Hydrogen-uptake (Hup) activity is implicated in the mitigation of energy losses associated with the biological nitrogen fixation process, and has been related to productivity increases in some legume hosts. However, in common bean (Phaseolus vulgaris L.) the expression of hydrogenase is rare. In this study an 18-kb hup gene cluster from Rhizobium leguminosarum bv. viciae encoding a NiFe hydrogenase was successfully transferred to three common bean rhizobial strains lacking hydrogenase activity (Hup−) but symbiotically very effective and used in commercial inoculants in Brazil: one strain originally from Colombia (Rhizobium tropici CIAT 899), and two strains from Brazil (R. tropici H 12 and Rhizobium freirei PRF 81). The inclusion of NiCl2 in the nutrient solution did not increase hydrogenase activity, indicating that common bean plants allow efficient nickel provision for hydrogenase synthesis in the bacteroids. The symbiotic performance—evaluated by nodulation, plant growth, N accumulation and seed production—of wild-type and Hup+ derivative strains was compared in experiments performed with cultivar Carioca under greenhouse conditions, in sterile substrate and in non-sterile soil. Statistically significant increases in one or more parameters were observed for all three Hup+ derivatives when compared to the respective wild-type strain. Differences were found mainly with the Brazilian strains, reaching impressive increases in nodule efficiency and seed total N content. The results highlight the potential of using Rhizobium Hup+ strains for the design of more energy-efficient inoculants for the common bean crop.

Rhizobium leguminosarum bv. viciae is a soil α-proteobacterium that establishes a diazotrophic symbiosis with different legumes of the Fabeae tribe. The number of genome sequences from rhizobial strains available in public databases is constantly increasing, although complete, fully annotated genome structures from rhizobial genomes are scarce. In this work, we report and analyse the complete genome of R. leguminosarum bv. viciae UPM791. Whole genome sequencing can provide new insights into the genetic features contributing to symbiotically relevant processes such as bacterial adaptation to the rhizosphere, mechanisms for efficient competition with other bacteria, and the ability to establish a complex signalling dialogue with legumes, to enter the root without triggering plant defenses, and, ultimately, to fix nitrogen within the host. Comparison of the complete genome sequences of two strains of R. leguminosarum bv. viciae, 3841 and UPM791, highlights the existence of different symbiotic plasmids and a common core chromosome. Specific genomic traits, such as plasmid content or a distinctive regulation, define differential physiological capabilities of these endosymbionts. Among them, strain UPM791 presents unique adaptations for recycling the hydrogen generated in the nitrogen fixation process.

Natural habitats containing high amounts of heavy metals provide a valuable source of bacteria adapted to deal with metal toxicity. A functional analysis of the population of legume endosymbiotic bacteria in an ultramafic soil was undertaken by studying a collection of Rhizobium leguminosarum bv viciae (Rlv) isolates obtained using pea as trap plant. One of the isolates, Rlv UPM1137, was selected on the basis of its higher tolerance to nickel and cobalt and presence of inducible mechanisms for such tolerance. A random transposon mutagenesis of Rlv UPM1137 allowed the generation of 14 transposant derivatives with increased nickel sensitivity; five of these transposants were also more sensitive to cobalt. Sequencing of the insertion sites revealed that one of the transposants (D2250) was affected in a gene homologous to the cation diffusion facilitator gene dmeF first identified in the metal-resistant bacterium Cupriavidus metallidurans CH34. The symbiotic performance of D2250 and two other transposants bearing single transposon insertions was unaffected under high-metal conditions, suggesting that, in contrast to previous observations in other Rlv strain, metal tolerance in UPM1137 under symbiotic conditions might be supported by functional redundancy between several mechanisms.

