Isolation and characterization of the lipopolysaccharides from Bradyrhizobium japonicum.

The lipopolysaccharide (LPS) of Bradyrhizobium japonicum 61A123 was isolated and partially characterized. Phenol-water extraction of strain 61A123 yielded LPS exclusively in the phenol phase. The water phase contained low-molecular-weight glucans and extracellular or capsular polysaccharides. The LPSs from B. japonicum 61A76, 61A135, and 61A101C were also extracted exclusively into the phenol phase. The LPSs from strain USDA 110 and its Nod- mutant HS123 were found in both the phenol and water phases. The LPS from strain 61A123 was further characterized by polyacrylamide gel electrophoresis, composition analysis, and 1H and 13C nuclear magnetic resonance spectroscopy. Analysis of the LPS by polyacrylamide gel electrophoresis showed that it was present in both high- and low-molecular-weight forms (LPS I and LPS II, respectively). Composition analysis was also performed on the isolated lipid A and polysaccharide portions of the LPS, which were purified by mild acid hydrolysis and gel filtration chromatography. The major components of the polysaccharide portion were fucose, fucosamine, glucose, and mannose. The intact LPS had small amounts of 2-keto-3-deoxyoctulosonic acid. Other minor components were quinovosamine, glucosamine, 4-O-methylmannose, heptose, and 2,3-diamino-2,3-dideoxyhexose. The lipid A portion of the LPS contained 2,3-diamino-2,3-dideoxyhexose as the only sugar component. The major fatty acids were beta-hydroxymyristic, lauric, and oleic acids. A long-chain fatty acid, 27-hydroxyoctacosanoic acid, was also present in this lipid A. Separation and analysis of LPS I and LPS II indicated that glucose, mannose, 4-O-methylmannose, and small amounts of 2,2-diamino-2,3-dideozyhexose and heptose were components of the core region of the LPS, whereas fucose, fucosmine, mannose, and small amounts of quinovosamine and glucosamine were components of the LPS O-chain region.     

Isolation and characterization of the DNA region encoding nodulation functions in Bradyrhizobium japonicum.

The DNA region encoding early nodulation functions of Bradyrhizobium japonicum 3I1b110 (I110) was isolated by its homology to the functionally similar region from Rhizobium meliloti. Isolation of a number of overlapping recombinant clones from this region allowed the construction of a restriction map of the region. The identified nodulation region of B. japonicum shows homology exclusively to those regions of R. meliloti and Rhizobium leguminosarum DNA known to encode early nodulation functions. The region of homology with these two fast-growing Rhizobium species was narrowed to an 11.7-kilobase segment. A nodulation-defective mutant of Rhizobium fredii USDA 201, strain A05B-2, was isolated and found to be defective in the ability to curl soybean root hairs. Some of the isolated recombinant DNA clones of B. japonicum were found to restore wild-type nodulation function to this mutant. Analysis of the complementation results allows the identification of a 1.8-kilobase region as essential for restoration of Hac function.     

Carbohydrate binding activities of Bradyrhizobium japonicum. II. Isolation and characterization of a galactose-specific lectin

Extracts of Bradyrhizobium japonicum were fractionated on Sepharose columns covalently derivatized with lactose. Elution of the material that was specifically bound to the affinity column with lactose yielded a protein of Mr approximately 38,000. Isoelectric focusing of this sample yielded two spots with pI values of 6.4 and 6.8. This protein specifically bound to galactose-containing glycoconjugates, but did not bind either to glucose or mannose. Derivatives of galactose at the C-2 position showed much weaker binding; there was an 18-fold difference in the relative binding affinities of galactose versus N-acetyl-D-galactosamine. These results indicate that we have purified a newly identified carbohydrate-binding protein from Bradyrhizobium japonicum, that can exquisitely distinguish galactose from its derivatives at the C-2 position.     

Bradyrhizobium japonicum mutants defective in nitrogen fixation and molybdenum metabolism.

