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mutational analysis of the bradyrhizobium japonicum common nod genes and further nod box-linked genomic dna regions.by insertional and deletional marker replacement mutagenesis the common nod region of bradyrhizobium japonicum was examined for the presence of additional, essential nodulation genes. an open reading frame located in the 800 bp large intergenic region between nodd1 and noda did not appear to be essential for nodulation of soybean. furthermore, a strain with a deletion of the nodi- and nodj-like genes downstream of nodc had a nod+ phenotype. a mutant with a 1.7 kb deletion immediately downstream ...19892710106
dual control of the bradyrhizobium japonicum symbiotic nitrogen fixation regulatory operon fixr nifa: analysis of cis- and trans-acting elements.aerobic expression of the fixr nifa operon in bradyrhizobium japonicum was shown to depend on a cis-acting, promoter-upstream dna sequence located between the -24/-12 promoter and position -86 relative to the transcription start site. an adenine at position -66 was essential for maximal expression. a chromosomal deletion of the upstream activator sequence (uas) led to a symbiotically defective phenotype which was typical of nifa mutants. b. japonicum crude extracts contained a protein that bound ...19892753853
cytoplasmic membrane systems involved in bacterium release into soybean nodule cells as studied with two bradyrhizobium japonicum mutant strains.two bradyrhizobium japonicum, tn5-induced, mutant strains, ml126 and ml150, were studied. both induce host cell division to form normal-sized nodules that do not fix nitrogen and whose cells have very few bacteroids (bar-). early-infection (15 days post infection) cells have much endoplasmic reticulum (er), numerous golgi bodies, and large vacuoles that are probably secondary lysosomes. later the cytoplasm of the host cells of both are dominated by hundreds of vesicles containing only finely fib ...19892759102
nucleotide sequence of a gene for indole-3-acetamide hydrolase from bradyrhizobium japonicum. 19892771653
critical spacing between two essential cysteine residues in the interdomain linker of the bradyrhizobium japonicum nifa protein.a special sequence motif in the bradyrhizobium japonicum nifa protein, consisting of two functionally essential cysteines separated by four other amino acids (cys-aa4-cys), has been proposed to be part of a potential metal-binding site [(1988) nucleic acids res. 16, 2207-2224]. using the techniques of oligonucleotide-directed mutagenesis, we report here that several of the four intervening amino acids can be replaced by others without loss of nifa function. the deletion of one amino acid to give ...19892792368
bradyrhizobium japonicum glnb, a putative nitrogen-regulatory gene, is regulated by ntrc at tandem promoters.the glnb gene from bradyrhizobium japonicum, the endosymbiont of soybeans (glycine max), was isolated and sequenced, and its expression was examined under various culture conditions and in soybean nodules. the b. japonicum glnb gene encodes a 12,237-dalton polypeptide that is highly homologous to the glnb gene products from klebsiella pneumoniae and escherichia coli. the gene is located directly upstream from glna (encoding glutamine synthetase), a linkage not observed in enteric bacteria. the g ...19892793830
rhizobium japonicum usda 191 has two nodd genes that differ in primary structure and function.several rhizobium genes (designated nod genes) are involved in early steps in nodule formation. here we present the results of dna sequence and functional analysis of two nodd genes from the symbiotic plasmid of usda 191, a fast-growing strain that forms nitrogen-fixing nodules on soybeans. both genes encoded full-length nodd-related polypeptides, which were 69% homologous to each other. one of these genes, nodd1, complemented a rhizobium trifolii nodd::tn5 mutant for clover nodulation; the othe ...19882826389
distinct structural features of the alpha and beta subunits of nitrogenase molybdenum-iron protein of clostridium pasteurianum: an analysis of amino acid sequences.nitrogenase is composed of two separately purified proteins, a molybdenum-iron (mofe) protein and an iron (fe) protein. structural genes (nifd and nifk) encoding alpha and beta subunits of the mofe protein of clostridium pasteurianum (cp) have been cloned and sequenced. the deduced amino acid sequences were analyzed for structures that could be related to the unique properties of the cp protein, particularly its low capacity to form an active enzyme with a heterologous fe protein. cp nifk is loc ...19882840948
