Identification and sequencing of a cytochrome P450 gene cluster from Bradyrhizobium japonicum.

Sequencing of a region from Bradyrhizobium japonicum previously shown to encode for cytochromes P450 revealed a cluster of three complete P450 genes (CYP112, CYP114, and CYP117) plus a partial P450 gene fragment (CYP115P). Present also are five additional open reading frames. The close positioning of the genes suggests that they comprise an operon. Although the biochemical function of the gene products is uncertain, the similarities to other genes suggests an operon involved in terpenoid synthesis. ORF3 has similarity to a [3Fe-4S] ferredoxin from Streptomyces griseolus. ORF4 has strong similarity to members of the short chain alcohol dehydrogenase family, including sterol dehydrogenases from enteric bacteria and to some plant 3-oxoacyl-(acyl carrier protein) reductases. ORF6 has strong similarity to prenyl transferases, including dimethylallyltranstransferase from Escherichia coli. ORF7 bears some similarity to plant genes for ent-kaurene synthase (a precursor of gibberellins), and to bacterial squalene-hopene cyclases. ORF8 has some similarity to a Streptomyces gene for synthesis of the cyclic sesquiterpene pentalenene. The 5' end of the mRNA transcript is 38-39 nucleotides downstream from the center of a motif that bears sequence homology to bacterial fnr promoters. A gus operon fusion to the promoter was expressed anaerobically and symbiotically 6-10-fold greater than aerobically.

Praja1, a novel gene encoding a RING-H2 motif in mouse development.

As part of a cloning strategy to identify genes involved in early mouse liver development we have isolated Praja1, a gene with similar sequences to the Drosophila melanogaster gene goliath (gl) which is involved in the fate of mesodermal cells ultimately forming gut musculatures, fat body, and the heart. Praja1 is a 2.1 kb gene encoding a putative 396 amino acid ORF and includes a COOH-terminal RING-H2 domain. Using the Jackson Laboratory BSS panel, we have localized Praja1 on chromosome X at 36 cM, which may be a candidate gene for mouse sla (sex linked sideroblastic anemia), near the X inactivation center gene, Xist. Northern blot analysis demonstrated three transcripts (3.1, 2.6 and 2.1 kb) in mRNA from adult mouse tissues brain, liver, and kidney as well as in mRNA from developing mouse embryos (days 7, 11, 15 and 17 post coitus, p.c.). In vitro transcription/translation yielded a product with an Mr of 59 kD. Immunohistochemical staining of in vitro liver explant cultures using a heterologous antibody against praja1 demonstrated cytoplasmic staining of cuboidal cells that have hepatocyte morphology and organization. The presence of the RING-H2 domain, a proline-rich region at the COOH-end, and regions rich in acidic amino acids, leads to the hypothesis that the Praja1 product is possibly involved in mediating protein-protein interactions, possibly as part of a protein sorting or transport pathway. This is strengthened by the similarity of Praja1 to rat Neurodap1, whose product has been shown to localize to the endoplasmic reticulum and golgi in brain.

Beta-glucan synthesis in Bradyrhizobium japonicum: characterization of a new locus (ndvC) influencing beta-(1-->6) linkages.

Bradyrhizobium japonicum synthesizes periplasmic cyclic beta-(1-->3),beta-(1-->6)-D-glucans during growth in hypoosmotic environments, and evidence is growing that these molecules may have a specific function during plant-microbe interactions in addition to osmoregulation. Site-directed Tn5 mutagenesis of the DNA region upstream of ndvB resulted in identification of a new gene (ndvC) involved in beta-(1--> 3), beta-(1-->6)-glucan synthesis and in nodule development. The predicted translation product was a polypeptide (ca. 62 kDa) with several transmembrane domains. It contained a sequence characteristic of a conserved nucleoside-sugar-binding motif found in many bacterial enzymes and had 51% similarity with a beta-glucanosyltransferase from Candida albicans. B. japonicum carrying a Tn5 insertion in ndvC resulted in synthesis of altered cyclic beta-glucans composed almost entirely of beta-(1--> 3)-glycosyl linkages. The mutant strain was only slightly sensitive to hypoosmotic growth conditions compared with the ndvB mutant, but it was severely impaired in symbiotic interactions with soybean (Glycine max). Nodulation was delayed by 8 to 10 days, and many small nodule-like structures apparently devoid of viable bacteria were formed. This finding suggests that the structure of the beta-glucan molecule is important for a successful symbiotic interaction, and beta-glucans may have a specific function in addition to their role in hypoosmotic adaptation.

