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PubMed2006

The evolutionary diversification of cyanobacteria: molecular-phylogenetic and paleontological perspectives.

Cyanobacteria have played a significant role in Earth history as primary producers and the ultimate source of atmospheric oxygen. To date, however, how and when the group diversified has remained unclear. Here, we combine molecular phylogenetic and paleontological studies to elucidate the pattern and timing of early cyanobacterial diversification. 16S rRNA, rbcL, and hetR genes were sequenced from 20 cyanobacterial strains distributed among 16 genera, with particular care taken to represent the known diversity of filamentous taxa. Unlike most other bacteria, some filamentous cyanobacteria evolved a degree of cell differentiation, producing both specialized cells for nitrogen fixation (heterocysts) and resting cells able to endure environmental stress (akinetes). Phylogenetic analyses support the hypothesis that cyanobacteria capable of cell differentiation are monophyletic, and the geological record provides both upper and lower bounds on the origin of this clade. Fossil akinetes have been identified in 1,650- to 1,400-mega-annum (Ma) cherts from Siberia, China, and Australia, and what may be the earliest known akinetes are preserved in approximately 2,100-Ma chert from West Africa. Geochemical evidence suggests that oxygen first reached levels that would compromise nitrogen fixation (and hence select for heterocyst differentiation) 2,450-2,320 Ma. Integrating phylogenetic analyses and geological data, we suggest that the clade of cyanobacteria marked by cell differentiation diverged once between 2,450 and 2,100 Ma, providing an internal bacterial calibration point for studies of molecular evolution in early organisms.

Base Sequence↗

PubMed2026

Whole-genome sequencing and characterization of Pseudomonas stutzeri P1 endophyte isolated from potato unveils plant growth-promoting and other traits.

Endophytic bacteria play an important role in plant growth promotion and stress tolerance, offering sustainable alternatives to chemical inputs in agriculture. In this study, an endophytic bacterial strain P1 was isolated and identified as Pseudomonas stutzeri, a plant-associated bacterium exhibiting multiple plant growth-promoting traits (PGPTs). Biochemical (qualitative and quantitative) and in vitro analyses demonstrated nitrogen fixation, phosphate solubilization, ammonia production, indole-3-acetic acid (IAA) production, biofilm formation, and tolerance to abiotic stresses, including salinity and drought. Furthermore, the P1 strain displayed strong biocontrol activity against the fungal pathogen Fusarium oxysporum f. sp. cumini, indicating its potential to mitigate biotic stress. Whole-genome sequencing generated a high-quality complete genome of 4,758,235 bp. Functional annotation showed enrichment of metabolic pathways associated with plant-microbe interactions and environmental adaptation. Further analyses using KEGG and PGPT-pred data confirmed the presence of genes associated with direct and indirect PGPT, such as nitrogen fixation, phosphate solubilization, biofilm formation, and stress tolerance. The genome also contained genes related to CAZymes, adhesion, and motility, highlighting a strong plant association, whereas the genome lacked major virulence factors and antimicrobial traits, supporting the non-pathogenic nature of the P1 strain. Overall, these findings demonstrate the potential of P1 as a promising bioinoculant candidate for sustainable agriculture in the potato sector.

PGPT-associated genes↗

PubMed1997

Evidence for multiple substrate-reduction sites and distinct inhibitor-binding sites from an altered Azotobacter vinelandii nitrogenase MoFe protein.

