Isolation of genes (nif/hup cosmids) involved in hydrogenase and nitrogenase activities in Rhizobium japonicum.

Recombinant cosmids containing a Rhizobium japonicum gene involved in both hydrogenase (Hup) and nitrogenase (Nif) activities were isolated. An R. japonicum gene bank utilizing broad-host-range cosmid pLAFR1 was conjugated into Hup- Nif- R. japonicum strain SR139. Transconjugants containing the nif/hup cosmid were identified by their resistance to tetracycline (Tcr) and ability to grow chemoautotrophically (Aut+) with hydrogen. All Tcr Aut+ transconjugants possessed high levels of H2 uptake activity, as determined amperometrically. Moreover, all Hup+ transconjugants tested possessed the ability to reduce acetylene (Nif+) in soybean nodules. Cosmid DNAs from 19 Hup+ transconjugants were transferred to Escherichia coli by transformation. When the cosmids were restricted with EcoRI, 15 of the 19 cosmids had a restriction pattern with 13.2-, 4.0-, 3.0-, and 2.5-kilobase DNA fragments. Six E. coli transformants containing the nif/hup cosmids were conjugated with strain SR139. All strain SR139 transconjugants were Hup+ Nif+. Moreover, one nif/hup cosmid was transferred to 15 other R. japonicum Hup- mutants. Hup+ transconjugants of six of the Hup- mutants appeared at a frequency of 1.0, whereas the transconjugants of the other nine mutants remained Hup-. These results indicate that the nif/hup gene cosmids contain a gene involved in both nitrogenase and hydrogenase activities and at least one and perhaps other hup genes which are exclusively involved in H2 uptake activity.     

Cloning and characterization of hydrogen uptake genes from Rhizobium leguminosarum.

A gene library of genomic DNA from the hydrogen uptake (Hup)-positive strain 128C53 of Rhizobium leguminosarum was constructed by using the broad-host-range mobilizable cosmid vector pLAFR1. The resulting recombinant cosmids contained insert DNA averaging 21 kilobase pairs (kb) in length. Two clones from the above gene library were identified by colony hybridization with DNA sequences from plasmid pHU1 containing hup genes of Bradyhizobium japonicum. The corresponding recombinant cosmids, pAL618 and pAL704, were isolated, and a region of about 28 kb containing the sequences homologous to B. japonicum hup-specific DNA was physically mapped. Further hybridization analysis with three fragments from pHU1 (5.9-kb HindIII, 2.9-kb EcoRI, and 5.0-kb EcoRI) showed that the overall arrangement of the R. leguminosarum hup-specific region closely parallels that of B. japonicum. The presence of functional hup genes within the isolated cosmid DNA was demonstrated by site-directed Tn5 mutagenesis of the 128C53 genome and analysis of the Hup phenotype of the Tn5 insertion strains in symbiosis with peas. Transposon Tn5 insertions at six different sites spanning 11 kb of pAL618 completely suppressed the hydrogenase activity of the pea bacteroids.     

Further evidence that two unique subunits are essential for expression of hydrogenase activity in Rhizobium japonicum.

Eight strains of Rhizobium lacking hydrogenase uptake (Hup) activity and 17 transconjugant strains carrying the hup cosmids pHU1, pHU52, or pHU53 (G. R. Lambert, M. A. Cantrell, F. J. Hanus, S. A. Russell, K. R. Haddad, and H. J. Evans, Proc. Natl. Acad. Sci. USA, 82:3232-3236, 1985) were screened for Hup activity and the presence of immunologically detectable hydrogenase polypeptides. Crude extracts of these strains were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot analysis with affinity-purified antibodies against the two subunits of purified hydrogenase (Mr 60,000 and 30,000). Derepressed transconjugants carrying the cosmid pHU52 were Hup+ and contained detectable levels of both hydrogenase subunit polypeptides. Non-derepressed strains, Hup- parent strains, and strains carrying cosmids other than pHU52 did not express Hup activity and contained no immunologically detectable protein. These data provide further evidence for the essential involvement of the smaller (Mr 30,000) subunit in the expression of hydrogenase activity in Rhizobium japonicum and suggest that the determinants for hydrogenase subunit synthesis are present on pHU52.     