Bradyrhizobium japonicum JH mutants deficient in molybdenum metabolism into the enzymes nitrogenase and nitrate reductase were isolated by using the vector pSUP1011, which carries transposon Tn5 (streptomycin and kanamycin resistance). Mutants in Mo metabolism were obtained at a frequency of 3.6 X 10(-3) (per Kan Strr colony). The mutants were detected by their poor ability to grow in nitrate-containing medium without added Mo. One of the mutant types required 10(5) times more molybdate than the wild type to obtain maximal nitrogen fixation activity. Double-reciprocal plots of Mo uptake versus concentration indicated that the wild-type strain had a high- and a lower-affinity component for Mo binding. Mutant strains JH-90 and JH-119 lacked the high-affinity Mo uptake component and were also clearly deficient in Mo accumulation into a nonexchangeable form. Nitrogenase activity as well as Mo uptake ability could be restored in strains JH-90 and JH-119 by the addition of the sterile supernatant fraction of the wild type. Therefore, mutant strains JH-90 and JH-119 appeared to be deficient in an extracellular Mo-binding factor produced by the wild type. Mutant strains JH-14 and JH-143 had Mo uptake kinetics like those of the wild type (both high- and low-affinity binding for Mo) and appeared to be deficient in intracellular Mo metabolism processes. The addition of the wild-type supernatant did not restore Mo uptake or nitrogenase activity in these strains.     

Isolation and characterization of low-indole-3-acetic acid-producing mutants from Bradyrhizobium elkanii

We isolated 11 low-indole-3-acetic acid (IAA)-producing mutants of Bradyrhizobium elkanii by Tn5 mutagenesis. The amount of IAA produced by each mutant was 2.2-13.6% of that of the wild-type. It was found by resting cell reactions that the biosynthetic step to convert indole-3-pyruvic acid to indole-3-acetaldehyde was blocked in all the mutants.     

Symbiotic properties of 5-methyltryptophan-resistant mutants of Bradyrhizobium japonicum

The effect that resistance to 5-methyltryptophan (MT) has on the symbiotic properties of B. japonicum was examined in a survey of fourteen clones. Resistance to MT often involves a mutational alteration in the regulation of tryptophan biosynthesis.Resistant clones (MT^R) were isolated from agar plates containing MT. In the selection process care was taken to avoid pigmented clones that are likely to accumulate large amounts of indole compounds or show increased tryptophan catabolism. Wild-type control clones (WT_c) were isolated from plates containing no selective agent. In greenhouse studies. Tracy-M soybean plants were inoculated with the two types of clones. After six weeks, plants which were inoculated with the MT resistant clones showed a much greater range of symbiotic effectiveness than did plants that received the control clones.While most MT-resistant clones were poor symbionts or unchanged in their symbiotic performance, one clone was obtained that had significantly improved symbiotic properties. The procedure may offer a way of selecting for clones with improved symbiotic performance. These results also indicate a link between tryptophan biosynthesis and symbiotic effectiveness.     

Isolation and characterization of the major nod factor of Bradyrhizobium japonicum strain 532C

Bradyrhizobium japonicum 532C nodulates soybean effectively under cool Canadian spring conditions and is used in Canadian commercial inoculants. The major lipo-chitooligosaccharide (LCO), bacteria-to-plant signal was characterized by HPLC, FAB-mass spectroscopy MALDI-TOF mass spectroscopy and revealed to be LCO Nod Bj-V (C18:1, MeFuc). This LCO is produced by type I strains of B. japonicum and is therefore unlikely to account for this strains superior ability to nodulate soybean under Canadian conditions. We also found that use of yeast extract mannitol medium gave similar results to that of Bergerson minimal medium.     

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.     

Peribacteroid membrane nodulin gene induction by Bradyrhizobium japonicum mutants

Seventeen translation products from Glycine max root mRNA precipitated with antiserum prepared against a peribacteroid membrane preparation from effective root nodules. Messenger RNA from fix ⁺ nodules coded for these 17 products plus 7 other nodule-specific polypeptides which bound to the antiserum. Of these 7 nodulins only 4 were present when nodules were infected with Bradyrhizobium japonicum 110 rif 15 2960, which induces the plant to produce empty peribacteroid membranes. In nodules infected with B. japonicum strains inducing either very short-lived or defective peribacteroid membrane, only 5 or 6, respectively, of these nodulins could be detected.From these results we hypothesize that the microsymbiont is responsible for the production of at least 4 different signals leading to peribacteriod membrane formation by the plant.     