characterization of the gene encoding glutamine synthetase i (glna) from bradyrhizobium japonicum.we have isolated the bradyrhizobium japonicum gene encoding glutamine synthetase i (glna) from a phage lambda library by using a fragment of the escherichia coli glna gene as a hybridization probe. the rhizobial glna gene has homology to the e. coli glna gene throughout the entire length of the gene and can complement an e. coli glna mutant when borne on an expression plasmid in the proper orientation to be transcribed from the e. coli lac promoter. high levels of glutamine synthetase activity c ...19852859270
expression of rhizobium japonicum nifh and nifdk operons can be activated by the klebsiella pneumonia nifa protein but not by the product of ntrc.rhizobium japonicum nifh'- and nifd'-'lacz fusions were constructed using the translational fusion vector pmc1403. beta-galactosidase activities from these fusion plasmids were measured in wild-type, ntra- and delta(ntrbc) escherichia coli strains carrying plasmids which overproduced the klebsiella pneumoniae nifa or ntrc gene products. in contrast to results reported in r. meliloti (ref. in the text) neither nifh nor nifd promoters were activated by the ntrc product. in the presence of nifa gen ...19852862569
involvement of glutamate in the respiratory metabolism of bradyrhizobium japonicum bacteroids.bradyrhizobium japonicum bacteroids were isolated anaerobically and supplied with 14c-labeled succinate, malate, aspartate, or glutamate for periods of up to 60 min in the presence of myoglobin to control the o2 concentration. succinate and malate were absorbed about twice as rapidly as glutamate and aspartate. conversion of substrate to co2 was most rapid for malate, followed by succinate, glutamate, and aspartate. when co2 production was expressed as a proportion of total carbon taken up, mala ...19872879829
acetoacetyl-coa thiolase of bradyrhizobium japonicum bacteroids: purification and properties.acetoacetyl-coa thiolase of bradyrhizobium japonicum bacteroids has been purified greater than 130-fold. the enzyme has a molecular weight of 180,000 +/- 15,000 and consists of four identical subunits of 44,000 +/- 2,000. the enzyme was specific for acetoacetyl-coa; ketodecanoyl-coa did not serve as a substrate. catalysis proceeds via a ping-pong mechanism. iodoacetamide effectively inhibited the enzyme but acetoacetyl-coa provided considerable protection against this compound. magnesium was fou ...19872883931
role of the bradyrhizobium japonicum ntrc gene product in differential regulation of the glutamine synthetase ii gene (glnii).we isolated the ntrc gene from bradyrhizobium japonicum, the endosymbiont of soybean (glycine max), and examined its role in regulating nitrogen assimilation. two independent ntrc mutants were constructed by gene replacement techniques. one mutant was unable to produce ntrc protein, while the other constitutively produced a stable, truncated ntrc protein. both ntrc mutants were unable to utilize potassium nitrate as a sole nitrogen source. in contrast to wild-type b. japonicum, the ntrc null mut ...19882903856
identification of the klebsiella pneumoniae glnb gene: nucleotide sequence of wild-type and mutant alleles.the glnb gene of klebsiella pneumoniae, which encodes the nitrogen regulation protein pii, has been cloned and sequenced. the gene encodes a 12429 dalton polypeptide and is highly homologous to the escherichia coli glnb gene. the sequences of a glnb mutation which causes glutamine auxotrophy and of a tn5 induced gln+ suppressor of this mutation were also determined. the glutamine auxotrophy was deduced to be the result of a modification of the uridylylation site of pii, and the suppression was s ...19882907369
protoporphyrinogen oxidation, a step in heme synthesis in soybean root nodules and free-living rhizobia.extracts of the crude bacteroid fraction of symbiotically grown bradyrhizobium japonicum were much more active in oxidizing protoporphyrinogen to protoporphyrin than were extracts of cells grown under free-living conditions, especially when assayed in atmospheres containing only traces of oxygen. this correlates with the higher heme content of the microaerophilic nodules. furthermore, the high level of oxidative activity in the crude bacteroid fraction was associated with an uncharacterized memb ...19892914857
repeated sequences similar to insertion elements clustered around the nif region of the rhizobium japonicum genome.two different repeated sequences (rss) were discovered in the rhizobium japonicum genome: rsrj alpha is 1126 base pairs long and is repeated 12 times; rsrj beta is approximately 950 base pairs long and is repeated at least 6 times. their arrangement in root nodule bacteroid dna is the same as in dna from bacteria grown in culture. deletion analysis showed that many copies of alpha and beta are clustered around the nitrogenase genes nifdk and nifh, or, in general, they are found within a genomic ...19852985537