Conserved Plasmid Hydrogen-Uptake (hup)-Specific Sequences within HupRhizobium leguminosarum Strains

Thirteen Rhizobium leguminosarum strains previously reported as H(2)-uptake hydrogenase positive (Hup) or negative (Hup) were analyzed for the presence and conservation of DNA sequences homologous to cloned Bradyrhizobium japonicum hup-specific DNA from cosmid pHU1 (M. A. Cantrell, R. A. Haugland, and H. J. Evans, Proc. Natl. Acad. Sci. USA 80:181-185, 1983). The Hup phenotype of these strains was reexamined by determining hydrogenase activity induced in bacteroids from pea nodules. Five strains, including H(2) oxidation-ATP synthesis-coupled and -uncoupled strains, induced significant rates of H(2)-uptake hydrogenase activity and contained DNA sequences homologous to three probe DNA fragments (5.9-kilobase [kb] HindIII, 2.9-kb EcoRI, and 5.0-kb EcoRI) from pHU1. The pattern of genomic DNA HindIII and EcoRI fragments with significant homology to each of the three probes was identical in all five strains regardless of the H(2)-dependent ATP generation trait. The restriction fragments containing the homology totalled about 22 kb of DNA common to the five strains. In all instances the putative hup sequences were located on a plasmid that also contained nif genes. The molecular sizes of the identified hup-sym plasmids ranged between 184 and 212 megadaltons. No common DNA sequences homologous to B. japonicum hup DNA were found in genomic DNA from any of the eight remaining strains showing no significant hydrogenase activity in pea bacteroids. These results suggest that the identified DNA region contains genes essential for hydrogenase activity in R. leguminosarum and that its organization is highly conserved within Hup strains in this symbiotic species.     

Cloning of DNA fragments carrying hydrogenase genes of Rhodopseudomonas capsulata

A cosmid library of Rhodopseudomonas capsulata DNA was constructed in Escherichia coli HB101 using the broad-host-range cosmid vector pLAFR1. More than ninety per cent of the clones in the bank contained cosmids with DNA inserts averaging 20 kilobase pairs in length. Mutants deficient in uptake hydrogenase (Hup-) were obtained from R. capsulata strain B10 by ethylmethylsulfonate (EMS) mutagenesis. The content of hydrogenase protein in Hup- mutant cells was tested by rocket immunoelectrophoresis. Hup- mutants (Rifr) were complemented with the clone bank by conjugation and, from the transconjugants selected by rifampicin and tetracycline resistance, Hup+ transconjugants were screened for the ability to grow photoautotrophically and to reduce methylene blue in a colony assay. The recombinant plasmid pAC57 restored hydrogenase activity in the Hup- mutants RCC8, RCC10, RCC12 and ST410 whereas pAG202 restored that of IR4. The cloned R. capsulata DNA insert of pAC57 gave 5 restriction fragments by cleavage with EcoRI endonuclease. Fragment 1 (7 kb) restored hydrogenase activity in Hup- mutant strains RCC12 and ST410 and fragment 5 (1.3 kb) in strains RCC8 and RCC10. Since the 2 cosmids pAC57 and pAG202 are different cosmids, as indicated by restriction analyses and absence of cross hybridization, it is concluded that at least two hup genes are required for the expression of hydrogenase activity in R. capsulata.     

Expression of Chromosome-integrated Hydrogen-uptake Genes in Cicer-Rhizobium

Integration of H₂-uptake (hup) gene cosmid pHU52 into the chromosomal DNA, conferred H₂- uptake activity on the Hup^- Cicer-Rhizobium strain G36–84 in the free-living state and in nodules. In five transconjugants (G36–84:: Tn5:: pHU52) derepressed for hup gene expression, the specific Hup activity ranged from 158 to 256 nmole H₂ hr^-1 mg-¹ protein which was 42 to 64% lower than the activity obtained in transconjugant with pHU52 as an episome. Integration of the cosmid significantly improved the relative efficiency of symbiotic N₂-fixation by imparting H₂-recycling capability to Hup^- Cicer-Rhizobium. Demonstration of Hup activity in the nodules of field grown chickpea plants suggests that the integrated hup genes are stably maintained in natural environment     