The arginine-277 residue of the alpha-subunit of the nitrogenase MoFe protein was targeted for substitution because it is (i) a close neighbor of alpha-cysteine-275, which is one of only two residues anchoring the FeMo cofactor to the polypeptide, and (ii) a component of a potential channel for entry/exit of substrates/products and for accepting FeMo cofactor during MoFe-protein maturation. Several of the eight mutant strains constructed were capable of good diazotrophic growth and also contained FeMo cofactor as indicated by its biologically unique S = 3/2 EPR spectrum. These observations indicate that the positively charged alpha-arginine-277 residue is not required for acceptance of the negatively charged FeMo cofactor by the separately synthesized, cofactor-deficient, apo-MoFe protein. The wide range of nitrogen-fixation phenotypes shown by these mutant strains generally correlated well with their C2H2- and proton-reduction activities, which range from 5 to 65% of wild-type activity. One notable exception is the histidine-substituted strain, DJ788 (alpha-277His). This strain, although unable to fix N2 and grow diazotrophically, elaborates an altered alpha-277His MoFe protein that catalyzes the reduction of the alternative substrates, C2H2, HCN, HN3, and protons. These observations are best explained if multiple redox levels are available to the MoFe protein but the alpha-277His MoFe protein is incapable of reaching the more-reduced redox levels required for nitrogen fixation. Under nonsaturating CO concentrations, the alpha-277His MoFe-protein-catalyzed reduction of C2H2 showed sigmoidal kinetics, which is consistent with inhibitor-induced cooperativity among two C2H4-evolving sites and indicates the presence of three sites, which can be simultaneously occupied, on the MoFe protein. Similar kinetics were not observed for alpha-277His MoFe-protein-catalyzed reduction of either HCN or HN3 with nonsaturating CO levels, indicating that these substrates are unlikely to share common binding sites with C2H2. Further, CN- did not induce cooperativity in C2H2 reduction and, therefore, CO and CN- are unlikely to share a common binding site. These changed substrate specificities, reinforced by changes in the FeMo-cofactor-derived S = 3/2 EPR spectrum, clearly indicate the importance of the alpha-277 residue in catalysis and the delicate control exerted on the properties of bound FeMo cofactor by its polypeptide environment.

Arginine↗

PubMed1985

Isolation of transfer-negative nif-plasmids (pCE1) and their integration into the chromosome of Escherichia coli with the help of phage Mu.

Strain JC5466 of Escherichia coli K12 harbouring the nitrogen fixation plasmid pCE1 was lysogenized with bacteriophage Mu cts, followed by partial induction and infection with bacteriophage PRD1. This made it possible to obtain transfer-defective derivatives of pCE1, carrying Mu prophage. These derivatives could be mobilized by using the helper plasmid pME400 and it was possible to segregate the helper plasmid from the donor plasmid in the transconjugants. By incubating the strains 302 and 328 at 42 degrees C, for induction of Mu prophage, derivatives with different plasmid contents could be obtained such as strains without plasmids, some with smaller or larger plasmids and others possessing plasmids without any visible alteration in size. Integration of the nitrogen-fixation (nif) genes into the chromosomes of the strains without plasmids and those containing a smaller plasmid, was confirmed by Southern hybridization using radioactive nifKDH DNA. Conjugation assays have shown that the plasmid is integrated into the chromosome as a unit but that it can also be excised.

Chromosomes, Bacterial↗

PubMed1983

Cloning and expression of a DNA fragment carrying a his nifA fusion and the nifBQ operon from a nif constitutive mutant of Klebsiella pneumoniae.

From the nifc mutant plasmid pPC868, previously shown to carry a DNA duplication responsible for the Nifc phenotype, a 10 kb HindIII fragment was cloned into the multicopy vector pBR325. Restriction analysis of the resulting plasmids and in vitro deleted derivatives confirmed that the mutation was a fusion between his and nifLA. The order was hisG-hisD'-'nifL-nifA so that nifA was transcribed under the control of the his promoter and the nifL gene was altered. In addition the cloned fragment contained the adjacent nifBQ operon, and complementation data revealed that the nifA, nifB and hisG genes were expressed. Synthesis of nifA product under the transcription control of the his (or cat [CmR]) promoter enabled complementation of nifA and nifB mutations either in the absence or the presence of ammonia, but did not restore nitrogen fixation in a glnF mutant. Therefore, the nifA gene product requires glnF for its positive control function in a manner analogous to ntrC. Protein content analysis of minicells containing various multicopy nif plasmids confirmed the genetic organization mentioned above. A new polypeptide of 51,500 daltons was found whose synthesis was observed at 30 degrees C but not at 37 degrees C. According to the physical map, this protein could be the nifB gene product. Our results are in agreement with nifB transcription being under the control of a thermolabile nifA product. Moreover we obtained results suggesting that the presence of multiple copies of a functional nifB gene inhibited nitrogen fixation.