slow-growing rhizobium japonicum comprises two highly divergent symbiotic types.we examined the interrelationships of the genomes of 10 slow-growing strains of rhizobium japonicum to provide a foundation for molecular genetic studies of these agriculturally important endosymbiotic bacteria of commercial soybeans. the degree of base substitution in and around known symbiotic genes (nif and presumptive nod), constitutively expressed genes (glna and reca), and two other cloned sequences was estimated from restriction site variation by using cloned dnas as hybridization probes ...19852989244
expression of symbiotic genes of rhizobium japonicum usda 191 in other rhizobia.a 200-megadalton plasmid was mobilized from rhizobium japonicum usda 191 to other rhizobium strains either that cannot nodulate soybeans or that form fix- nodules on certain cultivars. the symbiotic properties of the transconjugants indicate that both soybean specificity for nodulation and cultivar specificity for nitrogen fixation are plasmid encoded.19852989250
vectors for transposon mutagenesis of non-enteric bacteria.we have constructed a series of transposon delivery vectors derived from prk2013. since prk2013 has a broad host range transfer system and a cole1 replicon, it can be transferred to, but not replicated in, many non-enteric gram-negative bacteria. thus prk2013 provides an effective mechanism for the transient introduction of a transposon. delivery vectors containing tn7 (tmp str), tn10 (tet), tn10 hh104 (tet), or tn5-132 (tet) have been constructed. when transposition in caulobacter crescentus wa ...19852993823
inhibition of growth of rhizobium japonicum by cyclic gmp.exogenous cyclic guanosine-3',5'-monophosphate (cgmp) inhibited the growth of rhizobium japonicum at less than 100 microm. other nucleotides, including cyclic amp, cyclic imp, and cyclic cmp, had no inhibitory effect even at higher concentrations nor was the inhibition by cgmp reversed by cyclic amp. the inhibitory effect was independent of the carbon and nitrogen source(s) used. cgmp did not inhibit the growth of any other species of bacterium tested, including several fast-growing rhizobium sp ...19852997129
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. ...19852999080
proton motive force in washed cells of rhizobium japonicum and bacteroids from glycine max.the components of the proton motive force (delta p), namely the membrane potential and the transmembrane ph gradient, were measured in washed cells of rhizobium japonicum cc705 grown in cultures (5% o2-95% n2) in the presence of 10 mm kno3 and in bacteroids from glycine max. the delta p and its components remained reasonably constant in cells as well as in bacteroids at various stages of growth. the effects of uncouplers and atpase inhibitors on the delta p and its components were determined in ...19852999086
genetic locus in rhizobium japonicum (fredii) affecting soybean root nodule differentiation.a genetic locus in fast-growing rhizobium japonicum (fredii) usda 191 (fix+ on several contemporary soybean cultivars) was identified by random tn5 mutagenesis as affecting the development and differentiation of root nodules. this mutant (mu042) is prototrophic and shows no apparent alterations in its surface properties. it induces aberrant nodules, arrested at the same early level of differentiation, on all its host plants. an 8.1-kilobase ecori fragment containing tn5 was cloned from mu042. in ...19863009416
tn5-induced cytochrome mutants of bradyrhizobium japonicum: effects of the mutations on cells grown symbiotically and in culture.two bradyrhizobium japonicum cytochrome mutants were obtained by tn5 mutagenesis of strain lo and were characterized in free-living cultures and in symbiosis in soybean root nodules. one mutant strain, lo501, expressed no cytochrome aa3 in culture; it had wild-type levels of succinate oxidase activity but could not oxidize nadh or n,n,n',n'-tetramethyl-p-phenylenediamine (tmpd). the cytochrome content of lo501 root nodule bacteroids was nearly identical to that of the wild type, but the mutant e ...19873029019
inhibition of hydrogenase synthesis by dna gyrase inhibitors in bradyrhizobium japonicum.derepression of an uptake hydrogenase in bradyrhizobium japonicum is dependent on a microaerophilic environment. addition of dna gyrase inhibitors during depression of hydrogenase specifically prevented expression of the hydrogenase enzyme. antibodies to individual hydrogenase subunits failed to detect the protein after derepression in the presence of inhibitors, although there was no general inhibition of protein synthesis. the general pattern of proteins synthesized from 14c-labeled amino acid ...19873034865
a genetic locus essential for formate-dependent growth of bradyrhizobium japonicum.a genetic locus essential for the formate-dependent growth of bradyrhizobium japonicum was isolated by complementation of ethyl methanesulfonate-induced mutants with a cosmid gene library of b. japonicum dna. three related cosmids containing 18.7 kilobase pairs of b. japonicum dna in common were identified as being able to restore formate-dependent growth capability to mutants lacking either ribulosebisphosphate carboxylase or both ribulosebisphosphate carboxylase and phosphoribulokinase activit ...19873036781