Symbiotic Expression of Cosmid-Borne Bradyrhizobium japonicum Hydrogenase Genes

The expression of cosmid-borne Bradyrhizobium japonicum hydrogenase genes in alfalfa, clover, and soybean nodules harboring Rhizobium transconjugants was studied. Cosmid pHU52 conferred hydrogen uptake (Hup) activity in both free-living bacteria and in nodules on the different plant hosts, although in nodules the instability of the cosmid resulted in low levels of Hup activity. In contrast, cosmid pHU1, which does not confer Hup activity on free-living bacteria, gave a Hup⁺ phenotype in nodules on alfalfa and soybean. Nodules formed by B. japonicum USDA 123Spc(pHU1) recycled about 90% of nitrogenase-mediated hydrogen evolution. Both subunits of hydrogenase (30- and 60-kilodalton polypeptides) were detected in enzyme-linked immunosorbent assays of bacteroid preparations from nodules harboring B. japonicum strains with pHU1 or pHU52. Neither pHU53 nor pLAFR1 conferred detectable Hup activity in either nodules or free-living bacteria. Based on the physical maps of pHU1 and pHU52, it is suggested that a 5.5-kilobase EcoRI fragment unique to pHU52 contains a gene or part of a gene required for Hup activity in free-living bacteria but not in nodules. This conclusion is supported by the observation that two Tn5 insertions in the chromosome of B. japonicum USDA 122DES obtained by marker exchange with Tn5-mutagenized pHU1 abolished Hup activity in free-living bacteria but not in nodules.     

Increased Nitrogenase-Dependent H(2) Photoproduction by hup Mutants of Rhodospirillum rubrum

Transposon Tn5 mutagenesis was used to isolate mutants of Rhodospirillum rubrum which lack uptake hydrogenase (Hup) activity. Three Tn5 insertions mapped at different positions within the same 13-kb EcoRI fragment (fragment E1). Hybridization experiments revealed homology to the structural hydrogenase genes hupSLM from Rhodobacter capsulatus and hupSL from Bradyrhizobium japonicum in a 3.8-kb EcoRI-ClaI subfragment of fragment E1. It is suggested that this region contains at least some of the structural genes encoding the nickel-dependent uptake hydrogenase of R. rubrum. At a distance of about 4.5 kb from the fragment homologous to hupSLM, a region with homology to a DNA fragment carrying hypDE and hoxXA from B. japonicum was identified. Stable insertion and deletion mutations were generated in vitro and introduced into R. rubrum by homogenotization. In comparison with the wild type, the resulting hup mutants showed increased nitrogenase-dependent H(2) photoproduction. However, a mutation in a structural hup gene did not result in maximum H(2) production rates, indicating that the capacity to recycle H(2) was not completely lost. Highest H(2) production rates were obtained with a mutant carrying an insertion in a nonstructural hup-specific sequence and with a deletion mutant affected in both structural and nonstructural hup genes. Thus, besides the known Hup activity, a second, previously unknown Hup activity seems to be involved in H(2) recycling. A single regulatory or accessory gene might be responsible for both enzymes. In contrast to the nickel-dependent uptake hydrogenase, the second Hup activity seems to be resistant to the metal chelator EDTA.     

Integration of hup Genes into the Genome of Chickpea-Rhizobium Through Site-Specific Recombination

The hup gene fragment of cosmid pHU52 was integrated into the genome of chickpea-Rhizobium Rcd301 via site-specific homologous recombination. Two small fragments of genomic DNA of strain Rcd301 itself were provided to flank cloned hup genes to facilitate the integration. The hup insert DNA of cosmid pHU52 was Isolated as an Intact 30.2 kb fragment using EcoRI, and cloned on partially restricted cosmid clone pSPSm3, which carries a DNA fragment of strain Rcd301 imparting streptomycin resistance. One of the recombinant cosmid clones, pBSL 12 thus obtained was conjugally transferred to the strain Rcd30₁. The integration of hup gene fragment into the genomic DNA through site-specific homologous recombination, was ensured by introducing an incompatible plasmid, pPH1 JI. The integration was confirmed by Southern hybridization. The integrated hup genes were found to express ex plants in two such constructs BSL 12–1 and BSL 12–3.     

Revertible hydrogen uptake-deficient mutants of Rhizobium japonicum.