Base Sequence↗

PubMed1999

The signal transduction protein GlnK is required for NifL-dependent nitrogen control of nif gene expression in Klebsiella pneumoniae.

In Klebsiella pneumoniae, transcription of the nitrogen fixation (nif) genes is regulated in response to molecular oxygen or availability of fixed nitrogen by the coordinated activities of the nifA and nifL gene products. NifA is a nif-specific transcriptional activator, the activity of which is inhibited by interaction with NifL. Nitrogen control of NifL occurs at two levels: transcription of the nifLA operon is regulated by the global ntr system, and the inhibitory activity of NifL is controlled in response to fixed nitrogen by an unknown factor. K. pneumoniae synthesizes two PII-like signal transduction proteins, GlnB, which we have previously shown not to be involved in the response of NifL to fixed nitrogen, and the recently identified protein GlnK. We have now cloned the K. pneumoniae glnK gene, studied its expression, and shown that a null mutation in glnK prevents NifL from responding to the absence of fixed nitrogen, i.e., from relieving the inhibition of NifA activity. Hence, GlnK appears to be involved, directly or indirectly, in NifL-dependent regulation of nif gene expression in K. pneumoniae. Comparison of the GlnB and GlnK amino acid sequences from six species of proteobacteria identifies five residues (residues 3, 5, 52, 54, and 64) which serve to distinguish the GlnB and GlnK proteins.

Amino Acid Sequence↗

PubMed1992

Rhodobacter sphaeroides rdxA, a homolog of Rhizobium meliloti fixG, encodes a membrane protein which may bind cytoplasmic [4Fe-4S] clusters.

In the photosynthetic bacterium Rhodobacter sphaeroides, a chromosomal gene, rdxA, which encodes a 52-kDa protein, was found to be homologous to fixG, the first gene of a Rhizobium meliloti nitrogen fixation operon on the pSym plasmid (D. Kahn, M. David, O. Domergue, M.-L. Daveran, J. Ghai, P. R. Hirsch, and J. Batut, J. Bacteriol. 171:929-939, 1989). The deduced amino acid sequences of RdxA and FixG are 53% identical and 73% similar; sequence analyses suggested that each has five transmembrane helices and a central region resembling bacterial-type ferredoxins. Translational fusion proteins with an alkaline phosphatase reporter group were expressed in both R. sphaeroides and Escherichia coli and were used to assess the membrane topology of RdxA. Its ferredoxinlike sequence, which may bind two [4Fe-4S] centers, was found to be cytoplasmically located. Genetic disruptions showed that rdxA is not essential for nitrogen fixation in R. sphaeroides. Immediately downstream of rdxA, an open reading frame (ORFT2) that encoded a 48-kDa protein was found. This DNA sequence was not homologous to any region of the R. meliloti fixG operon. The N-terminal sequence of the ORFT2 gene product resembled amino acid sequences found in members of the GntR family of regulatory proteins (D. J. Haydon and J. R. Guest, FEMS Microbiol. Lett. 79:291-296, 1991). The rdxA gene was localized to the smaller of two R. sphaeroides chromosomes, upstream of and divergently transcribed from hemT, which encodes one of two 5-aminolevulinate synthase isozymes. The rdxA and hemT genes may share a transcriptional regulatory region. Southern hybridization analysis demonstrated the presence of an rdxA homolog on the R. sphaeroides large chromosome. The functions of this homolog, like those of rdxA, remain to be determined, but roles in oxidation-reduction processes are likely.

Amino Acid Sequence↗

PubMed1986

Nitrogen-fixing pseudomonads isolated from roots of plants grown in the canadian high arctic.

