capsulatus: duplication of a nifA-nifB region.

Rhodobacter capsulatus mutants unable to fix nitrogen were isolated by random transposon Tn5 mutagenesis. The Tn5 insertion sites of 30 Nif- mutants were mapped within three unlinked chromosomal regions designated A, B, and C. The majority of Tn5 insertions (21 mutants) map within nif region A, characterized by two ClaI fragments of 2.5 and 25 kilobases (kb). The 17-kb ClaI fragment of nif region B contains six nif::Tn5 insertions, and the three remaining mutations are located on a 32-kb ClaI fragment of nif region C. Hybridization experiments using all 17 Klebsiella pneumoniae nif genes individually as probes revealed homology to nifE, nifS, nifA, and nifB in nif region A. The nifHDK genes were localized in nif region B. About 2 kb away from this operon, a second copy of the DNA fragments homologous to nifA and nifB, originally found in nif region A, was identified.     

Structural and functional analysis of nitrogenase genes from the broad-host-range Rhizobium strain ANU240

The genes encoding the structural components of nitrogenase, nifH, nifD and nifK, from the fast-growing, broad-host-range Rhizobium strain ANU240 have been identified and characterized. They are duplicated and linked in an operon nifHDK in both copies. Sequence analysis of the nifH gene from each copy, together with partial sequence analysis of the nifD and nifK genes, and restriction endonuclease analysis suggested that the duplication is precise. Comparison of the Fe-protein sequence from strain ANU240 with that from other nitrogen-fixing organisms revealed that, despite its broad host range and certain physiological properties characteristic of Bradyrhizobium strains, ANU240 is more closely related to the narrow-host-range Rhizobium strains than to the broad-host-range Bradyrhizobium strains. The promoter regions of both copies of the nif genes contain the consensus sequence characteristic of nif promoters, and functional analysis of the two promoters suggested that both nif operons are transcribed in nodules.     

Sequencing and functional analysis of the nifENXorf1orf2 gene cluster of Herbaspirillum seropedicae

A 5.1-kb DNA fragment from the nifHDK region of H. seropedicae was isolated and sequenced. Sequence analysis showed the presence of nifENXorf1orf2 but nifTY were not present. No nif or consensus promoter was identified. Furthermore, orf1 expression occurred only under nitrogen-fixing conditions and no promoter activity was detected between nifK and nifE, suggesting that these genes are expressed from the upstream nifH promoter and are parts of a unique nif operon. Mutagenesis studies indicate that nifN was essential for nitrogenase activity whereas nifXorf1orf2 were not. High homology between the C-terminal region of the NifX and NifB proteins from H. seropedicae was observed. Since the NifX and NifY proteins are important for FeMo cofactor (FeMoco) synthesis, we propose that alternative proteins with similar activities exist in H. seropedicae.     

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.     

DNA hybridization analysis of the nif region of two methylotrophs and molecular cloning of nif-specific DNA.

DNA isolated from two diazotrophic methylotrophs, the obligate methanotroph Methylosinus sp. strain 6 and the methanol autotroph Xanthobacter sp. H4-14, hybridized to DNA fragments encoding nitrogen fixation (nif) genes from Klebsiella pneumoniae. This interspecific nif homology was limited to DNA fragments encoding the nitrogenase structural proteins (nifH, nifD, and nifK) and specific methylotroph DNA sequences. The hybridization patterns obtained with the two methylotrophs were dissimilar, indicating that the nif region of methylotrophs is not physically conserved. By using the K. pneumoniae nif structural genes as a probe, a fragment of nif DNA from each methylotroph was cloned and characterized. The DNA fragment from Methylosinus sp. 6 encoded two polypeptides of 57,000 and 34,000 molecular weight.     

The structural nif genes of the cyanobacteria Gloeothece sp. and Calothrix sp. share homology with those of Anabaena sp., but the Gloeothece genes have a different arrangement.

Probes carrying the Anabaena sp. strain PCC 7120 nitrogenase reductase (nifH) and nitrogenase (nifK and nifD) genes were hybridized to Southern blots of DNA from the unicellular, aerobic nitrogen-fixing cyanobacterium Gloeothece sp. strain PCC 6909 and from the filamentous cyanobacterium Calothrix sp. strain PCC 7601. These data suggest that the Gloeothece sp. nif structural proteins must be similar to those of other diazotrophs and that the ability for aerobic nitrogen fixation does not reside in the nif protein complex. We also found that the nif structural genes of Gloeothece sp. are clustered, whereas those of Calothrix sp. are arranged more like those of Anabaena sp.     