Bradyrhizobium japonicum cytochrome c550 is required for nitrate respiration but not for symbiotic nitrogen fixation.

Bradyrhizobium japonicum possesses three soluble c-type cytochromes, c550, c552, and c555. The genes for cytochromes c552 (cycB) and c555 (cycC) were characterized previously. Here we report the cloning, sequencing, and mutational analysis of the cytochrome c550 gene (cycA). A B. japonicum mutant with an insertion in cycA failed to synthesize a 12-kDa c-type cytochrome. This protein was detectable in the cycA mutant complemented with cloned cycA, which proves that it is the cycA gene product. The cycA mutant, a cycB-cycC double mutant, and a cycA-cycB-cycC triple mutant elicited N2-fixing root nodules on soybean (Nod+ Fix+ phenotype); hence, none of these three cytochromes c is essential for respiration supporting symbiotic N2 fixation. However, cytochrome c550, in contrast to cytochromes c552 and c555, was shown to be essential for anaerobic growth of B. japonicum, using nitrate as the terminal electron acceptor.

Nonpigmented and Bacteriochlorophyll-Containing Bradyrhizobia Isolated from Aeschynomene indica.

The legume genus Aeschynomene is unusual, since many species develop stem nodules and the bradyrhizobia isolated from these nodules produce bacteriochlorophyll (Bchl). Evidence is presented that the bradyrhizobia of Aeschynomene indica have wide distribution throughout the world, since A. indica was nodulated when grown in 58 soils collected in 14 different countries. Only 38 of 79 isolates tested synthesized Bchl and carotenoids during heterotrophic growth. Nine isolates produced Bchl constitutively, and cultures were pigmented after growth in the dark. The other isolates required light for Bchl production. The DNA from seven pigmented and three nonpigmented bradyrhizobia hybridized with a DNA probe containing the genes for the photosynthetic apparatus of Rhodobacter capsulatus, but DNA from two other nonpigmented isolates did not hybridize with this probe. A relationship between pigmentation in culture and symbiotic phenotype was not evident, since bradyrhizobia of both Bchl phenotypes nodulated stems of A. indica and formed nitrogen-fixing symbioses. Several isolates, which were ineffective on A. indica, probably do belong to the proposed cross-inoculation group 3 (D. Alazard, Appl. Environ. Microbiol. 50:732-734, 1985), since they did not nodulate Aeschynomene americana or Macroptilium atropurpureum. Since it has been suggested that extant rhizobia arose from photosynthetic ancestors (J. I. Sprent, p. 45-54, in P. M. Gresshoff, L. E. Roth, G. Stacey, and W. E. Newton, ed., Nitrogen Fixation: Achievements and Objectives, 1990), we propose that the nonpigmented isolates may represent an extant lineage of an intermediate evolutionary stage.

Identification and cloning of a cyclic beta-(1-->3), beta-(1-->6)-D-glucan synthesis locus from Bradyrhizobium japonicum.

A cosmid clone complementing a cyclic beta-(1-->2)-glucan biosynthesis (ndvB) mutant of Rhizobium meliloti was isolated from a Bradyrhizobium japonicum gene library. This clone specified synthesis of beta-(1-->3), beta-(1-->6)-linked glucans in R. meliloti. The complemented strain was osmotically tolerant and symbiotically competent on alfalfa. Thus, beta-(1-->3), beta-(1-->6)-glucans can substitute functionally for beta-(1-->2)-glucans in R. meliloti.

Cloning and Mutagenesis of a Cytochrome P-450 Locus from Bradyrhizobium japonicum That Is Expressed Anaerobically and Symbiotically.