Root-associated bacteria capable of reducing acetylene to ethylene (biological nitrogen fixation) were isolated from various native plants grown in the Canadian High Arctic. All the strains belonged to the genus Pseudomonas but varied in several physiological characteristics. The rates of acetylene reduction at 14 or 20 degrees C were higher than at 25 or 9 degrees C. Six strains reduced acetylene at 4 degrees C. All the strains exhibited chemotaxis to l-asparagine in semisolid agar at 4 to 25 degrees C. Eleven strains colonized roots of canola (Brassica campestris cv. Tobin) in field soil at population densities of log 4.3 to log 5.1 CFU/g of fresh root at 14 degrees C and log 4.0 to log 5.2 CFU/g of fresh root at 25 degrees C. Some of these nitrogen-fixing pseudomonad strains demonstrated a competitive advantage for root colonization over other rhizosphere bacteria at low temperatures. The combined capabilities of nitrogen fixation and root colonization by diazotrophic pseudomonads may be useful for the development of a biofertilizer inoculant for temperate and cold regions.

Journal Article↗

PubMed1989

Hydrogen inhibition of nitrogen reduction by nitrogenase in isolated soybean nodule bacteroids.

Dihydrogen, a by-product of biological nitrogen fixation, is a competitive inhibitor of N(2) reduction by nitrogenase. To evaluate the significance of H(2) inhibition in vivo, we have measured the apparent inhibition constant for H(2) inhibition of N(2) reduction in Bradyrhizobium japonicum bacteroids isolated from soybean nodules. The rate of N(2) reduction was measured as ammonia production by bacteroids incubated in a buffer containing 200 micromolar leghemoglobin and 10 millimolar succinate under 0.02 atmosphere O(2) and various concentrations of N(2) and H(2). The apparent inhibition constant for H(2) under these conditions was determined to be approximately 0.03 atmosphere. This relatively low value strengthens the proposal that H(2) inhibition of N(2) reduction may be a significant factor in lowering the efficiency of nitrogen fixation in legume nodules.

Journal Article↗

PubMed1987

Isolation and nitrogenase activity of vesicles from Frankia sp. strain EAN1pec.

Vesicles, specialized cell structures thought to be the site of nitrogen fixation in the actinorhizal bacteria, were isolated from Frankia sp. strain EAN1pec by using French pressure disruption of mycelia followed by differential and isopycnic gradient centrifugation. The isolated vesicles reduced acetylene when incubated anaerobically with Mg2+ ions, ATP, and dithionite. No nitrogenase activity was detected in the disrupted mycelial fractions. Vesicles permeabilized by freeze-thaw or detergents showed increased rates of acetylene reduction due to increased permeability of dithionite. The effect on nitrogenase activity of different ATP concentrations was the same in normal and permeabilized vesicles. The endogenous respiratory rate of vesicles was significantly lower than that of mycelia, and the respiration rate of vesicles did not increase following the addition of succinate. The low respiratory activity of vesicles and their apparent dependence on externally supplied ATP for acetylene reduction suggest that the energy and reducing power for nitrogen fixation may be supplied from the mycelia to which they are attached.

Anaerobiosis↗

PubMed2026

Genetic legacy effects in a mungbean-wheat rotation reveal potential to breed for system-level yield gains.

Legume crops provide protein-rich food, serve as critical disease breaks in cereal rotations, and contribute to soil fertility through symbiotic nitrogen fixation. However, crop improvement programs typically focus on within-crop performance rather than system-level benefits. We hypothesize that legacy effects (the influence of one crop's genotype on subsequent crop performance) are under genetic control and could be targeted in breeding programs. To test this, we evaluated how 309 genetically diverse mungbean genotypes influenced subsequent wheat performance. The mungbean panel was grown, followed by a single wheat cultivar sown in the same plots. Remarkably, wheat yield varied by nearly 1 t ha-1 (2.52-3.49 t ha-1), depending solely on the preceding mungbean genotype. Legacy effects showed moderate heritability (H2: 0.43-0.65), suggesting untapped genetic potential for breeding. However, these estimates were derived from a single site and season and require validation across environments. Analyses of mungbean traits, soil properties, and volatile organic compounds identified root architecture, symbiotic nitrogen fixation, and the soil microbiome as potential contributors to legacy effects, although these mechanisms remain to be tested directly. Haplotype mapping identified genomic regions in mungbean associated with wheat yield and, to a lesser extent, grain protein, revealing trade-offs between within-crop performance and legacy effects. Genetic simulations based on empirically derived marker effects compared genomic selection strategies targeting mungbean yield, wheat yield, or both simultaneously. A selection strategy placing equal weight on mungbean yield and subsequent wheat yield (50:50 weighting) achieved simultaneous gains in both crops (19.5% and 7.6%), highlighting the potential to breed for system-level productivity with reduced input requirements.

