Nucleotide sequence of Rhizobium meliloti nodulation genes

A Rhizobium meliloti DNA region, determining nodulation functions common in different Rhizobium species, has been delimited by directed Tn5 mutagenesis and its nucleotide sequence has been determined. The sequence data indicates three large open reading frames with the same polarity coding for three proteins of 196, 217 and 402 (or 426) amino acid residues, respectively. We suggest the existence of three nod genes on this region, which were designated as nodA, B and C, respectively. Comparison of the R. meliloti nodA, B, C nucleotide and amino acid sequences with those from R. leguminosarum, as reported in the accompanying paper, shows 69-72% homology, clearly demonstrating the high degree of conservation of common nod genes in these Rhizobium species.     

Recycling efficiency in hydrogenase uptake positive strains of Rhizobium leguminosarum

Essentially chlorophyll-free preparations of mitochondria from different tissues of the same plant can be obtained by a combined three step preparation procedure involving differential centrifugation, partition in aqueous polymeric two-phase system and centrifugation in a Percoll gradient. The polypeptide patterns of mitochondria from photosynthetic (leaves) and non-photosynthetic (petioles and roots) tissue from spinach were compared by use of SDS-electrophoresis.
About 35 polypeptides were found in leaf mitochondria with molecular weights from 14 to 103 kdalton. The polypeptide patterns of the membrane fractions and matrix fractions showed great differences. The membrane fractions contained significantly more polypeptide bands than the matrix fractions. The polypeptide patterns of mitochondria from photosynthetic and non-photosynthetic tissues showed some striking differences. The 15.9, 41.7, 50.7 and 101 kdalton polypeptides were clearly detected in leaf mitochondria but these polypeptides were not found or found in only small amounts in petiole and root mitochondria. The differences were mainly associated with the matrix fractions. Staining with 3,3',5,5'-tetramethylbenzidine and hydrogen peroxide for heme containing polypeptides showed that the polypeptides which differ do not contain heme.     

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.     

Screening method for the detection of uptake hydrogenase activity of Rhizobium leguminosarum bacteroids

Abstract A method has been developed for screening Rhizobium leguminosarum wild-type strains and mutants for uptake hydrogenase (Hup) activity, using H₂-dependent methylene blue reduction. For this purpose, a simple device has been constructed which allows the simultaneous screening of 6 strains and 6 controls. Bacteroids of R. leguminosarum isolated from pea root nodules were suspended in buffer containing methylene blue and inhibitors of dehydrogenases. The suspensions were first sparged with argon (to remove oxygen) and then with hydrogen.     

Comparison of characteristics of the nodX genes from various Rhizobium leguminosarum strains

We have analyzed the nucleotide sequences of the nodX genes from two strains of Rhizobium leguminosarum bv. viciae able to nodulate Afghan peas (strains A1 and Himalaya) and from two strains of R. leguminosarum bv. trifolii (ANU843 and CSF). The nodX genes of strains A1 and ANU843 were shown to be functional for the induction of nodules on Afghan peas. To analyze the cause of phenotypic differences of strain A1 and strain TOM we have studied the composition of the lipochitin-oligosaccharides (LCOs) produced by strain A1 after induction by the flavonoid naringenin or various pea root exudates. The structural analysis of the LCOs by mass spectrometry revealed that strain A1 synthesizes a family of at least 23 different LCOs. The use of exudates instead of naringenin resulted only in quantitative differences in the ratios of various LCOs produced.     

Nucleotide sequence of the ipaBCD structural genes of Shigella dysenteriae

A 9 kb EcoRI and two PstI fragments from the virulence plasmid of Shigella dysenteriae CG097 were shown to contain all ipa genes by probing with Shigella flexneri ipaB, -C, -D and -A gene probes. The DNA sequences of S. dysenteriae ipaBC genes were very similar to those of S. flexneri M90T and S. flexneri YSH6000, but ipaD differed by 22 codons from that of S. flexneri. The differences in ipaD may account for the different in vitro host specificities shown by S. dysenteriae and S. flexneri. The nucleotide composition of ipa genes revealed an unusually large number of codons that are rarely used in Escherichia coli chromosomal genes, indicating a different origin.     

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.     

Regulation of tryptophan genes in Rhizobium leguminosarum.

Twelve tryptophan auxotrophs of Rhizobium leguminosarum were characterized biochemically. They were grown in complex and minimal media with several carbon sources, in both limiting and excess tryptophan. Missing enzyme activities allowed assignment of all mutant to the trpE, trpD, trpB, or trpA gene, confirming earlier results with the same mutants (Johnston et al., Mol. Gen. Genet. 165:323-330, 1978). In regulatory experiments, only the first enzyme of the pathway, anthranilate synthase, responded (about 15-fold) to tryptophan excess or limitation.     

