The Bradyrhizobium Sp. LmicA16 Type VI Secretion System Is Required for Efficient Nodulation of Lupinus Spp.

Many bacteria of the genus Bradyrhizobium are capable of inducing nodules in legumes. In this work, the importance of a type VI secretion system (T6SS) in a symbiotic strain of the genus Bradyrhizobium is described. T6SS of Bradyrhizobium sp. LmicA16 (A16) is necessary for efficient nodulation with Lupinus micranthus and Lupinus angustifolius. A mutant in the gene vgrG, coding for a component of the T6SS nanostructure, induced less nodules and smaller plants than the wild-type (wt) strain and was less competitive when co-inoculated with the wt strain. A16 T6SS genes are organized in a 26-kb DNA region in two divergent gene clusters of nine genes each. One of these genes codes for a protein (Tsb1) of unknown function but containing a methyltransferase domain. A tsb1 mutant showed an intermediate symbiotic phenotype regarding vgrG mutant and higher mucoidity than the wt strain in free-living conditions. T6SS promoter fusions to the lacZ reporter indicate expression in nodules but not in free-living cells grown in different media and conditions. The analysis of nodule structure revealed that the level of nodule colonization was significantly reduced in the mutants with respect to the wt strain.

Novel putative Mesorhizobium and Ensifer genomospecies together with a novel symbiovar psoraleae nodulate legumes of agronomic interest grown in Tunisia.

Forty rhizobial strains were isolated from Lotus creticus, L. pusillus and Bituminaria bituminosa endemic to Tunisia, and they belonged to the Mesorhizobium and Ensifer genera based on 16S rDNA sequence phylogeny. According to the concatenated recA and glnII sequence-based phylogeny, four Bituminaria isolates Pb5, Pb12, Pb8 and Pb17 formed a monophyletic group with Mesorhizobium chacoense ICMP14587T, whereas four other strains Pb1, Pb6, Pb13 and Pb15 formed two separate lineages within the Ensifer genus. Among the L. pusillus strains, Lpus9 and Lpus10 showed a 96% identical nucleotide with Ensifer meliloti CCBAU83493T; whereas six other strains could belong to previously undescribed Mesorhizobium and Ensifer species. For L. creticus strains, Lcus37, Lcus39 and Lcus44 showed 98% sequence identity with Ensifer aridi JNVU TP6, and Lcus42 shared a 96% identical nucleotide with Ensifer meliloti CCBAU83493T; whereas another four strains were divergent from all the described Ensifer and Mesorhizobium species. The analysis of the nodC gene-based phylogeny identified four symbiovar groups; Mesorhizobium sp. sv. anthyllidis (Lpus3 and Lpus11 from L. pusillus, Lcus43 from L. creticus), Ensifer medicae sv. meliloti (four strains from L. creticus and two strains from L. pusillus), E. meliloti sv. meliloti (four from L. creticus, four from L. pusillus and four from B. bituminosa). In addition, four B. bituminosa strains (Pb5, Pb8, Pb12, and Pb17) displayed a distinctive nodC sequence distant from those of other symbiovars described to date. According to their symbiotic gene sequences and host range, the B. bituminosa symbionts (Pb5, Pb8, Pb12 and Pb17) would represent a new symbiovar of M. chacoense for which sv. psoraleae is proposed.

RNA sequencing and analysis of three Lupinus nodulomes provide new insights into specific host-symbiont relationships with compatible and incompatible Bradyrhizobium strains.

Nitrogen fixation in the legume root-nodule symbiosis has a critical importance in natural and agricultural ecosystems and depends on the proper choice of the symbiotic partners. However, the genetic determinism of symbiotic specificity remains unclear. To study this process, we inoculated three Lupinus species (L. albus, L. luteus, L. mariae-josephae), belonging to the under-investigated tribe of Genistoids, with two Bradyrhizobium strains (B. japonicum, B. valentinum) presenting contrasted degrees of symbiotic specificity depending on the host. We produced the first transcriptomes (RNA-Seq) from lupine nodules in a context of symbiotic specificity. For each lupine species, we compared gene expression between functional and non-functional interactions and determined differentially expressed (DE) genes. This revealed that L. luteus and L. mariae-josephae (nodulated by only one of the Bradyrhizobium strains) specific nodulomes were richest in DE genes than L. albus (nodulation with both microsymbionts, but non-functional with B. valentinum) and share a higher number of these genes between them than with L. albus. In addition, a functional analysis of DE genes highlighted the central role of the genetic pathways controlling infection and nodule organogenesis, hormones, secondary, carbon and nitrogen metabolisms, as well as the implication of plant defence in response to compatible or incompatible Bradyrhizobium strains.

