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...

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.

No abstract

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 TnS 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.Most hydrogen uptake hydrogenases are membrane-bound, heterodimeric iron-sulfur proteins containing nickel ([NiFe] hydrogenases) (for reviews, see references 9, 37, and 46). Bacteria forming nodules on legume roots synthesize uptake hydrogenases which recycle the H2 evolved by nitrogenase in the nodules (5, 27) and contribute to the overall efficiency of the N2 fixation process (6). These bacteria include Rhizobium leguminosarum bv. viciae and Bradyrhizobium japonicum, the microsymbionts of peas and soybeans, respectively. The genetic determinants for H2 uptake (hup genes) in R. leguminosarum bv. viciae UPM791 are clustered in a 20-kb DNA region of the symbiotic plasmid and have been isolated in cosmid pAL618 (24). This cosmid has the capacity to confer H2 uptake activity on Hup-strains of R. leguminosarum bv. viciae and Rhizobium etli in symbiosis with peas and beans, respectively (25). The DNA region spanning the H2 uptake gene cluster has been sequenced, and 17 genes, closely linked and oriented in the same direction, were identified (15,38,40). The first two genes, hupS and hupL, encode the hydrogenase structural polypeptides (14). The predicted proteins for the small (360 amino acid residues, including a leader peptide of 45 residues) and the large (596 amino acid residues) subunits are homologous to the corresponding hydrogenase structur...