Bacteria require nickel transporters for the synthesis of Ni-containing metalloenzymes in natural, low nickel habitats. In this work we carry out functional and topological characterization of Rhizobium leguminosarum HupE, a nickel permease required for the provision of this element for [NiFe] hydrogenase synthesis. Expression studies in the Escherichia coli nikABCDE mutant strain HYD723 revealed that HupE is a medium-affinity permease (apparent Km 227 ± 21 nM; Vmax 49 ± 21 pmol Ni(2+) min(-1) mg(-1) bacterial dry weight) that functions as an energy-independent diffusion facilitator for the uptake of Ni(ii) ions. This Ni(2+) transport is not inhibited by similar cations such as Mn(2+), Zn(2+), or Co(2+), but is blocked by Cu(2+). Analysis of site-directed HupE mutants allowed the identification of several residues (H36, D42, H43, F69, E90, H130, and E133) that are essential for HupE-mediated Ni uptake in E. coli cells. By using translational fusions to reporter genes we demonstrated the presence of five transmembrane domains with a periplasmic N-terminal domain and a C-terminal domain buried in the lipid bilayer. The periplasmic N-terminal domain contributes to stability and functionality of the protein.

In spite of potentially being an important source of rhizobial diversity and a key determinant of common bean productivity, there is a paucity of data on Rhizobium genetic variation and species composition in the important bean producing area of Chile and only one species has been documented (Rhizobium leguminosarum). In this study, 240 Rhizobium isolates from Torcaza bean (Phaseolus vulgaris L.) nodules established in the highest bean producing area in Chile (33°34′S–70°38′W and 37°36′S–71°47′W) were characterized by PCR-RFLP markers for nodC gene, revealing eight banding patterns with the polymorphic enzyme Hinf I. The locality of San Agustín de Aurora in Central Chile (35°32′S–71°29′W) had the highest level of diversity. Isolates were classified by species using PCR-RFLP markers for 16S rDNA gene and were confirmed by sequencing an internal fragment of the 16S rDNA gene. The results confirmed the presence of R. leguminosarum and three other species of rhizobia nodulating beans in South Central Chile (R. etli, R. tropici and R. leucaenae). R. tropici and R. leucaenae showed the least genetic variation and were most commonly identified in acid soils, while R. etli was the most common species in slightly acidic to moderately alkaline soils, with higher levels of organic matter content. R. leguminosarum was identified in almost all soils, was the most genetically diverse, and was the most common, being documented in soils with pH that ranged between 5.3 and 8.2, and with organic matter content between 2.1 and 4 %.

Background: [NiFe] hydrogenase biosynthesis requires the interaction among multiple accessory proteins for metal cofactor assembly. Results: Combined bioinformatic protein modeling and mutant analysis on HupK and HypC proteins identify key residues required for hydrogenase maturation. Conclusion: HypC-HupK interaction is a relevant step for hydrogenase biosynthesis in the presence of oxygen. Significance: The results expand our knowledge on the mechanism of hydrogenase biosynthesis in aerobic bacteria. [NiFe] hydrogenases are key enzymes for the energy and redox metabolisms of different microorganisms. Synthesis of these metalloenzymes involves a complex series of biochemical reactions catalyzed by a plethora of accessory proteins, many of them required to synthesize and insert the unique NiFe(CN)2CO cofactor. HypC is an accessory protein conserved in all [NiFe] hydrogenase systems and involved in the synthesis and transfer of the Fe(CN)2CO cofactor precursor. Hydrogenase accessory proteins from bacteria-synthesizing hydrogenase in the presence of oxygen include HupK, a scaffolding protein with a moderate sequence similarity to the hydrogenase large subunit and proposed to participate as an intermediate chaperone in the synthesis of the NiFe cofactor. The endosymbiotic bacterium Rhizobium leguminosarum contains a single hydrogenase system that can be expressed under two different physiological conditions: free-living microaerobic cells (∼12 μm O2) and bacteroids from legume nodules (∼10–100 nm O2). We have used bioinformatic tools to model HupK structure and interaction of this protein with HypC. Site-directed mutagenesis at positions predicted as critical by the structural analysis have allowed the identification of HupK and HypC residues relevant for the maturation of hydrogenase. Mutant proteins altered in some of these residues show a different phenotype depending on the physiological condition tested. Modeling of HypC also predicts the existence of a stable HypC dimer whose presence was also demonstrated by immunoblot analysis. This study widens our understanding on the mechanisms for metalloenzyme biosynthesis in the presence of oxygen.