A new symbiotic cluster on the pSym megaplasmid of Rhizobium meliloti 2011 carries a functional fix gene repeat and a nod locus.

A 290-kilobase (kb) region of the Rhizobium meliloti 2011 pSym megaplasmid, which contains nodulation genes (nod) as well as genes involved in nitrogen fixation (nif and fix), was shown to carry at least six sequences repeated elsewhere in the genome. One of these reiterated sequences, about 5 kb in size, had previously been identified as part of a cluster of fix genes located 220 kb downstream of the nifHDK promoter. Deletion of the reiterated part of this fix cluster does not alter the symbiotic phenotype. Deletion of the second copy of this reiterated sequence, which maps on pSym 40 kb upstream of the nifHDK promoter, also has no effect. Deletion of both of these copies however leads to a Fix- phenotype, indicating that both sequences carry functionally reiterated fix gene(s). The fix copy 40 kb upstream of nifHDK is part of a symbiotic cluster which also carries a nod locus, the deletion of which produces a marked delay in nodulation.     

Molecular cloning of nif DNA from Azotobacter vinelandii.

Two clones which contained nif DNA were isolated from a clone bank of total EcoRI-digested Azotobacter vinelandii DNA. The clones carrying the recombinant plasmids were identified by use of the 32P-labeled 6.2-kilobase (kb) nif insert from pSA30 (which contains the Klebsiella pneumoniae nifK, nifD, and nifH genes) as a hybridization probe. Hybridization analysis with fragments derived from the nif insert of pSA30 showed that the 2.6-kb insert from one of the plasmids (pLB1) contains nifK whereas the 1.4-kb insert from the other plasmid (pLB3) contains nifD. Marker rescue tests using genetic transformation indicated that the 2.6-kb A. vinelandii nif fragment contains the wild-type alleles for the nif-6 and nif-38 mutations carried by Nif- strains UW6 and UW38. The 1.4-kb insert contains the wild-type allele for the nif-10 mutation carried by Nif- strain UW10.     

Reiteration of genes involved in symbiotic nitrogen fixation by fast-growing Rhizobium japonicum.

By using cloned Rhizobium meliloti nodulation (nod) genes and nitrogen fixation (nif) genes, we found that the genes for both nodulation and nitrogen fixation were on a plasmid present in fast-growing Rhizobium japonicum strains. Two EcoRI restriction fragments from a plasmid of fast-growing R. japonicum hybridized with nif structural genes of R. meliloti, and three EcoRI restriction fragments hybridized with the nod clone of R. meliloti. Cross-hybridization between the hybridizing fragments revealed a reiteration of nod and nif DNA sequences in fast-growing R. japonicum. Both nif structural genes D and H were present on 4.2- and 4.9-kilobase EcoRI fragments, whereas nifK was present only on the 4.2-kilobase EcoR2 fragment. These results suggest that the nif gene organizations in fast-growing and in slow-growing R. japonicum strains are different.     

Cloning and characterization of hydrogen uptake genes from Rhizobium leguminosarum.

A gene library of genomic DNA from the hydrogen uptake (Hup)-positive strain 128C53 of Rhizobium leguminosarum was constructed by using the broad-host-range mobilizable cosmid vector pLAFR1. The resulting recombinant cosmids contained insert DNA averaging 21 kilobase pairs (kb) in length. Two clones from the above gene library were identified by colony hybridization with DNA sequences from plasmid pHU1 containing hup genes of Bradyhizobium japonicum. The corresponding recombinant cosmids, pAL618 and pAL704, were isolated, and a region of about 28 kb containing the sequences homologous to B. japonicum hup-specific DNA was physically mapped. Further hybridization analysis with three fragments from pHU1 (5.9-kb HindIII, 2.9-kb EcoRI, and 5.0-kb EcoRI) showed that the overall arrangement of the R. leguminosarum hup-specific region closely parallels that of B. japonicum. The presence of functional hup genes within the isolated cosmid DNA was demonstrated by site-directed Tn5 mutagenesis of the 128C53 genome and analysis of the Hup phenotype of the Tn5 insertion strains in symbiosis with peas. Transposon Tn5 insertions at six different sites spanning 11 kb of pAL618 completely suppressed the hydrogenase activity of the pea bacteroids.     