Cytochromes P-450, which in many organisms participate in the metabolism of a variety of endobiotic and xenobiotic substances, are synthesized by symbiotic bacteroids of Bradyrhizobium japonicum. Polyclonal antibodies were raised against two cytochromes P-450 (CYP112 and CYP114) purified from bacteroids. A lambda gt11 expression clone of B. japonicum USDA 110 DNA that reacted with the anti-CYP112 antibody was obtained and was used to screen a library of USDA 110 genomic DNA in pLAFR1 for a clone of the P-450 locus. Forced expression of subclones of the P-450 locus in Escherichia coli produced polypeptides that reacted with either the anti-CYP112 antibody or the anti-CYP114 antibody; no cross-reactivity was evident. A Western blot (immunoblot) analysis showed that neither protein was present in free-living aerobically grown B. japonicum cells, but that both proteins were present in cells grown anaerobically, as well as in bacteroids. A mutant strain disrupted in the CYP112 locus produced neither CYP112 nor CYP114, indicating that the mutation was polar for CYP114. The mutant produced effective nodules on soybeans, even though the bacteroids contained no detectable P-450. This suggests that the cytochromes P-450 which we examined are not involved in an essential symbiotic function.

Isolation and characterization of an ndvB locus from Rhizobium fredii.

A gene (ndvB) in Rhizobium meliloti that is essential for nodule development in Medicago sativa (alfalfa), specifies synthesis of a large membrane protein. This protein appears to be an intermediate in beta-1,2-glucan synthesis by the microsymbiont. Southern hybridization analysis showed strong homology between an ndvB (chvB) probe and genomic DNA of R. fredii but not from Bradyrhizobium japonicum. A cosmid clone containing the putative ndvB locus was isolated from a Rhizobium fredii gene library. The cosmid clone which complemented R. meliloti ndvB mutants for synthesis of beta-1,2-glucans and effective nodulation of alfalfa was mapped and subcloned. Fragment-specific Tn5 mutagenesis followed by homologous recombination into the R. fredii genome indicated that the region was essential for beta-1,2-glucan synthesis and for formation of an effective symbiosis with Glycine max (soybean).

Isolation and Characterization of a Competition-Defective Bradyrhizobium japonicum Mutant.

Tn5 mutagenesis was coupled with a competition assay to isolate mutants of Bradyrhizobium japonicum defective in competitive nodulation. A double selection procedure was used, screening first for altered extracellular polysaccharide production (nonmucoid colony morphology) and then for decreased competitive ability. One mutant, which was examined in detail, was deficient in acidic polysaccharide and lipopolysaccharide production. The wild-type DNA region corresponding to the Tn5 insertion was isolated, mapped, and cloned. A 3.6-kb region, not identified previously as functioning in symbiosis, contained the gene(s) necessary for complementation of the mutation. The mutant was motile, grew normally on minimal medium, and formed nodules on soybean plants which fixed almost as much nitrogen as the wild type during symbiosis.

Characterization of cytochromes c550 and c555 from Bradyrhizobium japonicum: cloning, mutagenesis, and sequencing of the c555 gene (cycC).

The major soluble c-type cytochromes in cultured cells of Bradyrhizobium japonicum USDA 110 comprised a CO-reactive c555 (Mr, approximately 15,500) and a non-CO-reactive c550 (Mr, approximately 12,500). Levels of cytochrome per gram of soluble protein in aerobic, anaerobic, and symbiotic cells were 32, 21, and 30 nmol, respectively, for c555 and 31, 44, and 65 nmol, respectively, for c550. The midpoint redox potentials (Em,7) of the purified cytochromes were +236 mV for c555 and +277 mV for c550. The CO reactivity of c555 was pH dependent, with maximal reactivity at pH 10 or greater. Rabbit antiserum was produced against purified c555 and used to screen a B. japonicum USDA 110 genomic DNA expression library in lambda gt11 for a downstream portion of the c555 gene (cycC). This sequence was then used to probe a cosmid library for the entire c555 locus. The nucleotide sequence shows an open reading frame of 149 amino acids, with an apparent signal sequence at the N terminus and a heme-binding site near the C terminus. The deduced amino acid sequence is similar to those of the cytochromes c556 of Rhodopseudomonas palustris and Agrobacterium tumefaciens. The cycC gene was mutagenized by insertion of a kanamycin resistance cassette and homologously recombined into the B. japonicum genome. The resulting mutant made no c555 but made normal amounts of c550. The levels of membrane cytochromes were unaffected. The mutant and wild type exhibited identical phenotypes when used to nodulate plants of soybean (Glycine max L. Merr.), with no significant differences in nodule number, nodule mass, or total amount of N2 fixed.

