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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

Uptake hydrogenase activity and ATP formation in Rhizobium leguminosarum bacteroids.

The role of uptake hydrogenase was studied in Rhizobium leguminosarum bacteroids from the nodules of Pisum sativum L. cv. Homesteader. Uptake hydrogenase activity, measured by the 3H2 uptake method, was dependent on O-consumption and was similar to H2 uptake measured by gas chromatography. Km for O2 of 0.0007 atm (0.0709 kPa) and a Km for H2 of 0.0074 atm (0.7498, kPa) were determined. H2 increased the rate of endogenous respiration by isolates with uptake hydrogenase (Hup+) but had no effect on an isolate lacking uptake hydrogenase (Hup-). A survey of 14 Hup+ isolates indicated a wide range of H2 uptake activities. Four of the isolates tested had activities similar to or higher than those found in two Hup+ Rhizobium japonicum strains. H2 uptake was strongly coupled to ATP formation in only 5 of the 14 isolates. H2 increased the optimal O2 level of C2H2 reduction by 0.01 atm and permitted enhanced C2H2 reduction at O2 levels above the optimum in both a coupled and an uncoupled isolate. At suboptimal O2 concentrations a small enhancement of C2H2 reduction by H2 was seen in two out of three isolates in which H2 oxidation was coupled to ATP formation. Thus, the main function of uptake hydrogenase in R. leguminosarum appears to be in the protection of nitrogenase from O2 damage.     

Construction of a broad host range cosmid cloning vector and its use in the genetic analysis of Rhizobium mutants

We have constructed a cosmid derivative of the low copy-number broad host-range cloning vector pRK290 (Ditta et al., 1980) by inserting a 1.6-kb Bg/II fragment containing lambda cos into the unique Bg/II site in pRK290. The new vector, pLAFR1, is 21.6 kb long, confers tetracycline resistance, contains a unique EcoRI site, and can be mobilized into and stably replicates within many Gram-negative hosts. We constructed a clone bank of Rhizobium meliloti DNA in pLAFR1 using a partial EcoRI digest. The mean insert size was 23.1 kb. When the clone bank was mated (en masse) from Escherichia coli to various R. meliloti auxotrophic mutants, tetracycline-resistant (Tcr) transconjugants were obtained at frequencies ranging from 0.1 to 0.8, and among these, prototrophic colonies were obtained at frequencies ranging from 0.001 to 0.007. pLAFR1 cosmids were mobilized from R. meliloti prototrophic colonies into E. coli and then reintroduced into R. meliloti auxotrophs. In most cases, 100% of these latter Tcr transconjugants were prototrophic.     

The identification, characterization, sequencing and mutagenesis of the genes (hupSL) encoding the small and large subunits of the H2-uptake hydrogenase of Azotobacter chroococcum

The structural genes (hupSL) of the membrane-bound NiFe-containing H2-uptake hydrogenase (Hup) of Azotobacter chroococcum were identified by oligonucleotide screening and sequenced. The small subunit gene (hupS) encodes a signal sequence of 34 amino acids followed by a 310-amino-acid, 34156D protein containing 12 cysteine residues. The large subunit gene (hupL) overlaps hupS by one base and codes for a predicted 601-amino-acid, 66433D protein. There are two regions of strong homology with other Ni hydrogenases: a Cys-Thr-Cys-Cys-Ser motif near the N-terminus of HupS and an Asp-Pro-Cys-Leu-Ala-Cys motif near the carboxy-terminus of HupL. Strong overall homology exists between Azotobacter, Bradyrhizobium japonicum and Rhodobacter capsulatus Hup proteins but less exists between the Azotobacter proteins and hydrogenases from Desulfovibrio strains. Mutagenesis of either hupS or hupL genes of A. chroococcum yielded Hup- phenotypes but some of these mutants retained a partial H2-evolving activity. Hybridization experiments at different stages of gene segregation confirmed the multicopy nature of the Azotobacter genome.     

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.     

6-cyanopurine, a color indicator useful for isolating mutations in the nif (nitrogen fixation) genes of Klebsiella pneumoniae.