Further analysis of nitrogen fixation (nif) genes in Azotobacter chroococcum: identification and expression in Klebsiella pneumoniae of nifS, nifV, nifM, and nifB genes and localization of nifE/N-, nifU-, nifA- and fixABC-like genes

The results presented extend previous investigations on the genetics of nitrogen fixation in Azotobacter chroococcum and indicate that nif- and fix-like DNA is located in at least five different regions of the genome. Region I contains functional copies of nifS,V and M, as well as nifH, D and K, all of which complemented mutants of Klebsiella pneumoniae. In addition, nifE- and/or nifN-like and nifU-like DNA is located in this region. The organization of the nif cluster in region I closely resembles that of K. pneumoniae. though spread over 22 kb as compared with 14 kb. Region II contains a functional nifB gene, which complemented a K. pneumoniae nifB mutant, and seems to be adjacent to ap nifA-like gene. Region III harbours nifH*, encoding a second nitrogenase Fe-protein. Region IV contains a reiteration of nifE- on and/or nifN-like sequences, and DNA homologous to Rhizobium meliloti fixABC is present in region V. The apparent complexity of nifDNA in A. chroococcum is probably related to the two systems for N2-fixation pr present in this organism.     

The pleiotropic nature of symbiotic regulatory mutants: Bradyrhizobium japonicum nifA gene is involved in control of nif gene expression and formation of determinate symbiosis

In the slow-growing soybean symbiont, Bradyrhizobium japonicum (strain 110), a nifA-like regulatory gene was located immediately upstream of the previously mapped fixA gene. By interspecies hybridization and partial DNA sequencing the gene was found to be homologous to nifA from Klebsiella pneumoniae and Rhizobium meliloti, and to a lesser extent, also to ntrC from K. pneumoniae. The B. japonicum nifA gene product was shown to activate B. japonicum and K. pneumoniae nif promoters (using nif::lacZ translational fusions) both in Escherichia coli and B. japonicum backgrounds. In the heterologous E. coli system activation was shown to be dependent on the ntrA gene product. Site-directed insertion and deletion/replacement mutagenesis revealed that nifA is probably the promoter-distal cistron within an operon. NifA- mutants were Fix- and pleiotropic: (i) they were defective in the synthesis of several proteins including the nifH gene product (nitrogenase Fe protein); the same proteins had been known to be repressed under aerobic growth of B. japonicum but derepressed at low O2 tension; (ii) the mutants had an altered nodulation phenotype inducing numerous, small, widely distributed soybean nodules in which the bacteroids were subject to severe degradation. These results show that nifA not only controls nitrogenase genes but also one or more genes involved in the establishment of a determinate, nitrogen-fixing root nodule symbiosis.     

Relationship between tryptophan biosynthesis and indole-3-acetic acid production in Azospirillum: identification and sequencing of a trpGDC cluster

Summary Screening the tryptophan (Trp)-dependent indole-3-acetic acid (IAA) production of different Azospirillum species revealed that A. irakense KA3 released 10 times less IAA into the medium than A. brasilense Sp7. A cosmid library of strain Sp7 was transferred into A. irakense KA3 with the aim of characterizing genes involved in IAA biosynthesis. Trp-dependent IAA production was increased in two transconjugants which both contained an identical 18.5 kb HindIII fragment from Sp7. After Tn5 mutagenesis, cosmids carrying Tn5 insertions at 36 different positions of the 18.5 kb fragment were isolated and transferred into strain KA3. IAA production by the recipient strains was screened by HPLC. The Tn5 insertions of 4 clones with decreased IAA production were mapped on a 2 kb Sall — SphI fragment. Recombination of Tn5 insertions at this locus into the genome of strain Sp7 led to Trp auxotrophic mutants. A 5.2 kb EcoRI — SalI fragment including the kb SalI — SphI fragment was sequenced and six open reading frames were identified. Three of them were clustered and their deduced amino acid sequences showed significant similarity to TrpG, TrpD and TrpC, which are enzymes involved in tryptophan biosynthesis. One of the remaining open reading frames probably encodes an acetyltransferase. The region responsible for the enhanced Trp-dependent IAA production in strain KA3 corresponded to trpD, coding for the phosphoribosyl anthranilate transferase.