We have developed mutants of Rhizobium japonicum which are deficient in H2 uptake capacity (Hup-) and which spontaneously revert to the parent type at a frequency consistent with that of a single-point mutation (ca. 1.0 x 10(-09)). The mutagenesis by nitrous acid and the selection of the Hup- phenotype by using penicillin and chemolithotrophy as enrichment for chemolithotrophy-deficient strains are described. Two mutants retain low but reproducible levels of ribulose bisphosphate-dependent CO2 fixation when grown on a low-carbon medium under an atmosphere of 1% O2, 4% H2, 5% CO2, and 90% N2. Neither O2 nor the artificial electron acceptors phenazine methosulfate or methylene blue supported detectable H2 uptake by the free-living Hup- mutants or by their bacteroids. Plant growth experiments under bacteriologically controlled conditions were conducted to assess the mutants' performance as inocula for soybean plants. Plants inoculated with Hup- strains had lower dry weights and contained less total N than did plants inoculated with the parent Hup+ strain. Use of either the Hup- mutants or the Hup+ parent strain as inocula, however, did not significantly affect the acetylene-reducing activity or the fresh weight of nodules. These results, obtained with apparently isogenic lines of H2 uptake-deficient R. japonicum, provide strong support for a beneficial role of the H2 uptake phenotype in legume symbiosis.     

A histone-like protein of Helicobacter pylori protects DNA from stress damage and aids host colonization

Genomic DNA in a bacterial cell is folded into a compact structure called a nucleoid, and nucleoid-associated proteins are responsible for proper assembly of active higher-order genome structures. The human gastric pathogen Helicobacter pylori express a nucleoid-associated protein encoded by the hup gene, which is the homolog to the Escherichia coli histone-like protein HU. An H. pylori hup mutant strain (X47 hup:cat) showed a defect in stationary phase survival. The X47 hup:cat mutant was more sensitive to the DNA damaging agent mitomycin C, and displayed a decreased frequency of DNA recombination, indicating Hup plays a significant role in facilitating DNA recombinational repair. The X47 hup:cat mutant was also sensitive to both oxidative and acid stress, conditions that H. pylori commonly encounters in the host. The hup mutant cells survived significantly (7-fold) less upon exposure to macrophages than the wild type strain. In a mouse infection model, the hup mutant strain displayed a greatly reduced ability to colonize host stomachs. The geometric means of colonization number for the wild type and hup mutant were 6×10(5) and 1.5×10(4)CFU/g stomachs, respectively. Complementation of the hup strain by chromosomal insertion of a functional hup gene restored oxidative stress resistance, DNA transformation frequency, and mouse colonization ability to the wild type level. We directly demonstrated that the purified His-tagged H. pylori Hup protein can protect (in vitro) an H. pylori-derived DNA fragment from oxidative damage.     

根瘤菌吸氢酶基因转移的研究

通过三亲本杂交将含有花生根瘤菌吸氢基因的质粒pZ 55(Tcr)转入不吸氢的花生根瘤菌Ra 34等菌株 (Hup- ,Nif+,Apr)中 ,筛选到既具有吸氢又具有固氮能力的花生根瘤菌结合株Rz34 2。在自生和共生条件下 ,结合株均可表达高吸氢和高固氮活性。以结合株Rz34 2接种的花生植株叶片的干重比不接种的、接种受体株Ra34(Hup- )和接种对照菌株L8 3(Hup+,Nif+)的分别高 6.2 %、7.6%和 6.3% ;种子的含氮量分别高 8 9%、1.00 %和 6.0 % ;产量分别高 188%、1 0 5%和 1 0 7%。研究结果表明 ,以含吸氢基因的结合株接种花生能提高根瘤菌与花生的共生固氮效率 ,增加作物的产量。     

Nif- Hup- mutants of Rhizobium japonicum.

Two H2 uptake-negative (Hup-) Rhizobium japonicum mutants were obtained that also lacked symbiotic N2 fixation (acetylene reduction) activity. One of the mutants formed green nodules and was deficient in heme. Hydrogen oxidation activity in this mutant could be restored by the addition of heme plus ATP to crude extracts. Bacteroid extracts from the other mutant strain lacked hydrogenase activity and activity for both of the nitrogenase component proteins. Hup+ revertants of the mutant strains regained both H2 uptake ability and nitrogenase activity.     

Hydrogenase genes are uncommon and highly conserved in Rhizobium leguminosarum bv. viciae

A screening for hydrogen uptake (hup) genes in Rhizobium leguminosarum bv. viciae isolates from different locations within Spain identified no Hup+ strains, confirming the scarcity of the Hup trait in R. leguminosarum. However, five new Hup+ strains were isolated from Ni-rich soils from Italy and Germany. The hup gene variability was studied in these strains and in six available strains isolated from North America. Sequence analysis of three regions within the hup cluster showed an unusually high conservation among strains, with only 0.5-0.6% polymorphic sites, suggesting that R. leguminosarum acquired hup genes de novo in a very recent event.     