Genetic diversity of indigenous rhizobial symbionts of the Lupinus mariae-josephae endemism from alkaline-limed soils within its area of distribution in Eastern Spain.

The genomic diversity of a collection of 103 indigenous rhizobia isolates from Lupinus mariae-josephae (Lmj), a recently described Lupinus species endemic to alkaline-limed soils from a restricted habitat in Eastern Spain, was investigated by molecular methods. Isolates were obtained from soils of four geographic locations in the Valencia province that harbored the known Lmj plant populations. Using an M13 RAPD fingerprinting technique, 19 distinct RAPD profiles were identified. Phylogenetic analysis based on 16S rDNA and the housekeeping genes glnII, recA and atpD showed a high diversity of native Bradyrhizobium strains that were able to establish symbiosis with Lmj. All the strains grouped in a clade unrelated to strains of the B. canariense and B. japonicum lineages that establish symbioses with lupines in acid soils of the Mediterranean area. The phylogenetic tree based on concatenated glnII, recA and atpD gene sequences grouped the Lmj isolates in six different operational taxonomic units (OTUs) at the 93% similarity level. These OTUs were not associated to any specific geographical location, and their observed divergence predicted the existence of different Bradyrhizobium genomic species. In contrast, phylogenetic analysis of symbiotic genes based on nodC and nodA gene sequences, defined only two distinct clusters among the Lmj strains. These two Lmj nod gene types were largely distinct from nod genes of bradyrhizobia nodulating other Old World lupine species. The singularity and large diversity of these strains in such a small geographical area makes this an attractive system for studying the evolution and adaptation of the rhizobial symbiont to the plant host.

Biodiversity of uptake hydrogenase systems from legume endosymbiotic bacteria.

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.

Genetics and biotechnology of the H(2)-uptake [NiFe] hydrogenase from Rhizobium leguminosarum bv. viciae, a legume endosymbiotic bacterium.

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.

Engineering the Rhizobium leguminosarum bv. viciae hydrogenase system for expression in free-living microaerobic cells and increased symbiotic hydrogenase activity.

Rhizobium leguminosarum bv. viciae UPM791 induces hydrogenase activity in pea (Pisum sativum L.) bacteroids but not in free-living cells. The symbiotic induction of hydrogenase structural genes (hupSL) is mediated by NifA, the general regulator of the nitrogen fixation process. So far, no culture conditions have been found to induce NifA-dependent promoters in vegetative cells of this bacterium. This hampers the study of the R. leguminosarum hydrogenase system. We have replaced the native NifA-dependent hupSL promoter with the FnrN-dependent fixN promoter, generating strain SPF25, which expresses the hup system in microaerobic free-living cells. SPF25 reaches levels of hydrogenase activity in microaerobiosis similar to those induced in UPM791 bacteroids. A sixfold increase in hydrogenase activity was detected in merodiploid strain SPF25(pALPF1). A time course induction of hydrogenase activity in microaerobic free-living cells of SPF25(pALPF1) shows that hydrogenase activity is detected after 3 h of microaerobic incubation. Maximal hydrogen uptake activity was observed after 10 h of microaerobiosis. Immunoblot analysis of microaerobically induced SPF25(pALPF1) cell fractions indicated that the HupL active form is located in the membrane, whereas the unprocessed protein remains in the soluble fraction. Symbiotic hydrogenase activity of strain SPF25 was not impaired by the promoter replacement. Moreover, bacteroids from pea plants grown in low-nickel concentrations induced higher levels of hydrogenase activity than the wild-type strain and were able to recycle all hydrogen evolved by nodules. This constitutes a new strategy to improve hydrogenase activity in symbiosis.