Role of uptake hydrogenase in providing reductant for nitrogenase in Rhizobium leguminosarum bacteroids

The role of uptake hydrogenase in providing reducing power to nitrogenase was investigated in Rhizobium leguminosarum bacteroids from nodules of Pisum sativum L. (cv. Homesteader). H₂ increased the rate of C₂H₂ reduction in the absence of added substrates. Malate also increased nitrogenase (C₂H₂) activity while decreasing the effect of H₂. At exogenous malate concentrations above 0.05 mM no effect of H₂ was seen. Malate appeared to be more important as a source of reductant than of ATP. When iodoacetate was used to minimize the contribution of endogenous substrates to nitrogenase activity in an isolate in which H₂ uptake was not coupled to ATP formation, H₂ increased the rate of C₂H₂ reduction by 77%. In the presence of iodoacetate, an ATP-generating system did not enhance C₂H₂ reduction, but when H₂ was also included, the rate of C₂H₂ reduction was increased by 280% over that with the ATP-generating system alone. The data suggest that, under conditions of substrate starvation, the uptake hydrogenase in R. leguminosarum could provide reductant as well as ATP in an isolate in which the H₂ uptake is coupled to ATP formation, to the nitrogenase complex.     

DNA sequence of the Rhizobium leguminosarum nodulation genes nodAB and C required for root hair curling

A 3.2kb fragment of DNA cloned from Rhizobium leguminosarum has been shown to contain the genes necessary for the induction of root hair curling, the first observed step in the infection of leguminous plants by R. leguminosarum. The DNA sequence of this region has been determined and three open reading frames were identified: genes corresponding to these open reading frames have been called nodA, nodB and nodC and are transcribed in that order. Mutations within the nodC gene completely blocked root hair curling. However, a subcloned fragment containing only the nodC gene did not induce normal root hair curling (although some branching was observed), indicating that the nodA and B genes may also be required for normal root hair curling. From an analysis of the predicted amino acid sequences of the nodAB and C genes it appeared unlikely that their products are secreted; therefore it is concluded that the induction of root hair curling could be due to a secreted metabolite.     

Nitrate reductase activity in nodules of pea inoculated with hydrogenase positive and hydrogenase negative strains of Rhizobium leguminosarum

The nitrate reductase (NR, EC 1.6.6.1) activity in root nodules formed by hydrogenase positive (Hup⁺) and hydrogenase negative (Hup⁻) Rhizobium leguminosarum strains was examined in symbioses with the pea cultivar Alaska ( Pisum sativum L.), Rates of activity were determined by the in vivo assay in nodules from plants that were only N₂-dependent or grown in the presence of 2 m M KNO₃. The rates varied widely among strains, regardless of the Hup phenotype of the R. leguminosarum strain used for inoculation, but the overall results indicated that nodules formed by Hup⁻ strains accumulated more nitrite in the incubation medium than did those with Hup⁻ phenotypes. Total plant dry weight and reduced nitrogen content of pea plants grown in the presence of 2 m M KNO₃ and inoculated with single Hup⁺ and Hup⁻ R. leguminosarum strains were statistically different among some strains. These observations suggest that the possible advantages derived from the presence of the Hup system on whole plant growth may be counteracted by the higher rates of NR activity in the Hup⁻ strains in the R. leguminosarum -pea symbiosis.     

Host-range related structural features of the acidic extracellular polysaccharides of Rhizobium trifolii and Rhizobium leguminosarum

Proton nuclear magnetic resonance (1H NMR) and fast atom bombardment mass spectrometric analyses were performed on enzymatically derived oligosaccharides from the acidic excreted polysaccharides (EPS) from representative bacterial strains of the pea-nodulating symbiont, Rhizobium leguminosarum (128C53, 128C63, and 300) and the clover-nodulating symbiont, Rhizobium trifolii (NA-30, ANU843, 0403, TA-1, LPR5035, USDA20.102, and 4S). The results revealed structural similarities and differences between EPS of these two species. Octasaccharide units containing galactose, glucuronic acid, alpha-L-threo-hex-4-enopyranosyluronic acid, and glucose in a molar ratio of 1:1:1:5 were obtained from the EPS of the three R. leguminosarum strains and had the same primary glycosyl sequence and location of pyruvate, acetate, and 3-hydroxybutyrate substituents. About 80% of the galactose residues were acylated with 3-hydroxybutyrate, and there were two acetyl groups per repeating unit distributed between the 2 glucose residues of the main chain-derived sequence of the octasaccharides. In contrast, the R. trifolii strains had varied EPS structures, each of which differed from the common R. leguminosarum EPS structure. The EPS from one group of R. trifolii strains (0403 and LPR5035) most closely resembled the R. leguminosarum EPS but differed in that a lower number of galactose and glucose residues were substituted by 3-hydroxybutyryl and acetyl groups, respectively. The EPS from a second group of R. trifolii strains (ANU843, TA-1, and NA-30) was even more different than the R. leguminosarum EPS. These R. trifolii octasaccharides bore a single acetyl group on O-3 of the glucuronic acid residue. In addition, the level of acylation by 3-hydroxybutyryl groups was 50% of that present in the R. leguminosarum EPS. The remaining two strains of R. trifolii (USDA20.102 and 4S) had very different patterns of acylation to each other and to all of the other strains. The EPS from strain USDA20.102 practically lacked 3-hydroxybutyryl groups and had a unique degree and pattern of acetylation. The oligomers from the EPS of R. trifolii strain 4S completely lacked 3-hydroxybutyryl groups and galactose. The latter EPS contained only one O-1-carboxyethylidene group and had a different degree and pattern of acetylation. Interestingly, these two latter strains differ from the other R. trifolii strains in nodulation rates on rare clover species in the Trifolium cross-inoculation group. Thus, we define several groups of R. trifolii based upon their EPS structures and establish their similarities and distinct differences with the EPS of R. leguminosarum.(ABSTRACT TRUNCATED AT 400 WORDS)     

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.