crop rotations↗

PubMed2001

[IRepeat sequences of genomes of Rhizobium and Sinorhizobium meliloti: a comparative analysis].

Amongst prokaryotic genomes, those of nitrogen-fixing members of the Rhizobiaceae family are relatively large (6-9 Mb), often include mega-plasmids of 1.5-2 Mb, and contain numerous families of repeated DNA sequences. Although most essential nodulation and nitrogen fixation genes are well characterized, these represent only a small fraction of the DNA content. Little is known about the detailed structure of rhizobial genomes. With the development of sequencing techniques and new bio-informatic tools such studies become possible, however. Using the 2275 shotgun sequences of ANU265 (a derivative of NGR234 cured of pNGR234a), we have identified numerous families of repeats. Amongst these, the 58-bp-long NGRREP-4 represents the third most abundant DNA sequence after the RIME1 and RIME2 repeats, all of which are also found in Sinorhizobium meliloti. Surprisingly, studies on the distribution of these elements showed that in proportion to its size, the chromosome of NGR234 carries many more RIME modules than pNGR234a or pNGR234b. Together with the presence in NGR234 and S. meliloti 1021 of an insertion sequence (IS) element more conserved than essential nodulation and nitrogen fixation genes, these results give new insights into the origin and evolution of rhizobial genomes.

Base Sequence↗

PubMed1997

Negative autoregulation of the Rhizobium meliloti fixK gene is indirect and requires a newly identified regulator, FixT.

fixK genes are crp/fnr homologues that have been discovered in diverse Rhizobium spp., in which they are usually essential for symbiotic nitrogen fixation. One recurrent function of fixK genes in rhizobia is to activate the transcription of operons required for respiration in the microoxic environment of the nodule. In a similar manner to its Escherichia coli crp and fnr homologues, R. meliloti fixK regulates its own expression negatively. However, we demonstrate here that fixK negative autoregulation is not direct and, instead, involves a newly identified gene, fixT, the expression of which depends on fixK. Inactivation of fixT resulted in derepression of fixK expression under free-living microoxic conditions. Furthermore, constitutively expressed fixT strongly repressed fixK-lacZ expression in the absence of a functional fixK gene. Several lines of evidence indicate that fixT is active via its protein product FixT. FixT does not resemble any protein present in databases so far. Nodules induced by a fixT mutant were Fix+, thus demonstrating that fixT is not essential for symbiotic nitrogen fixation.

Amino Acid Sequence↗

PubMed1999

Isolation and characterization of alfalfa-nodulating rhizobia present in acidic soils of central argentina and uruguay