Fractionation of the beta-Linked Glucans of Bradyrhizobium japonicum and Their Response to Osmotic Potential.

Bradyrhizobium japonicum USDA 110 synthesized both extracellular and periplasmic polysaccharides when grown on mannitol minimal medium. The extracellular polysaccharides were separated into a high-molecular-weight acidic capsular extracellular polysaccharide fraction (90% of total hexose) and three lower-molecular-weight glucan fractions by liquid chromatography. Periplasmic glucans, extracted from washed cells with 1% trichloroacetic acid, gave a similar pattern on liquid chromatography. Linkage analysis of the major periplasmic glucan fractions demonstrated mainly 6-linked glucose (63 to 68%), along with some 3,6- (8 to 18%), 3- (9 to 11%), and terminal (7 to 8%) linkages. The glucose residues were beta-linked as shown by H-nuclear magnetic resonance analysis. Glucan synthesis by B. japonicum cells grown on mannitol medium with 0 to 350 mM fructose as osmolyte was measured. Fructose at 150 mM or higher inhibited synthesis of periplasmic and extracellular 3- and 6-linked glucans but had no effect on the synthesis of capsular acidic extracellular polysaccharides.

Exopolysaccharide-Deficient Mutants of Rhizobium fredii HH303 Which Are Symbiotically Effective.

Nineteen Tn5-induced mutants of Rhizobium fredii HH303 defective in acidic exopolysaccharide synthesis were isolated by screening for lack of Calcofluor fluorescence. They were grouped by complementation analysis by using Rhizobium meliloti cosmids carrying exo genes. All of the 19 mutants were symbiotically effective or partially effective, indicating that the major bacterial acidic exopolysaccharide of this strain of R. fredii may not be required for symbiotic development in the soybean.

Synthesis of Exopolysaccharide by Bradyrhizobium japonicum during Growth on Hydroaromatic Substrates.

The hydroaromatic acids shikimate and quinate, which may be available as carbon sources in the soil, supported production of only low levels of acidic exopolysaccharide by Bradyrhizobium japonicum. Exopolysaccharide production (micrograms per 10 cells) was 4.9 on quinate and 4.5 on shikimate; in comparison, it was 128 on adipate, 18 on l-arabinose, and 39 on d-glucose.

Genetic Diversity in Bradyrhizobium japonicum Serogroup 123 and Its Relation to Genotype-Specific Nodulation of Soybean.

The genetic diversity among 20 field isolates of Bradyrhizobium japonicum serogroup 123 was examined by using restriction endonuclease digestions, one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis of total cell proteins, Southern hybridization analysis of nif and nod genes, and intrinsic antibiotic resistance profiles. All of the isolates were previously separated into three broad nodulation classes (low, medium, and high) based on their ability to form symbioses with specific soybean genotypes. Results of our studies indicate that there is a relationship between these three genotype-specific nodulation classes and groupings that have been made based on genomic DNA digestion patterns, sodium dodecyl sulfate-protein profiles, and Southern hybridizations to a nifHD gene probe. Intrinsic antibiotic resistance profiles and nodAB gene hybridizations were not useful in determining interrelationships between isolates and nodulation classes. Southern hybridizations revealed that two of the isolates had reiterated nod genes; however, there was no correlation between the presence of extra nodAB genes and the nodulation classes or symbiotic performance on permissive soybean genotypes. Hybridizations with the nif gene probe indicated that there is a relationship among serogroup, nodulation class, and the physical organization of the genome.

Decreased Exopolysaccharide Synthesis by Anaerobic and Symbiotic Cells of Bradyrhizobium japonicum.