Iron, zinc, copper, molybdenum and some other transition metals are essential nutrients (Fraustro da Silva and Williams 2001). They are constitutive elements of around half of the proteins of a typical cell (Andreini et al. 2008), where they may act as structural elements, such as zinc in the zinc fi nger domain, but mostly as key elements in the active site of enzymes involved in almost every physiological process, from oxidative respiration to photosynthesis. These elements are growth-limiting nutrients for autotrophic organisms, since they form very stable complexes, resulting in low solubility and making their uptake diffi cult (Ruel and Bouis 1998, Fung et al. 2000, Grotz and Guerinot 2006). Moreover, living beings cannot accumulate high amounts of essential transition metals, since these elements can catalyze the production of free radicals in Fenton-style reactions or compete with other metals for the active site of metalloenzymes (Goldstein et al. 1993, Ranquet et al. 2007, Macomber and Imlay 2009). This is also the basis of the toxic effect of non-biogenic metals such as cadmium, lead or mercury.

ABSTRACT A gene encoding a homolog to the cation diffusion facilitator protein DmeF from Cupriavidus metallidurans has been identified in the genome of Rhizobium leguminosarum UPM791. The R. leguminosarum dmeF gene is located downstream of an open reading frame (designated dmeR ) encoding a protein homologous to the nickel- and cobalt-responsive transcriptional regulator RcnR from Escherichia coli . Analysis of gene expression showed that the R. leguminosarum dmeRF genes are organized as a transcriptional unit whose expression is strongly induced by nickel and cobalt ions, likely by alleviating the repressor activity of DmeR on dmeRF transcription. An R. leguminosarum dmeRF mutant strain displayed increased sensitivity to Co(II) and Ni(II), whereas no alterations of its resistance to Cd(II), Cu(II), or Zn(II) were observed. A decrease of symbiotic performance was observed when pea plants inoculated with an R. leguminosarum dmeRF deletion mutant strain were grown in the presence of high concentrations of nickel and cobalt. The same mutant induced significantly lower activity levels of NiFe hydrogenase in microaerobic cultures. These results indicate that the R. leguminosarum DmeRF system is a metal-responsive efflux mechanism acting as a key element for metal homeostasis in R. leguminosarum under free-living and symbiotic conditions. The presence of similar dmeRF gene clusters in other Rhizobiaceae suggests that the dmeRF system is a conserved mechanism for metal tolerance in legume endosymbiotic bacteria.

Background [NiFe] hydrogenases are enzymes that catalyze the oxidation of hydrogen into protons and electrons, to use H 2 as energy source, or the production of hydrogen through proton reduction, as an escape valve for the excess of reduction equivalents in anaerobic metabolism. Biosynthesis of [NiFe] hydrogenases is a complex process that occurs in the cytoplasm, where a number of auxiliary proteins are required to synthesize and insert the metal cofactors into the enzyme structural units. The endosymbiotic bacterium Rhizobium leguminosarum requires the products of eighteen genes ( hupSLCDEFGHIJKhypABFCDEX ) to synthesize an active hydrogenase. hupF and hupK genes are found only in hydrogenase clusters from bacteria expressing hydrogenase in the presence of oxygen. Results HupF is a HypC paralogue with a similar predicted structure, except for the C-terminal domain present only in HupF. Deletion of hupF results in the inability to process the hydrogenase large subunit HupL, and also in reduced stability of this subunit when cells are exposed to high oxygen tensions. A Δ hupF mutant was fully complemented for hydrogenase activity by a C-terminal deletion derivative under symbiotic, ultra low-oxygen tensions, but only partial complementation was observed in free living cells under higher oxygen tensions (1% or 3%). Co-purification experiments using Strep Tag-labelled HupF derivatives and mass spectrometry analysis indicate the existence of a major complex involving HupL and HupF, and a less abundant HupF-HupK complex. Conclusions The results indicate that HupF has a dual role during hydrogenase biosynthesis: it is required for hydrogenase large subunit processing and it also acts as a chaperone to stabilize HupL when hydrogenase is synthesized in the presence of oxygen.