Generation of new hydrogen-recycling Rhizobiaceae strains by introduction of a novel hup minitransposon.

Hydrogen evolution by nitrogenase is a source of inefficiency for the nitrogen fixation process by the Rhizobium-legume symbiosis. To develop a strategy to generate rhizobial strains with H(2)-recycling ability, we have constructed a Tn5 derivative minitransposon (TnHB100) that contains the ca. 18-kb H(2) uptake (hup) gene cluster from Rhizobium leguminosarum bv. viciae UPM791. Bacteroids from TnHB100-containing strains of R. leguminosarum bv. viciae PRE, Bradyrhizobium japonicum, R. etli, and Mesorhizobium loti expressed high levels of hydrogenase activity that resulted in full recycling of the hydrogen evolved by nitrogenase in nodules. Efficient processing of the hydrogenase large subunit (HupL) in these strains was shown by immunoblot analysis of bacteroid extracts. In contrast, Sinorhizobium meliloti, M. ciceri, and R. leguminosarum bv. viciae UML2 strains showed poor expression of the hup system that resulted in H(2)-evolving nodules. For the latter group of strains, no immunoreactive material was detected in bacteroid extracts using anti-HupL antiserum, suggesting a low level of transcription of hup genes or HupL instability. A general procedure for the characterization of the minitransposon insertion site and removal of antibiotic resistance gene included in TnHB100 has been developed and used to generate engineered strains suitable for field release.

A novel autoregulation mechanism of fnrN expression in Rhizobium leguminosarum bv viciae.

The fnrN gene from Rhizobium leguminosarum UPM791 controls microaerobic expression of both nitrogen fixation and hydrogenase activities in symbiotic cells. Two copies of fnrN are present in this strain, one chromosomal (fnrN1) and the other located in the symbiotic plasmid (fnrN2). Their expression was studied by cloning the regulatory regions in lacZ promoter-probe vectors. The fnrN genes were found to be autoregulated: they are expressed only at basal levels under aerobic conditions; they are highly expressed under microaerobic conditions; and they are expressed at basal levels in the double mutant DG2 (fnrN1 fnrN2) under any condition. The promoters of both genes contain two FnrN-binding sequences (anaeroboxes), centred at positions -12.5 (proximal anaerobox) and -44.5 (distal anaerobox). Expression analysis and gel retardation experiments with fnrN1-derivative promoter mutants altered in key bases of the anaerobox sequences demonstrated that binding of FnrN1 to the distal anaerobox is necessary for microaerobic activation of transcription, and that binding of FnrN1 to the proximal anaerobox results in transcriptional repression. The apparent affinity of FnrN1 for the proximal anaerobox was fivefold lower than for the distal anaerobox, resulting in repression of transcription of fnrN1 only at high-FnrN1 concentrations. This positive and negative autoregulation mechanism ensures an equilibrated expression of fnrN in response to microaerobic conditions.

Nickel availability and hupSL activation by heterologous regulators limit symbiotic expression of the Rhizobium leguminosarum bv. viciae hydrogenase system in Hup(-) rhizobia.

A limited number of Rhizobium and Bradyrhizobium strains possess a hydrogen uptake (Hup) system that recycles the hydrogen released from the nitrogen fixation process in legume nodules. To extend this ability to rhizobia that nodulate agronomically important crops, we investigated factors that affect the expression of a cosmid-borne Hup system from Rhizobium leguminosarum bv. viciae UPM791 in R. leguminosarum bv. viciae, Rhizobium etli, Mesorhizobium loti, and Sinorhizobium meliloti Hup(-) strains. After cosmid pAL618 carrying the entire hup system of strain UPM791 was introduced, all recipient strains acquired the ability to oxidize H(2) in symbioses with their hosts, although the levels of hydrogenase activity were found to be strain and species dependent. The levels of hydrogenase activity were correlated with the levels of nickel-dependent processing of the hydrogenase structural polypeptides and with transcription of structural genes. Expression of the NifA-dependent hupSL promoter varied depending on the genetic background, while the hyp operon, which is controlled by the FnrN transcriptional regulator, was expressed at similar levels in all recipient strains. With the exception of the R. etli-bean symbiosis, the availability of nickel to bacteroids strongly affected hydrogenase processing and activity in the systems tested. Our results indicate that efficient transcriptional activation by heterologous regulators and processing of the hydrogenase as a function of the availability of nickel to the bacteroid are relevant factors that affect hydrogenase expression in heterologous rhizobia.

