Azotobacter vinelandii is a soil bacterium related to the Pseudomonas genus that fixes nitrogen under aerobic conditions while simultaneously protecting nitrogenase from oxygen damage. In response to carbon availability, this organism undergoes a simple differentiation process to form cysts that are resistant to drought and other physical and chemical agents. Here we report the complete genome sequence of A. vinelandii DJ, which has a single circular genome of 5,365,318 bp. In order to reconcile an obligate aerobic lifestyle with exquisitely oxygen-sensitive processes, A. vinelandii is specialized in terms of its complement of respiratory proteins. It is able to produce alginate, a polymer that further protects the organism from excess exogenous oxygen, and it has multiple duplications of alginate modification genes, which may alter alginate composition in response to oxygen availability. The genome analysis identified the chromosomal locations of the genes coding for the three known oxygen-sensitive nitrogenases, as well as genes coding for other oxygen-sensitive enzymes, such as carbon monoxide dehydrogenase and formate dehydrogenase. These findings offer new prospects for the wider application of A. vinelandii as a host for the production and characterization of oxygen-sensitive proteins.

Plant microbiome and its manipulation herald a new era for plant biotechnology with the potential to benefit sustainable crop production. However, studies evaluating the diversity, structure and impact of the microbiota in economic important crops are still rare. Here we describe a comprehensive inventory of the structure and assemblage of the bacterial and fungal communities associated with sugarcane. Our analysis identified 23,811 bacterial OTUs and an unexpected 11,727 fungal OTUs inhabiting the endophytic and exophytic compartments of roots, shoots, and leaves. These communities originate primarily from native soil around plants and colonize plant organs in distinct patterns. The sample type is the primary driver of fungal community assemblage, and the organ compartment plays a major role in bacterial community assemblage. We identified core bacterial and fungal communities composed of less than 20% of the total microbial richness but accounting for over 90% of the total microbial relative abundance. The roots showed 89 core bacterial families, 19 of which accounted for 44% of the total relative abundance. Stalks are dominated by groups of yeasts that represent over 12% of total relative abundance. The core microbiome described here comprise groups whose biological role underlies important traits in plant growth and fermentative processes.

Molybdate- and ATP-dependent in vitro synthesis of the iron-molybdenum cofactor (FeMo-co) of nitrogenase requires the protein products of at least the nifB, nifN, and nifE genes. Extracts of FeMo-co-negative mutants of Klebsiella pneumoniae and Azotobacter vinelandii with lesions in different genes can be complemented for FeMo-co synthesis. Both K. pneumoniae and A. vinelandii dinitrogenase (component I) deficient in FeMo-co can be activated by FeMo-co synthesized in vitro. Properties of the partially purified dinitrogenase activated by FeMo-co synthesized in vitro were comparable to those of dinitrogenase from the wild-type organism; e.g., ratios of acetylene- to nitrogen-reduction activities, as well as those of acetylene reduction activities to EPR spectrum peak height at g = 3.65, were very similar. A. vinelandii mutants UW45 and CA30 have mutations in a gene functionally equivalent to nifB of K. pneumoniae.

Iron is critical for symbiotic nitrogen fixation (SNF) as a key component of multiple ferroproteins involved in this biological process. In the model legume Medicago truncatula, iron is delivered by the vasculature to the infection/maturation zone (zone II) of the nodule, where it is released to the apoplast. From there, plasma membrane iron transporters move it into rhizobia-containing cells, where iron is used as the cofactor of multiple plant and rhizobial proteins (e.g. plant leghemoglobin and bacterial nitrogenase). MtNramp1 (Medtr3g088460) is the M. truncatula Natural Resistance-Associated Macrophage Protein family member, with the highest expression levels in roots and nodules. Immunolocalization studies indicate that MtNramp1 is mainly targeted to the plasma membrane. A loss-of-function nramp1 mutant exhibited reduced growth compared with the wild type under symbiotic conditions, but not when fertilized with mineral nitrogen. Nitrogenase activity was low in the mutant, whereas exogenous iron and expression of wild-type MtNramp1 in mutant nodules increased nitrogen fixation to normal levels. These data are consistent with a model in which MtNramp1 is the main transporter responsible for apoplastic iron uptake by rhizobia-infected cells in zone II.