ABSTRACT Synthesis of the hydrogen uptake (Hup) system in Rhizobium leguminosarum bv. viciae requires the function of an 18-gene cluster ( hupSLCDEFGHIJK - hypABFCDEX ). Among them, the hupE gene encodes a protein showing six transmembrane domains for which a potential role as a nickel permease has been proposed. In this paper, we further characterize the nickel transport capacity of HupE and that of the translated product of hupE2 , a hydrogenase-unlinked gene identified in the R. leguminosarum genome. HupE2 is a potential membrane protein that shows 48% amino acid sequence identity with HupE. Expression of both genes in the Escherichia coli nikABCDE mutant strain HYD723 restored hydrogenase activity and nickel transport. However, nickel transport assays revealed that HupE and HupE2 displayed different levels of nickel uptake. Site-directed mutagenesis of histidine residues in HupE revealed two motifs (HX 5 DH and FHGX[AV]HGXE) that are required for HupE functionality. An R. leguminosarum double mutant, SPF22A ( hupE hupE2 ), exhibited reduced levels of hydrogenase activity in free-living cells, and this phenotype was complemented by nickel supplementation. Low levels of symbiotic hydrogenase activity were also observed in SPF22A bacteroid cells from lentil ( Lens culinaris L.) root nodules but not in pea ( Pisum sativum L.) bacteroids. Moreover, heterologous expression of the R. leguminosarum hup system in bacteroid cells of Rhizobium tropici and Mesorhizobium loti displayed reduced levels of hydrogen uptake in the absence of hupE . These data support the role of R. leguminosarum HupE as a nickel permease required for hydrogen uptake under both free-living and symbiotic conditions.

A new and sensitive methodology based on normal phase HPLC has been developed for the speciation of nickel in low-complexity plant extracts. The method combines a silica stationary phase column, a 9:1 (v/v) hexane:ethanol mixture as mobile phase, and the detection of nickel complexes by either UV or MS. The developed methodology was applied to the speciation of nickel complexes in the cytoplasm of pea root nodules. Results obtained indicate that nickel citrate and nickel malate accounts for 99% of nickel present in pea nodule cytoplasm fraction. The low detection limit of the method (<0.2 nM) enables nickel speciation in non-hyperaccumulator plants.

The legume host affects the expression of Rhizobium leguminosarum hydrogenase activity in root nodules. High levels of symbiotic hydrogenase activity were detected in R. leguminosarum bacteroids from different hosts, with the exception of lentil (Lens culinaris). Transcription analysis showed that the NifA-regulated R. leguminosarum hydrogenase structural gene promoter (P(1)) is poorly induced in lentil root nodules. Replacement of the P(1) promoter by the FnrN-dependent promoter of the fixN gene restored transcription of hup genes in lentil bacteroids, but not hydrogenase activity. In the P(fixN)-hupSL strain, additional copies of the hup gene cluster and nickel supplementation to lentil plants increased bacteroid hydrogenase activity. However, the level of activity in lentil still was significantly lower than in pea bacteroids, indicating that an additional factor is impairing hydrogenase expression inside lentil nodules. Immunological analysis revealed that lentil bacteroids contain reduced levels of both hydrogenase structural subunit HupL and nickel-binding protein HypB. Altogether, results indicate that hydrogenase expression is affected by the legume host at the level of both transcription of hydrogenase structural genes and biosynthesis or stability of nickel-related proteins HypB and HupL, and suggest the existence of a plant-dependent mechanism that affects hydrogenase activity during the symbiosis by limiting nickel availability to the bacteroid.

ABSTRACT Bradyrhizobium sp. ( Lupinus ) and Bradyrhizobium sp. ( Vigna ) mutants in which hydrogenase ( hup ) activity was affected were constructed and analyzed. Vigna unguiculata plants inoculated with the Bradyrhizobium sp. ( Vigna ) hup mutant showed reduced nitrogenase activity and also a significant decrease in nitrogen content, suggesting a relevant contribution of hydrogenase activity to plant yield.