Identification and characterization of hupT, a gene involved in negative regulation of hydrogen oxidation in Bradyrhizobium japonicum.

The Bradyrhizobium japonicum hupT gene was sequenced, and its gene product was found to be homologous to NtrB-like histidine kinases. A hupT mutant expresses higher levels of hydrogenase activity than the wild-type strain under hydrogenase-inducing conditions (i.e., microaerobiosis plus hydrogen, or symbiosis), whereas in noninduced hupT cells, hupSL expression is derepressed but does not lead to hydrogenase activity. We conclude that HupT is involved in the repression of HupSL synthesis at the transcriptional level but that enzymatic activation requires inducing conditions.

Rhizobium leguminosarum bv. viciae hypA gene is specifically expressed in pea (Pisum sativum) bacteroids and required for hydrogenase activity and processing.

Rhizobium leguminosarum bv. viciae strain UPM791 induces in symbiosis with peas the synthesis of a nickel-containing hydrogenase which recycles the hydrogen evolved by nitrogenase. The genes required for synthesis of this hydrogenase, hupSLCDEFGHIJKhypABFCDEX, are clustered in the symbiotic plasmid. Analysis of a hypA-deficient mutant showed that HypA is essential for symbiotic hydrogenase activity and required for correct processing of the hydrogenase large subunit. Unlike other microoxically induced hyp genes, the hypA gene was only expressed in pea bacteroids from its own promoter. The hypA mRNA 5' end was mapped 109 bp upstream of the translational start codon. This distinct pattern of expression suggests a different role for HypA and the remaining Hyp proteins in hydrogenase synthesis.

FnrN controls symbiotic nitrogen fixation and hydrogenase activities in Rhizobium leguminosarum biovar viciae UPM791.

Rhizobium leguminosarum bv. viciae UPM791 contains a second copy of the fnrN gene, which encodes a redox-sensitive transcriptional activator functionally homologous to Escherichia coli Fnr. This second copy (fnrN2) is located in the symbiotic plasmid, while fnrN1 is in the chromosome. Isolation and sequencing of the fnrN2 gene revealed that the deduced amino acid sequence of FnrN2 is 87.5% identical to the sequence of FnrN1, including a conserved cysteine-rich motif characteristic of Fnr-like proteins. Individual R. leguminosarum fnrN1 and fnrN2 mutants exhibited a Fix+ phenotype and near wild-type levels of nitrogenase and hydrogenase activities in pea (Pisum sativum L.) nodules. In contrast, an fnrN1 fnrN2 double mutant formed ineffective nodules lacking both nitrogenase and hydrogenase activities. Unlike the wild-type strain and single fnrN1 or fnrN2 mutants, the fnrN1 fnrN2 double mutant was unable to induce micro-oxic or bacteroid activation of the hypBFCDEX operon, which encodes proteins essential for hydrogenase synthesis. In the search for symbiotic genes that could be controlled by FnrN, a fixNOQP operon, putatively encoding a micro-oxically induced, bacteroid-specific cbb3-type terminal cytochrome oxidase, was isolated from strain UPM791 and partially sequenced. The fixNOQP operon was present in a single copy located in the symbiotic plasmid, and an anaerobox was identified in the fixN promoter region. Consistent with this, a fixNOQP'-lacZ fusion was shown to be highly induced in micro-oxic cells of the wild-type strain. A high level of micro-oxic induction was also observed in single fnrN1 and fnrN2 mutants, but no detectable induction was observed in the fnrN1 fnrN2 double mutant. The lack of expression of fixNOQP in the fnrN1 fnrN2 double mutant is likely to cause the observed Fix- phenotype. These data demonstrate that, contrary to the situation in other rhizobia, FnrN controls both hydrogenase and nitrogenase activities of R. leguminosarum bv. viciae UPM791 in the nodule and suggest that this strain lacks a functional fixK gene.

