Genetic organization of the hydrogen uptake (hup) cluster from Rhizobium leguminosarum

Leyva, Antonio; Palacios, José Luis Calvo; Murillo, Jesús; Ruiz-Argüeso, Tomás

doi:10.1128/jb.172.3.1647-1655.1990

Cited by 47 publications

(56 citation statements)

References 41 publications

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“…viciae (25,36), indicating that the potential for hydrogenase activity of this species has been underestimated in the past. Nickel-dependent stimulation of the activity of the nickel-containing enzymes urease and hydrogenase has been reported previously for soybeans grown in low-nickel soil (2).…”

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“…viciae mutant strains examined are able to transcribe and translate the hydrogenase structural genes at levels at least similar to those found in the wild-type strain but are unable to process the hydrogenase subunits even when supplied with large amounts of nickel. Taking into account that the TnS insertions in AL25, AL26, and AL18 and ALl map in different transcriptional units (25), it can be concluded that several accessory genes, at least one from each of the clusters hupGHIJ, hupK hypA, and hypBFCDE, are involved in the nickel-dependent processing of pea bacteroid hydrogenase subunits. DISCUSSION The data presented in this work show that nickel availability to pea plants grown in nitrogen-free nutrient solutions limits the hydrogenase activity of pea bacteroids from R. leguminosarum Hup+ strains.…”

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“…viciae hydrogenase small subunit (see Materials and Methods) and antibodies raised against the B. japonicum hydrogenase large subunit, which were previously shown to be cross-reactive with the homologous polypeptide from R. leguminosarum bv. viciae (25).…”

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“…Cosmid pAL618 is a pLAFR1 derivativ.4 containing the .hydrogenase gene cluster from strain UPM791 in a DNA insert of 20 kb (24). This cosmid was introduced into UPM791 by triparental mating as previously described (25). Strains were routinely grown on yeast mannitol medium (25) (41).…”

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“…This cosmid was introduced into UPM791 by triparental mating as previously described (25). Strains were routinely grown on yeast mannitol medium (25) (41). Pea bacteroids were prepared from 24-day-old nodulated plants and analyzed amperometrically for H2 uptake hydrogenase activity (41).…”

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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

et al. 1994

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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...

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