6-Cyanopurine (6-CP) can be used as a color indicator for certain classes of nif (N2 fixation) mutations in Klebsiella pneumoniae. Under N2-fixing conditions, Nif+ colonies and most Nif- colonies are purple on media containing 6-CP. Twenty-two Nif- mutants with altered color on medium containing 6-CP were isolated. All white mutants contained mutations in the regulatory genes, nifAA-nifL. Mutants which were more darkly colored than the wild type had mutations distributed among six nif genes. Medium with 6-CP was used to isolate Nif- mutants with deletions internal to the nif genes, and 6-CP was used to identify strains depressed for nitrogenase synthesis in the presence of NH4+.     

Identification and Characterization of two nitrogen fixation regulatory regions, nifA and nfrX, in Azotobacter vinelandii and Azotobacter chroococcum

Five Tn5-induced Nif- mutants of Azotobacter vinelandii were characterized as regulatory mutants because they were restored to Nif+ by the introduction of constitutively expressed nifA from Klebsiella pneumoniae. The mutants fell into two different classes on the basis of hybridization to a Rhizobium leguminosarum nifA gene probe and by complementation with cosmids isolated from pLAFRI gene banks of A. vinelandii and Azotobacter chroococcum. One mutant, MV3, was located in or near a nifA gene. The others, MV12, MV16, MV18 and MV26, defined a new regulatory gene, which has been called nfrX. The lack of expression of different nif-lacZ fusions confirmed the regulatory phenotype of all five mutant strains. The ability of both nifA and nfrX mutants to grow on nitrogen-free medium with vanadium, but not on medium with molybdenum, suggests that neither gene is required for expression of the alternative V-containing nitrogenase of A. vinelandii. A fragment carrying Tn5 and flanking DNA from MV3 was used as a probe to isolate the nifA region of A. chroococcum. Ligation of two adjacent EcoRI fragments of A. chroococcum yielded an intact nifA gene that activated expression of nifH-lac fusions and also restored MV3 to Nif+. The four nfrX mutants were complemented by pLAFR1 cosmids pLV163 and pLC121. The nfrX gene was subcloned from pLV163 and located within a 3.2 kb fragment. To determine whether nfrX might be found in other nitrogen-fixing organisms, DNA from 13 different species was hybridized to an nfrX probe. The failure to observe hybridization suggests that nfrX may be specific to nif regulation in Azotobacter.     

Cloning and characterization of a Bacteroides conjugal tetracycline-erythromycin resistance element by using a shuttle cosmid vector.

The Bacteroides conjugal tetracycline resistance (Tcr) elements appear not to be plasmids. In many cases, resistance to erythromycin (Emr) is cotransferred with Tcr. Using a newly constructed shuttle cosmid, pNJR1, we cloned 44 to 50 kilobase pairs of a conjugal Tcr Emr element on overlapping cosmid clones. Cosmid libraries were made in Escherichia coli with DNA from the original clinical Bacteroides thetaiotaomicron DOT strain containing Tcr Emr-DOT or from a Bacteroides uniformis Tcr Emr-DOT transconjugant strain. The cosmid clones were mobilized from E. coli into B. uniformis in groups of 10 to 20 per filter mating, with selection for Tcr or Emr transconjugants. The Tcr and Emr genes were cloned both separately and together on 30-kilobase-pair fragments. Several of the Tcr clones also contained transfer genes that permitted self-transfer of the cosmid from B. uniformis donors to E. coli or B. uniformis recipients. Neither the Tcr nor the Emr gene conferred resistance on E. coli, and the transfer-proficient clones did not self-transfer out of E. coli. Southern blot analysis was used to compare DNA from independently isolated Bacteroides strains carrying conjugal Tcr or Tcr Emr elements and their respective B. uniformis transconjugants. Results of these analyses indicate that there are large regions of homology, including regions outside the Tcr and Emr genes, but that the elements are not identical. Some Tcr clones contained a region which hybridized to chromosomal DNA from the wild-type B. uniformis recipient strain that did not carry the Tcr Emr-DOT element. This region of homology appeared not to be a junction fragment. It was not required in a Bacteroides recipient for successful transfer of the Tcr Emr element. Although we are not sure we have cloned a junction fragment between the Tcr Emr-DOT element and the B. uniformis chromosome, the preliminary function and restriction map appears to be linear.