Experiments were conducted to determine whether symbiotic bacteroids of Bradyrhizobium japonicum produce exopolysaccharide within soybean (Glycine max [L.] Merr. cv ;Lee 74') nodules. B. japonicum strains RT2, a derivative of USDA 110 with resistance to streptomycin and rifampicin, and RT176-1, a mutant deficient in exopolysaccharide synthesis, were used. Although aerobically cultured RT2 produced 1550 micrograms of exopolysaccharide per 10(10) cells, root nodules formed by RT2 contained only 55.7 micrograms of polysaccharide per 10(10) bacteroids, indicating that little exopolysaccharide synthesis occurred within the nodules. The polysaccharide level of RT2 nodules was about equal to that of nodules containing the exopolysaccharide mutant RT176-1 (61.0 micrograms per 10(10) bacteroids). Gas chromatographic analysis showed that the sugar composition of polysaccharide from nodules of RT2 or RT176-1 was almost the same as that of polysaccharide from unnodulated root tissue, but differed strikingly from that of rhizobial exopolysaccharide from aerobic cultures. Thus, the host plant and not the bacteroids was probably the source of most or all of the polysaccharide in the nodule extracts. Also, bacteroids from nodules failed to bind soybean lectin, confirming the absence of an exopolysaccharide capsule.To test the hypothesis that this reduced synthesis of exopolysaccharide by bacteroids is related to the low free O(2) concentration within nodules, strain RT2 was grown on l-arabinose/succinate/glutamate/nitrate medium both aerobically and anaerobically. Anaerobiosis caused a 92% reduction in total exopolysaccharide synthesis, with amounts averaging only 123 micrograms per 10(10) cells. Anaerobically cultured cells also failed to bind soybean lectin. These results suggest that the low free O(2) content of the nodules may be responsible for the reduced exopolysaccharide synthesis by the bacteroids.

Amino Acids Translocated from Turgid and Water-stressed Barley Leaves: I. Phloem Exudation Studies.

The phloem exudation technique of King and Zeevaart (Plant Physiol 1974 53: 96-103) was modified for use with barley plants, to investigate the effect of water stress upon amino acid translocation at seedling and grainfilled stages.Seedling leaves and flag leaves from unstressed and moderately water-stressed plants exuded (14)CO(2) assimilates, sugars, and amino acids when their sheaths were cut and immersed in a 5 millimolar solution of Na(2)EDTA (pH 7.0). By including PEG 6000 (-10 bars) in the Na(2)EDTA solution, leaves severed from moderately water-stressed plants could be maintained in a wilted state. Such leaves produced about as much exudate as turgid leaves of unstressed plants.The following observations suggest a phloem origin for most of the exudate. Exudation was markedly stimulated by light and by CO(2) enrichment. The release of NO(3) (-) declined after cutting, and did not parallel exudation of (14)CO(2) assimilates, sugar, and amino acids. The relative quantities and specific radioactivities of sugars and amino acids in the exudate differed from those of sugars and amino acids extracted from sheath tissue.Major amino acids in exudate from unstressed seedling and flag leaves were glutamine, glutamate, serine, alanine, and aspartate; proline was virtually absent. Exudate from water-stressed leaves contained relatively more serine, and also some proline and gamma-aminobutyric acid.

Amino Acids Translocated from Turgid and Water-stressed Barley Leaves : II. Studies with N and C.

Movement of labeled amino acids from leaf blades to sheaths was followed after supplying (13)NH(3) gas, (14)CO(2), or (14)C-amino-acids to attached blades of barley plants.Blades of turgid and wilted plants fed (13)NH(3) (at about 120 to 700 microliters per liter) had incorporated (13)N mainly into free glutamine and glutamate after 30 minutes, and turgid blades had exported 1 to 3% of the assimilated (13)N to the sheaths, mostly as glutamine and glutamate. Wilted blades exported less of the assimilated (13)N than turgid blades even though they exported (14)CO(2) assimilates as actively as turgid blades.When substrate amounts (about 0.28 micromole) of [(14)C]glutamate and [(14)C]proline were applied to turgid and wilted blades, these amino acids entered the phloem and were translocated at velocities similar to those for (14)CO(2) assimilates (about 0.2 centimeter per minute). Wilted blades metabolized tracer amounts of [(14)C]glutamate to glutamine and proline, and exported (14)C in the form of these three amino acids. Approximate calculations of mass transfer rates of glutamate, glutamine, and proline made for wilted blades indicated that glutamine and glutamate together carried 76 micrograms of N per day, whereas proline carried only about 9 micrograms of N per day.