Bradyrhizobium japonicum mutants defective in root-nodule bacteroid development and nitrogen fixation

The genome of the slow-growing Bradyrhizobium japonicum (strain 110) was mutagenized with transposon Tn5. A total of 1623 kanamycin/streptomycin resistant derivatives were screened in soybean infection tests for nodulation (Nod) and symbiotic nitrogen fixation (Fix). In this report we describe 14 strains possessing a stable, reproducible Nod⁺Fix^- phenotype. These strains were also grown under microaerobic culture conditions to test them for free-living nitrogen fixation activity (Nif). In addition to strains having reduced Fix and Nif activities, there were also strains that had reduced symbiotic Fix activity but were Nif⁺ ex planta.Analysis of the genomic structure revealed that the majority of the strains had a single Tn5 insertion without any further apparent physical alteration. A few strains had additional insertions (by Tn5 or IS50), or a deletion, or had cointegrated part of the vector used for Tn5 mutagenesis. One of the insertions was found in a known nif gene (nifD) whereas all other mutations seem to affect different, hitherto unknown genes or operons.Several mutant strains had an altered nodulation phenotype, inducing numerous, small, widely distributed nodules. Light and electron microscopy revealed that most of these mutants were defective in different stages of bacteroid development and/or bacteroid persistence. The protein patterns of the mutants were inspected by two-dimensional gel electrophoresis after labelling microaerobic cultures with l-(³⁵S)methionine. Of particular interest were mutants lacking a group of proteins the synthesis of which was known to be under oxygen control. Such strains can be regarded as potential regulatory mutants.     

Properties of streptomycin dependent non-nodulating mutants of cowpea rhizobia and Bradyrhizobium japonicum

We have isolated and characterized mutants from cowpea rhizobia strains JRW3 and IRC256 and Bradyrhizobium japonicum USDA110, which show dependence on streptomycin (Sm) for growth. In the presence of Sm, the majority of the Sm^D (streptomycin dependent) mutants showed cross-resistance to other aminoglycoside antibiotics and some showed no growth at 37°C and 40°C. When nodulation abilities of Sm^D mutants (derived from all three strains) were examined, most of them (> 91%) showed non-nodulating phenotypes to their respective hosts. Preliminary biochemical and genetic characterization indicated that drug-uptake function was altered in Sm^D mutant, and the wild type strain JRW3 could be transformed to streptomycin dependent by Sm^D DNA.     

Nickel uptake in Bradyrhizobium japonicum.

Free-living Bradyrhizobium japonicum grown heterotrophically with 1 microM 63Ni2+ accumulated label. Strain SR470, a Hupc mutant, accumulated almost 10-fold more 63Ni2+ on a per-cell basis than did strain SR, the wild type. Nongrowing cells were also able to accumulate nickel over a 2-h period, with the Hupc mutant strain SR470 again accumulating significantly more 63Ni2+ than strain SR. These results suggest that this mutant is constitutive for nickel uptake as well as for hydrogenase expression. The apparent Kms for nickel uptake in strain SR and strain SR470 were found to be similar, approximately 26 and 50 microM, respectively. The Vmax values, however, were significantly different, 0.29 nmol of Ni/min per 10(8) cells for SR and 1.40 nmol of Ni/min per 10(8) cells for SR470. The uptake process was relatively specific for nickel; only Cu2+ and Zn2+ (10 microM) were found to appreciably inhibit the uptake of 1 microM Ni, while a 10-fold excess of Mg2+, Co2+, Fe3+, or Mn2+ did not affect Ni2+ uptake. The lack of inhibition by Mg2+ indicates that nickel is not transported by a magnesium uptake system. Nickel uptake was also inhibited by cold (53% inhibition at 4 degrees C) and slightly by the ionophores nigericin and carbonyl cyanide m-chlorophenylhydrazone. Other ionophores did not appreciably affect nickel uptake, even though they significantly stimulated O2 uptake. The cytochrome c oxidase inhibitors azide, cyanide, and hydroxylamine did not inhibit Ni2+ uptake, even at concentrations (of cyanide and hydroxylamine) that inhibited O2 uptake. The addition of oxidizable substrates such as succinate or gluconate did not increase nickel uptake, even though they increased respiratory activity. Nickel update showed a pH dependence with an optimum at 6.0. Most (approximately 85%) of the 63Ni2+ taken up in 1 min by strain SR470 was not exchangeable with cold nickel.     