Uptake hydrogenases in legume endosymbiotic bacteria recycle hydrogen produced during the nitrogen fixation process in legume nodules. Despite the described beneficial effect on plant productivity, the hydrogen oxidation capability is not widespread in the Rhizobiaceae family. Characterization of hydrogenase gene clusters in strains belonging to Rhizobium, Bradyrhizobium and Azorhizobium reveals a similar overall genetic organization along with important differences in gene regulation. In addition, phylogenetic analysis of hup genes indicates distinct evolutionary origins for hydrogenase genes in Rhizobia.

A limited number of strains belonging to several genera of Rhizobiaceae are capable of expressing a hydrogenase system that allows partial or full recycling of hydrogen evolved by nitrogenase, thus increasing the energy efficiency of the nitrogen fixation process. This review is focused on the genetics and biotechnology of the hydrogenase system from Rhizobium leguminosarum bv. viciae, a frequent inhabitant of European soils capable of establishing symbiotic association with peas, lentils, vetches and other legumes.

Expression of several virulence factors in the plant pathogen bacterium Ralstonia solanacearum is controlled by a complex regulatory network, at the center of which is PhcA. We provide genetic evidence that PhcA also represses the expression of hrp genes that code for the Type III protein secretion system, a major pathogenicity determinant in this bacterium. The repression of hrp genes in complete medium is relieved in a phcA mutant and two distinct signals, a quorum-sensing signal and complex nitrogen sources, appear to trigger this PhcA-dependent repression. This control of hrp gene expression by PhcA is realized at the level of the HrpG regulatory protein.

In this work, we report the cloning and sequencing of the Azorhizobium caulinodans ORS571 hydrogenase gene cluster. Sequence analysis revealed the presence of 20 open reading frames hupTUVhypFhupSLCDFGHJK hypABhupRhypCDEhupE. The physical and genetic organization of A. caulinodans ORS571 hydrogenase system suggests a close relatedness to that of Rhodobacter capsulatus. In contrast to the latter species, a gene homologous to Rhizobium leguminosarum hupE was identified downstream of the hyp operon. A hupSL mutation drastically reduced the high levels of hydrogenase activity induced by the A. caulinodans ORS571 wild-type strain in symbiosis with Sesbania rostrata plants. However, no significant effects on dry weight and nitrogen content of S. rostrata plants inoculated with the hupSL mutant were observed in plant growth experiments.

Synthesis of the Rhizobium leguminosarum [NiFe] hydrogenase requires the participation of 16 accessory genes (hupCDEFGHIJKhypABFCDEX) besides the genes encoding the structural proteins (hupSL). Transcription of hupSL is controlled by a -24/-12-type promoter (P(1)), located upstream of hupS and regulated by NifA. In this work, a second -24/-12-type promoter (P(3)), located upstream of the hupG gene and transcribing hupGHIJ genes in R. leguminosarum pea (Pisum sativum L.) bacteroids, has been identified in the hup gene cluster. Promoter P(3) was also active in R. leguminosarum free-living cells, as evidenced by genetic complementation of hydrogenase mutants. Both NifA and NtrC activated P(3) expression in the heterologous host Klebsiella pneumoniae. Also, P(3) activity was highly stimulated by K. pneumoniae NifA in Escherichia coli. This NifA activation of P(3) expression only required the sigma(54)-binding site, and it was independent of any cis-acting element upstream of the sigma(54) box, which suggests a direct interaction of free NifA with the RNA polymerase holoenzyme. P(3)-dependent hupGHIJ expression in pea nodules started in interzone II/III, spanned through nitrogen-fixing zone III, and was coincident with the NifA-dependent nifH expression pattern. However, P(3) was dispensable for hupGHIJ transcription and hydrogenase activity in pea bacteroids due to transcription initiated at P(1). This fact and the lack of an activator recruitment system suggest that P(3) plays a secondary role in symbiotic hupGHIJ expression.