Immunological homology between the membrane-bound uptake hydrogenases of Rhizobium japonicum and Escherichia coli.

Two polypeptides present in aerobic and anaerobic cultures of Escherichia coli HB101 were shown to cross-react with antibodies to the 30- and 60-kilodalton (kDa) subunits of the uptake hydrogenase of Rhizobium japonicum. The cross-reactive polypeptides in a series of different E. coli strains are of Mrs ca. 60,000 and 30,000, and both polypeptides are present in proportion to measurable hydrogen uptake (Hup) activity (r = 0.95). The 60-kDa polypeptide from E. coli HB101 comigrated on native gels with detectable Hup activity. The exact role of the 30-kDa polypeptide in E. coli is unclear. E. coli MBM7061, a natural Hup- variant, grown anaerobically or aerobically lacked detectable Hup activity and failed to cross-react with the antisera against the hydrogenase from R. japonicum. Anaerobically cultured E. coli MBM7061, however, did express formate hydrogenlyase activity, indicating that the hydrogenases involved in the oxygen-dependent activation of hydrogen and the formate-dependent evolution of hydrogen are biochemically distinct.     

Transposon Tn5-Generated Bradyrhizobium japonicum Mutants Unable To Grow Chemoautotrophically with H(2)

Twelve Tn5-induced mutants of Bradyrhizobium japonicum unable to grow chemoautotrophically with CO(2) and H(2) (Aut) were isolated. Five Aut mutants lacked hydrogen uptake activity (Hup). The other seven Aut mutants possessed wild-type levels of hydrogen uptake activity (Hup), both in free-living culture and symbiotically. Three of the Hup mutants lacked hydrogenase activity both in free-living culture and as nodule bacteroids. The other two mutants were Hup only in free-living culture. The latter two mutants appeared to be hypersensitive to repression by oxygen, since Hup activity could be derepressed under 0.4% O(2). All five Hup mutants expressed both ex planta and symbiotic nitrogenase activities. Two of the seven Aut Hup mutants expressed no free-living nitrogenase activity, but they did express it symbiotically. These two strains, plus one other Aut Hup mutant, had CO(2) fixation activities 20 to 32% of the wild-type level. The cosmid pSH22, which was shown previously to contain hydrogenase-related genes of B. japonicum, was conjugated into each Aut mutant. The Aut Hup mutants that were Hup both in free-living culture and symbiotically were complemented by the cosmid. None of the other mutants was complemented by pSH22. Individual subcloned fragments of pSH22 were used to complement two of the Hup mutants.     

Conserved Plasmid Hydrogen-Uptake (hup)-Specific Sequences within Hup+Rhizobium leguminosarum Strains

Thirteen Rhizobium leguminosarum strains previously reported as H₂-uptake hydrogenase positive (Hup⁺) or negative (Hup⁻) were analyzed for the presence and conservation of DNA sequences homologous to cloned Bradyrhizobium japonicum hup-specific DNA from cosmid pHU1 (M. A. Cantrell, R. A. Haugland, and H. J. Evans, Proc. Natl. Acad. Sci. USA 80:181-185, 1983). The Hup phenotype of these strains was reexamined by determining hydrogenase activity induced in bacteroids from pea nodules. Five strains, including H₂ oxidation-ATP synthesis-coupled and -uncoupled strains, induced significant rates of H₂-uptake hydrogenase activity and contained DNA sequences homologous to three probe DNA fragments (5.9-kilobase [kb] HindIII, 2.9-kb EcoRI, and 5.0-kb EcoRI) from pHU1. The pattern of genomic DNA HindIII and EcoRI fragments with significant homology to each of the three probes was identical in all five strains regardless of the H₂-dependent ATP generation trait. The restriction fragments containing the homology totalled about 22 kb of DNA common to the five strains. In all instances the putative hup sequences were located on a plasmid that also contained nif genes. The molecular sizes of the identified hup-sym plasmids ranged between 184 and 212 megadaltons. No common DNA sequences homologous to B. japonicum hup DNA were found in genomic DNA from any of the eight remaining strains showing no significant hydrogenase activity in pea bacteroids. These results suggest that the identified DNA region contains genes essential for hydrogenase activity in R. leguminosarum and that its organization is highly conserved within Hup⁺ strains in this symbiotic species.