DNA sequence and mutational analysis of rhizobitoxine biosynthesis genes in Bradyrhizobium elkanii.

We cloned and sequenced a cluster of genes involved in the biosynthesis of rhizobitoxine, a nodulation enhancer produced by Bradyrhizobium elkanii. The nucleotide sequence of the cloned 28.4-kb DNA region encompassing rtxA showed that several open reading frames (ORFs) were located downstream of rtxA. A large-deletion mutant of B. elkanii, USDA94 Delta rtx::Omega 1, which lacks rtxA, ORF1 (rtxC), ORF2, and ORF3, did not produce rhizobitoxine, dihydrorhizobitoxine, or serinol. The broad-host-range cosmid pLAFR1, which contains rtxA and these ORFs, complemented rhizobitoxine production in USDA94 Delta rtx::Omega 1. Further complementation experiments involving cosmid derivatives obtained by random mutagenesis with a kanamycin cassette revealed that at least rtxA and rtxC are necessary for rhizobitoxine production. Insertional mutagenesis of the N-terminal and C-terminal regions of rtxA indicated that rtxA is responsible for two crucial steps, serinol formation and dihydrorhizobitoxine biosynthesis. An insertional mutant of rtxC produced serinol and dihydrorhizobitoxine but no rhizobitoxine. Moreover, the rtxC product was highly homologous to the fatty acid desaturase of Pseudomonas syringae and included the copper-binding signature and eight histidine residues conserved in membrane-bound desaturase. This result suggested that rtxC encodes dihydrorhizobitoxine desaturase for the final step of rhizobitoxine production. In light of results from DNA sequence comparison, gene disruption experiments, and dihydrorhizobitoxine production from various substrates, we discuss the biosynthetic pathway of rhizobitoxine and its evolutionary significance in bradyrhizobia.     

Isolation of a factor stimulatory to Bradyrhizobium japonicum in broth culture

Previous experiments indicated that water extracts of lambsquarters (Chenopodium album) and green foxtail (Setaria viridis), among others, stimulated growth of Bradyrhizobium japonicum in broth culture. Water extracts of lambsquarters shoots collected before or after anthesis were equally stimulatory. The stimulatory effect of extracts of lambsquarters when heated to 100°C for 30 min or autoclaved for 15 min was reduced by about 20% compared to untreated extracts. Extracts of green foxtail were less affected by higher temperature under similar conditions. Extracts of both green foxtail and lambsquarters completely lost their stimulatory effect following exposure to aerial microflora for 120 h. Water extract of lambsquarters shoots was more stimulatory than methanol extract, and neither ether nor butanol extracts resulted in stimulation. Both shoots and roots of lambsquarters and green foxtail were sequentially extracted first by water followed by methanol and vice-versa. The bioassay of these extracts indicated that there could be two components of the growth factor-one, larger component is soluble in water, the other, smaller component is soluble in methanol. After fractionation of the crude aqueous extract of lambsquarters shoots by four organic solvents, the residual aqueous extract retained the growth factor. Dialysis of the residual aqueous extract of lambsquarters shoots through a membrane (MWCO 1000) indicated that the molecular weight of the growth factor was less than 1000. The fraction having molecular weight <1000 was separated by paper chromatography using 6% acetic acid as developer. The fraction with Rf 0.91 showed the highest stimulation of the bacterium.     

Isolation and characterization of unusual gin mutants.

Site-specific inversion of the G segment in phage Mu DNA is promoted by two proteins, the DNA invertase Gin and the host factor FIS. Recombination occurs if the recombination sites (IR) are arranged as inverted repeats and a recombinational enhancer sequence is present in cis. Intermolecular reactions as well as deletions between direct repeats of the IRs rarely occur. Making use of a fis- mutant of Escherichia coli we have devised a scheme to isolate gin mutants that have a FIS independent phenotype. This mutant phenotype is caused by single amino acid changes at five different positions of gin. The mutant proteins display a whole set of new properties in vivo: they promote inversions, deletions and intermolecular recombination in an enhancer- and FIS-independent manner. The mutants differ in recombination activity. The most active mutant protein was analysed in vitro. The loss of site orientation specificity was accompanied with the ability to recombine even linear substrates. We discuss these results in connection with the role of the enhancer and FIS protein in the wild-type situation.     