Proline Accumulation in Water-stressed Barley Leaves in Relation to Translocation and the Nitrogen Budget.

Mobilization of N from leaves of barley (Hordeum vulgare L.) during water stress, and the role of proline as a mobilized species, were examined in plants at the three-leaf stage. The plants responded to water stress by withdrawing about 25% of the total reduced N from the leaf blades via phloem translocation. Most of this N loss was during the first 2 days while translocation of (14)C-photosynthate out of the stressed blade still remained active. Free proline accumulation in the blade was initially slow, and became more rapid during the 2nd day of stress. Although a major free amino acid, proline accounted for only about 5% of the total N (soluble + insoluble) retained in severely stressed blades. When the translocation pathway in water-stressed leaves was interrupted just below the blade by a heat girdle, a cold jacket, or by blade excision, N loss from the blade was prevented and proline began to accumulate rapidly on 1st day of stress. Little free proline accumulated in the blades until after the ability to translocate was lost. Proline was, however, probably not a major species of N translocated during stress, because proline N accumulation in heat-girdled stressed leaves was five times slower than the rate of total N export from intact blades.

Light stimulation of proline synthesis in water-stressed barley leaves.

The effect of light on [(14)C]glutamate conversion to free proline during water stress was studied in attached barley (Hordeum vulgare L.) leaves which had been trimmed to 10 cm in length. Plants at the three-leaf stage were stressed by flooding the rooting medium with polyethylene glycol 6000 (osmotic potential-19 bars) for up to 3 d. During this time the free proline content of 10-cm second leaves rose from about 0.02 to 2 µmol/leaf while free glutamate content remained steady at about 0.6 µmol/leaf. In stressed leaves, the amount of [(14)C]glutamate converted to proline in a 3-h period of light or darkness was taken to reflect the in-vivo rate of proline biosynthesis because the following conditions were met: (a) free-glutamate levels were not significantly different in light and darkness; (b) both tracer [(14)C]-glutamate and [(14)C]proline were rapidly absorbed; (c) rates of [(14)C]proline oxidation and incorporation into protein were very slow. As leaf water potential fell, more [(14)C]glutamate was converted to proline in both light and darkness, but at any given water potential in the range-12 to-20 bars, illuminated leaves converted twice as much [(14)C]glutamate to proline.

Proteases and Peptidases of Castor Bean Endosperm: Enzyme Characterization and Changes during Germination.

The endosperm of castor bean seeds (Ricinus communis L.) contains two -SH-dependent aminopeptidases, one hydrolyzing l-leucine-beta-naphthylamide optimally at pH 7.0, and the other hydrolyzing l-proline-beta-naphthylamide optimally at pH 7.5. After germination the endosperm contains in addition an -SH-dependent hemoglobin protease, a serine-dependent carboxypeptidase, and at least two -SH-dependent enzymes hydrolyzing the model substrate alpha-N-benzoyl-dl-arginine-beta-naphthylamide (BANA). The carboxypeptidase is active on a variety of N-carbobenzoxy dipeptides, especially N-carbobenzoxy-L-phenylalanine-l-alanine and N-carbobenzoxy-l-tyrosine-l-leucine. The pH optima for the protease, carboxypeptidase, and BANAase acivities are 3.5 to 4.0, 5.0 to 5.5, and 6 to 8, respectively.The two aminopeptidases increased about 4-fold in activity during the first 4 days of growth, concurrent with the period of rapid depletion of storage protein. Activities then declined as the endosperm senesced, but were still evident after 6 days. Senescence was complete by day 7 to 8. Hemoglobin protease, carboxypeptidase, and BANAase activities appeared in the endosperm at day 2 to 3, and reached peak activity at day 5 to 6.The data indicate that the aminopeptidases are involved in the early mobilization of endosperm storage protein, whereas protease, carboxypeptidase, and BANAase may take part in later turnover and/or senescence.