We describe the isolation and characterization of alfalfa-nodulating rhizobia from acid soils of different locations in Central Argentina and Uruguay. A collection of 465 isolates was assembled, and the rhizobia were characterized for acid tolerance. Growth tests revealed the existence of 15 acid-tolerant (AT) isolates which were able to grow at pH 5.0 and formed nodules in alfalfa with a low rate of nitrogen fixation. Analysis of those isolates, including partial sequencing of the genes encoding 16S rRNA and genomic PCR-fingerprinting with MBOREP1 and BOXC1 primers, demonstrated that the new isolates share a genetic background closely related to that of the previously reported Rhizobium sp. Or191 recovered from an acid soil in Oregon (B. D. Eardly, J. P. Young, and R. K. Selander, Appl. Environ. Microbiol. 58:1809-1815, 1992). Growth curves, melanin production, temperature tolerance, and megaplasmid profiles of the AT isolates were all coincident with these characteristics in strain Or191. In addition to the ability of all of these strains to nodulate alfalfa (Medicago sativa) inefficiently, the AT isolates also nodulated the common bean and Leucaena leucocephala, showing an extended host range for nodulation of legumes. In alfalfa, the time course of nodule formation by the AT isolate LPU 83 showed a continued nodulation restricted to the emerging secondary roots, which was probably related to the low rate of nitrogen fixation by the largely ineffective nodules. Results demonstrate the complexity of the rhizobial populations present in the acidic soils represented by a main group of N2-fixing rhizobia and a second group of ineffective and less-predominant isolates related to the AT strain Or191.

Journal Article↗

PubMed2006

New insights on glyphosate mode of action in nodular metabolism: Role of shikimate accumulation.

The short-term effects of the herbicide glyphosate (1.25-10 mM) on the growth, nitrogen fixation, carbohydrate metabolism, and shikimate pathway were investigated in leaves and nodules of nodulated lupine plants. All glyphosate treatments decreased nitrogenase activity rapidly (24 h) after application, even at the lowest and sublethal dose used (1.25 mM). This early effect on nitrogenase could not be related to either damage to nitrogenase components (I and II) or limitation of carbohydrates supplied by the host plant. In fact, further exposure to increasing glyphosate concentrations (5 mM) and greater time after exposure (5 days) decreased nodule starch content and sucrose synthase (SS; EC 2.4.1.13) activity but increased sucrose content within the nodule. These effects were accompanied by a great inhibition of the activity of phosphoenolpyruvate carboxylase (PEPC; EC 4.1.1.31). There were remarkable and rapid effects on the increase of shikimic and protocatechuic (PCA) acids in nodules and leaves after herbicide application. On the basis of the role of shikimic acid and PCA in the regulation of PEPC, as potent competitive inhibitors, this additional effect provoked by glyphosate on 5-enolpyruvylshikimic-3-phosphate synthase enzyme (EPSPS; EC 2.5.1.19) inhibition would divert most PEP into the shikimate pathway, depriving energy substrates to bacteroids to maintain nitrogen fixation. These findings provide a new explanation for the effectiveness of glyphosate as a herbicide in other plant tissues, for the observed differences in tolerance among species or cultivars, and for the transitory effects on glyphosate-resistant transgenic crops under several environmental conditions.

Carbohydrate Metabolism↗

PubMed1993

Regulation of the kinase activity of heme protein FixL from the two-component system FixL/FixJ of Rhizobium meliloti.

The Rhizobium meliloti two-component system FixL/FixJ regulates nitrogen fixation in response to oxygen during symbiosis. FixJ is a transcriptional activator of critical nif and fix promoters; its in vivo activity is enhanced by FixL in diminished oxygen (David, M., Daveran, M.-L., Batut, J., Dedieu, A., Domergue, O., Ghai, J., Hertig, C., Boistard, P., and Kahn, D. (1988) Cell 54, 671-683; Virts, E. L., Stanfield, S. W., Helinski, D. R., and Ditta, G. S. (1988) Proc. Natl. Acad. Sci. U.S.A. 85, 3062-3065). FixL* is a soluble truncated version of FixL that contains heme; it catalyzes its autophosphorylation and the phosphorylation of FixJ (Gilles-Gonzalez, M.A., Ditta, G. S., and Helinski, D. R. (1991) Nature 350, 170-172). We examine the kinetics of phosphoryl transfer in this system. First, there is a slow autophosphorylation of FixL* in ATP that is accelerated in the absence of oxygen and in the presence of Mn2+. This reaction is reversible, i.e. phospho-FixL* reacts with ADP to generate ATP. Since the reverse reaction is faster, most FixL* is not phosphorylated at equilibrium. Next, there is a rapid phosphoryl transfer directly from phospho-FixL* to FixJ that is unaffected by oxygen. Finally, phospho-FixJ is hydrolyzed; this reaction is very fast and not controlled by oxygen. We propose that in addition to the oxygen signal previously noted in vivo, energy charge and manganese concentration are also indicators of symbiosis that impact on the induction of nitrogen fixation genes.