Rapid and efficient selection of recombinant site-directed mutants of Bradyrhizobium japonicum by colony hybridization

Abstract Due to the high incidence of spontaneous antibiotic resistance and slow growth of Bradyrhizobium japonicum strains, screening for site-directed mutants is cumbersome and time-consuming. A rapid method for selection of recombinant site-directed mutants of B. japonicum was developed. A kanamycin (Km) and a spectinomycin (Sp) cassette were each used to replace DNA fragments in the chromosome by homologous recombination. The primary new features of this method involve a simple plate selection for the antibiotic (Km or Sp) resistant mutants, then colony streaking, and lysis for DNA hybridization on a nitrocellulose filter enabling direct identification of the recombinant site-directed mutants. This method has permitted us to quickly and easily identify a large number of positive recombinant mutants from a large number of indicidual colonies. The procedure eliminates the need to first isolate genomic DNA from each mutant for Southern hybridization. All of the tested site-directed mutants from this method were confirmed to exhibit the expected mutant phenotype.     

Nickel accumulation and storage in Bradyrhizobium japonicum.

Hydrogenase-derepressed (chemolithotrophic growth conditions) and heterotrophically grown cultures of Bradyrhizobium japonicum accumulated nickel about equally over a 3-h period. Both types of cultures accumulated nickel primarily in a form that was not exchangeable with NiCl2, and they accumulated much more Ni than would be needed for the Ni-containing hydrogenase. The nickel accumulated by heterotrophically incubated cultures could later be mobilized to allow active hydrogenase synthesis during derepression in the absence of nickel, while cells both grown and derepressed without nickel had low hydrogenase activities. The level of activity in cells grown with Ni and then derepressed without nickel was about the same as that in cultures derepressed in the presence of nickel. The Ni accumulated by heterotrophically grown cultures was associated principally with soluble proteins rather than particulate material, and this Ni was not lost upon dialyzing an extract containing the soluble proteins against either Ni-containing or EDTA-containing buffer. However, this Ni was lost upon pronase or low pH treatments. The soluble Ni-binding proteins were partially purified by gel filtration and DEAE chromatography. They were not antigenically related to hydrogenase peptides. Much of the 63Ni eluted as a single peak of 48 kilodaltons. Experiments involving immunoprecipitation of 63Ni-containing hydrogenase suggested that the stored source of Ni in heterotrophic cultures that could later be mobilized into hydrogenase resided in the nonexchangeable Ni-containing fraction rather than in loosely bound or ionic forms.     

Molybdate transport by Bradyrhizobium japonicum bacteroids.

Bacteroid suspensions of Bradyrhizobium japonicum USDA 136 isolated from soybeans grown in Mo-deficient conditions were able to transport molybdate at a nearly constant rate for up to 1 min. The apparent Km for molybdate was 0.1 microM, and the Vmax was about 5 pmol/min per mg (dry weight) of bacteroid. Supplementation of bacteroid suspensions with oxidizable carbon sources did not markedly increase molybdate uptake rates. Anaerobically isolated bacteroids accumulated twice as much Mo in 1 h as aerobically isolated cells did, but the first 5 min of molybdate uptake was not dependent on the isolation condition with respect to O2. Respiratory inhibitors such as cyanide, azide, and hydroxylamine did not appreciably affect molybdate uptake, even at concentrations that inhibited O2 uptake. The uncouplers carbonyl cyanide m-chlorophenylhydrazone (CCCP) and carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP) and the ionophores nigericin and monensin significantly inhibited molybdate uptake. The electrogenic ionophores valinomycin and gramicidin stimulated molybdate uptake. Rapid pH shift experiments indicated that molybdate transport depends on a transmembrane proton gradient (delta pH), and it is probably transported electroneutrally as H2MoO4. Most of the 99MoO4(2-) taken up was not exchangeable with a 100-fold excess of unlabeled MoO4(2-). Tungstate was a competitive inhibitor of molybdate uptake, with a Ki of 0.034 microM, and vanadate inhibited molybdate uptake slightly.