Hydrogenase genes from Rhizobium leguminosarum bv. viciae are controlled by the nitrogen fixation regulatory protein nifA.

Rhizobium leguminosarum bv. viciae expresses an uptake hydrogenase in symbiosis with peas (Pisum sativum) but, unlike all other characterized hydrogen-oxidizing 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 -24/-12 type promoter (hupSp). A regulatory region located in the -173 to -88 region was essential for promoter activity in R. leguminosarum. Activation of hupSp 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. NifA-dependent hupSp activity in heterologous hosts was also absolutely dependent on the RpoN sigma-factor and on integration host factor. Proteins immunologically related to integration host factor were identified in R. leguminosarum. The data suggest that hupSp is structurally and functionally similar to nitrogen fixation promoters. The requirement to coordinate nitrogenase-dependent H2 production and H2 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 H2-rich environment for rhizobia, the legume nodule.

The hydrogenase gene cluster of Rhizobium leguminosarum bv. viciae contains an additional gene (hypX), which encodes a protein with sequence similarity to the N10-formyltetrahydrofolate-dependent enzyme family and is required for nickel-dependent hydrogenase processing and activity.

Plasmid pAL618 contains the genetic determinants for H2 uptake (hup) from Rhizobium leguminosarum bv. viciae, including a cluster of 17 genes named hupSLCDEFGHIJK-hypABFCDE. A 1.7-kb segment of insert DNA located downstream of hypE has now been sequenced, thus completing the sequence of the 20441-bp insert DNA in plasmid pAL618. An open reading frame (designated hypX) encoding a protein with a calculated M(r) of 62300 that exhibits extensive sequence similarity with HoxX from Alcaligenes eutrophus (52% identity) and Bradyrhizobium japonicum (57% identity) was identified 10 bp downstream of hypE. Nodule bacteroids produced by hypX mutants in pea (Pisum sativum L.) plants grown at optimal nickel concentrations (100 microM) for hydrogenase expression, exhibited less than 5% of the wild-type levels of hydrogenase activity. These bacteroids contained wild-type levels of mRNA from hydrogenase structural genes (hupSL) but accumulated large amounts of the immature form of HupL protein. The Hup-deficient mutants were complemented for normal hydrogenase activity and nickel-dependent maturation of HupL by a hypX gene provided in trans. From expression analysis of hypX-lacZ fusion genes, it appears that hypX gene is transcribed from the FnrN-dependent hyp promoter, thus placing hypX in the hyp operon (hypBFCDEX). Comparisons of the HypX/HoxX sequences with those in databases provided unexpected insights into their function in hydrogenase synthesis. Similarities were restricted to two distinct regions in the HypX/HoxX sequences. Region I, corresponding to a sequence conserved in N10-formyltetrahydrofolate-dependent enzymes involved in transferring one-carbon units (C1), was located in the N-terminal half of the protein, whereas region II, corresponding to a sequence conserved in enzymes of the enoyl-CoA hydratase/isomerase family, was located in the C-terminal half. These similarities strongly suggest that HypX/HoxX have dual functions: binding of the C1 donor N10-formyltetrahydrofolate and transfer of the C1 to an unknown substrate, and catalysis of a reaction involving polarization of the C = O bond of an X-CO-SCoA substrate. These results also suggest the involvement of a small organic molecule, possibly synthesized with the participation of an X-CO-SCoA precursor and of formyl groups, in the synthesis of the metal-containing active centre of hydrogenase.

Identification of a gene for a chemoreceptor of the methyl-accepting type in the symbiotic plasmid of Rhizobium leguminosarum bv. viciae UPM791.

The 4 kb DNA region located immediately upstream of the Rhizobium leguminosarum bv. viciae UPM791 hydrogen structural genes was sequenced and found to encode a chemoreceptor of the methyl-accepting type, the first to be described in a rhizobial symbiotic plasmid. Two additional open reading frames were found. Their protein products showed sequence homology to dehydrogenases and isomerases involved in the metabolism of aromatic compounds. Mutant analysis showed that this region is not required for hydrogenase activity.

The hypBFCDE operon from Rhizobium leguminosarum biovar viciae is expressed from an Fnr-type promoter that escapes mutagenesis of the fnrN gene.