Adenosine Diphosphate↗

PubMed2000

Differential regulation of fixN-reiterated genes in Rhizobium etli by a novel fixL-fixK cascade.

Among the complexities in the regulation of nitrogen fixation in the Rhizobiaceae are reiteration of regulatory components as well as variant roles for each component between species. For Rhizobium etli CFN42, we reported that the symbiotic plasmid (pCFN42d) contains a key regulatory gene (fixKd) and genes for a symbiotic cytochrome oxidase (fixNOQPd). Here we discuss the occurrence of reiteration of these genes (fixKf and fixNOQPf) and the finding of an unusual fixL homolog on a plasmid previously considered cryptic (pCFN42f). The structure of the deduced FixL polypeptide is suggestive of a fusion of the receiver and transmitter modules of a two-component regulatory system as described in R. leguminosarum bv. viciae VF39. Gene fusion analysis, coupled with mutation of each regulatory element, revealed that free-living expression of FixKf was dependent fully on FixL. In contrast, synthesis of FixKd was not detected under the conditions tested. The FixKf protein is needed for microaerobic expression of both fixN reiterations, whereas the FixKd protein appears to be dispensable. Interestingly, expression of the fixN reiterations exhibits a differential dependence for FixL, where transcription of fixNf was suppressed in the absence of FixL but expression of fixNd still showed significant levels. This suggests the existence of a FixL-independent mechanism for expression of the fixNd reiteration. Surprisingly, mutations in fixL, fixKd, or fixKf (either singly or in combination) did not alter symbiotic effectiveness. A mutation in fixNd (but not in fixNf) was, however, severely affected, indicating a differential role for these reiterations in nitrogen fixation.

Artificial Gene Fusion↗

PubMed1990

A Rhizobium leguminosarum mutant defective in symbiotic iron acquisition.

Iron acquisition by symbiotic Rhizobium spp. is essential for nitrogen fixation in the legume root nodule symbiosis. Rhizobium leguminosarum 116, an ineffective mutant strain with a defect in iron acquisition, was isolated after nitrosoguanidine mutagenesis of the effective strain 1062. The pop-1 mutation in strain 116 imparted to it a complex phenotype, characteristic of iron deficiency: the accumulation of porphyrins (precursors of hemes) so that colonies emitted a characteristic pinkish-red fluorescence when excited by UV light, reduced levels of cytochromes b and c, and wild-type growth on high-iron media but low or no growth in low-iron broth and on solid media supplemented with the iron scavenger dipyridyl. Several iron(III)-solubilizing agents, such as citrate, hydroxyquinoline, and dihydroxybenzoate, stimulated growth of 116 on low-iron solid medium; anthranilic acid, the R. leguminosarum siderophore, inhibited low-iron growth of 116. The initial rate of 55Fe uptake by suspensions of iron-starved 116 cells was 10-fold less than that of iron-starved wild-type cells. Electron microscopic observations revealed no morphological abnormalities in the small, white nodules induced by 116. Nodule cortical cells were filled with vesicles containing apparently normal bacteroids. No premature degeneration of bacteroids or of plant cell organelles was evident. We mapped pop-1 by R plasmid-mediated conjugation and recombination to the ade-27-rib-2 region of the R. leguminosarum chromosome. No segregation of pop-1 and the symbiotic defect was observed among the recombinants from these crosses. Cosmid pKN1, a pLAFR1 derivative containing a 24-kilobase-pair fragment of R. leguminosarum DNA, conferred on 116 the ability to grow on dipyridyl medium and to fix nitrogen symbiotically. These results indicate that the insert cloned in pKN1 encodes an element of the iron acquisition system of R. leguminosarum that is essential for symbiotic nitrogen fixation.

Chromosome Mapping↗