The products of the Rhizobium leguminosarum hyp gene cluster are necessary for synthesis of a functional uptake [NiFe] hydrogenase system in symbiosis with pea plants, and at least for HypB and HypF, a role in hydrogenase-specific nickel metabolism has been postulated (L. Rey, J. Murillo, Y. Hernando, E. Hidalgo, E. Cabrera, J. Imperial, and T. Ruiz-Argiieso, Mol. Microbiol. 8:471-481, 1993). The R. leguminosarum hypB gene product has been overexpressed in Escherichia coli and purified by immobilized nickel chelate affinity chromatography in a single step. The purified recombinant HypB protein was able to bind 3.9 + 0.1 Ni2+ ions per HypB monomer in solution. Co2+, Cu2+, and Zn2+ ions competed with Ni2+ with increasing efficiency.Monospecific HypB antibodies were raised and used to show that HypB is synthesized in R. eguminosarum microaerobic vegetative cells and pea bacteroids but not in R. leguminosarum aerobic cells. HypB protein synthesized by R. kguminosarum microaerobic vegetative cells could also be isolated by immobilized nickel chelate affinity chromatography. A histidine-rich region at the amino terminus of the protein (23-HGHHHH DGHHDHDHDHDHHRGDHEHDDHHH-54) is proposed to play a role in nickel binding, both in solution and in chelated form.Rhizobium leguminosarum bv. viciae possesses an H2 uptake system that is able to oxidize H2 generated by the nitrogenase complex as a byproduct of the N2 reduction reaction (8,40). This system consists of an uptake [NiFe] hydrogenase and accessory proteins, and it is only expressed in the plant symbiotic state. The main features of the system have been studied by our laboratory in the Pisum-pea bacteroid symbiosis. The genetic determinants for the H2 uptake system are clustered in a 15-kb DNA region (hup region) in the symbiotic plasmid (21,22). This region has been sequenced, and 17 potential genes have been identified. The first six genes constitute the hydrogenase structural operon and include the genes hupS and hupL, encoding the hydrogenase polypeptides (13), and four additional genes, hupCDEF (14). A five-gene cluster containing hupGHIJK has been identified downstream the hydrogenase structural operon (38 Purification of the HypB protein by Ni(II)-NTA-agarose chromatography. The Ni(II)-nitrilotriacetic acid (NTA)-agarose matrix was obtained from Diagen (Dusseldorf, Germany), and the manufacturer's recommendations for its use (12) were followed, with minor modifications as follows.(i) Denaturing conditions. Frozen cells from a 100-ml induced culture were lysed in the presence of 6 M guanidineHCl (3.5 ml), and cell extracts were applied to an Ni(II)-NTAagarose column (2 by 0.8 cm). Proteins were stepwise eluted by means of buffers of decreasing pH, 8.0, 6.3, 5.9, and 4.5, all of which contained 8 M urea, at a flow rate of 0.5 ml min-1. Fractions (1 ml) were collected, and portions were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Coomassie blue staining (18).(ii) Nondenaturing conditions. All the manipulations were carri...

Nitrogenase catalyses the ATP-dependent reduction of N2 to NH3, and is composed of two proteins, dinitrogenase (MoFe protein or component I) and dinitrogenase reductase (Fe protein or component II). Dinitrogenase contains a unique prosthetic group (iron-molybdenum cofactor, FeMoco) comprised of Fe, Mo and S, which has been proposed as the site of N2 reduction. Biochemical and genetic studies of Nif- (nitrogen fixation) mutants of Klebsiella pneumoniae which are defective in nitrogen fixation, have shown that the nifB, nifQ, nifN, nifE and nifV genes are required for the biosynthesis of FeMo-co. Recently, a system for in vitro synthesis of FeMoco was described. The assay requires at least the nifB, nifN and nifE gene products, and a low-molecular-weight factor (V factor) produced in the presence of the nifV gene product. We have used this system to study FeMoco biosynthesis. We report here the isolation of V factor and identify it as homocitric acid ([R]2-hydroxy-1,2,4-butanetricarboxylic acid).

When apodinitrogenase (lacking FeMo-co) was activated with FeMo-co synthesized in vitro in the presence of 3H-labeled homocitrate, label was incorporated into dinitrogenase. The physical association of the label with FeMo-co was demonstrated by reisolation and purification of the cofactor from dinitrogenase. The presence of homocitrate in FeMo-co was established by NMR analysis of the organic acid extracted from dinitrogenase. Quantitation of homocitrate in dinitrogenase showed it to be present at a 1:1 ratio with molybdenum.

Rhizobium leguminosarum bv. viciae expresses an uptake hydrogenase in symbiosis with peas (Pisum sativum) but, unlike all other characterized hydrogenoxidizing bacteria, cannot express it in free-living conditions. The hydrogenase-specific transcriptional activator gene hoxA described in other species was shown to have been inactivated in R. leguminosarum by accumulation of frameshift and deletion mutations. Symbiotic transcription of hydrogenase structural genes hupSL originates from a ؊24͞؊12 type promoter (hupS p ). A regulatory region located in the ؊173 to ؊88 region was essential for promoter activity in R. leguminosarum. Activation of hupS p was observed in Klebsiella pneumoniae and Escherichia coli cells expressing the K. pneumoniae nitrogen fixation regulator NifA, and in E. coli cells expressing R. meliloti NifA. This activation required direct interaction of NifA with the essential ؊173 to ؊88 regulatory region. However, no sequences resembling known NifA-binding sites were found in or around this region. NifA-dependent activation was also observed in R. etli bean bacteroids. NifAdependent hupS p activity in heterologous hosts was also absolutely dependent on the RpoN -factor and on integration host factor. Proteins immunologically related to integration host factor were identified in R. leguminosarum. The data suggest that hupS p is structurally and functionally similar to nitrogen fixation promoters. The requirement to coordinate nitrogenase-dependent H 2 production and H 2 oxidation in nodules might be the reason for the loss of HoxA in R. leguminosarum and the concomitant NifA control of hup gene expression. This evolutionary acquired control would ensure regulated synthesis of uptake hydrogenase in the most common H 2 -rich environment for rhizobia, the legume nodule.