Pea (Pisum sativum L.) bacteroids produced by Rhizobium leguminosarum bv. viciae UPM791 synthesize a membrane-bound (NiFe) hydrogenase which oxidizes H2 arising from the nitrogen fixation process in root nodules. Synthesis of the active enzyme requires the products of the structural genes hupSL and an array of accessory proteins from at least 15 additional genes, including the gene cluster hypABFCDE, likely involved in nickel metabolism. Unlike the hupSL genes, which are expressed only in symbiosis, the hypBFCDE operon was also activated in vegetative cells in response to low pO2 in the culture medium. In microaerobic cells and in bacteroids, transcription of the hypBFCDE operon occurred from a promoter, P5b, with a transcription initiation site located 190 bp upstream of the ATG start codon of hypB, within the coding sequence of hypA. Transcription start site 5b was preceded by an Fnr box (anaerobox), 5'-TTGAgccatgTCAA-3', centered at position -39.5. Expression of the P5b promoter in the heterologous Rhizobium meliloti bacterial host was dependent on the presence of an active fixK gene. A 2.6-kb EcoRI fragment was isolated from an R. leguminosarum bv. viciae UPM791 gene bank by complementing an R. meliloti FixK- mutant. Sequencing of this DNA fragment identified an fnrN gene, and cassette insertion mutagenesis demonstrated that R. leguminosarum bv. viciae fnrN is able to replace the R. meliloti fixK gene for activation of both the R. leguminosarum bv. viciae hypBFCDE operon and the R. meliloti fix genes. However, bacteroids from a genomic FnrN- mutant of R. leguminosarum bv. viciae exhibited wild-type levels of hydrogenase activity. Microaerobic expression of P(5b) was reduced to ca. 50% of the wild-type level in the FnrN(-) mutant. These results indicate that hyp gene expression escapes mutagenesis of the fnrN gene and suggest the existence of a second fnr-like gene in R. leguminosarum by. viciae. Southern blot analysis with an fnrN internal probe revealed the presence of a second genomic region with homology to fnrN.

Purification of Rhizobium leguminosarum HypB, a nickel-binding protein required for hydrogenase synthesis.

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-Argüeso, 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. leguminosarum microaerobic vegetative cells and pea bacteroids but not in R. leguminosarum aerobic cells. HypB protein synthesized by R. leguminosarum 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.

Nickel availability to pea (Pisum sativum L.) plants limits hydrogenase activity of Rhizobium leguminosarum bv. viciae bacteroids by affecting the processing of the hydrogenase structural subunits.

Rhizobium leguminosarum bv. viciae UPM791 induces the synthesis of an [NiFe] hydrogenase in pea (Pisum sativum L.) bacteroids which oxidizes the H2 generated by the nitrogenase complex inside the root nodules. The synthesis of this hydrogenase requires the genes for the small and large hydrogenase subunits (hupS and hupL, respectively) and 15 accessory genes clustered in a complex locus in the symbiotic plasmid. We show here that the bacteroid hydrogenase activity is limited by the availability of nickel to pea plants. Addition of Ni2+ to plant nutrient solutions (up to 10 mg/liter) resulted in sharp increases (up to 15-fold) in hydrogenase activity. This effect was not detected when other divalent cations (Zn2+, Co2+, Fe2+, and Mn2+) were added at the same concentrations. Determinations of the steady-state levels of hupSL-specific mRNA indicated that this increase in hydrogenase activity was not due to stimulation of transcription of structural genes. Immunoblot analysis with antibodies raised against the large and small subunits of the hydrogenase enzyme demonstrated that in the low-nickel situation, both subunits are mainly present in slow-migrating, unprocessed forms. Supplementation of the plant nutrient solution with increasing nickel concentrations caused the conversion of the slow-migrating forms of both subunits into fast-moving, mature forms. This nickel-dependent maturation process of the hydrogenase subunits is mediated by accessory gene products, since bacteroids from H2 uptake-deficient mutants carrying Tn5 insertions in hupG and hupK and in hypB and hypE accumulated the immature forms of both hydrogenase subunits even in the presence of high nickel levels.