transposon-induced symbiotic mutants of bradyrhizobium japonicum: isolation of two gene regions essential for nodulation.two strains of the soybean endosymbiont bradyrhizobium japonicum, usda 110 and 61 a101 c, were mutagenized with transposon tn5. after plant infection tests of a total of 6,926 kanamycin and streptomycin resistant transconjugants, 25 mutants were identified that are defective in nodule formation (nod-) or nitrogen fixation (fix-). seven nod- mutants were isolated from strain usda110 and from strain 61 a101 c, 4 nod- mutants and 14 fix- mutants were identified. subsequent auxotrophic tests on thes ...19873037278
conformational changes in the membrane-bound hydrogenase of bradyrhizobium japonicum. evidence that the redox state of the enzyme affects its accessibility to protease and membrane-impermeant reagents.the sensitivity of the membrane-bound hydrogenase of bradyrhizobium japonicum to inactivation by proteases and membrane-impermeant protein modification reagents was compared under hydrogen versus oxygen. in membrane vesicles, the half-life of enzyme inactivation by trypsin of the h2-reduced enzyme was approximately 10 min, whereas o2-oxidized enzyme was much less sensitive to trypsin inactivation (half-life of over 90 min). diazobenzene sulfonate (dabs) affected the enzyme activity in a manner s ...19883053719
nucleotide sequence of the genetic loci encoding subunits of bradyrhizobium japonicum uptake hydrogenase.an indispensable part of the hydrogen-recycling system in bradyrhizobium japonicum is the uptake hydrogenase, which is composed of 34.5- and 65.9-kda subunits. the gene encoding the large subunit is located on a 5.9-kilobase fragment of the h2-uptake-complementing cosmid phu52 [zuber, m., harker, a.r., sultana, m.a. & evans, h.j. (1986) proc. natl. acad. sci. usa 83, 7668-7672]. we have now determined that the structural genes for both subunits are present on this fragment. two open reading fram ...19883054886
selenium increases hydrogenase expression in autotrophically cultured bradyrhizobium japonicum and is a constituent of the purified enzyme.we have investigated the effect of added selenite on autotrophic growth and the time course of hydrogen oxidation derepression in bradyrhizobium japonicum 122des cultured in a medium purified to remove selenium compounds. in addition, hydrogenase was purified to near homogeneity and examined for the specific incorporation of se into the enzyme. the addition of se at 0.1 microm significantly increased total cell protein and hydrogenase specific activity of harvested cells. also, the addition of s ...19883056905
cloning and sequencing of the genes encoding the large and the small subunits of the h2 uptake hydrogenase (hup) of rhodobacter capsulatus.the structural genes (hup) of the h2 uptake hydrogenase of rhodobacter capsulatus were isolated from a cosmid gene library of r. capsulatus dna by hybridization of bradyrhizobium japonicum. the r. capsulatus genes were localized on a 3.5 kb hindiii fragment. the fragment, cloned onto plasmid pac76, restored hydrogenase activity and autotrophic growth of the r. capsulatus mutant jp91, deficient in hydrogenase activity (hup-). the nucleotide sequence, determined by the dideoxy chain termination me ...19883067084
kinetics of monoclonal antibody production in low serum growth medium.factors affecting growth and monoclonal antibody production in vitro by a mouse-mouse hybridoma cell line have been investigated in a series of studies. the goal was to maximize antibody yields and demonstrate that antibodies can be produced efficiently on a large-scale in fermentors. this initial report describes (i) development of a radial immunodiffusion assay for accurate determination of antibody levels in culture, (ii) a culture medium formulation that allowed for reduction in the amount o ...19863080528
activation of the bradyrhizobium japonicum nifh and nifdk operons is dependent on promoter-upstream dna sequences.previous analysis of b. japonicum nifh'- and nifd'-'lacz translational fusions showed that these promoters could be activated by the k. pneumoniae nifa plus the e. coli ntra gene products. to study the functions of the dna 5' to these promoters, plasmids carrying deletions in this region were constructed and analyzed in vivo in a heterologous system consisting of an e. coli (ntra+) background with a plasmid that constitutively expresses the k. pneumoniae nifa gene. activation of the b. japonicum ...19863086837
expression of uptake hydrogenase and hydrogen oxidation during heterotrophic growth of bradyrhizobium japonicum.strains i-110 ars, sr, usda 136, usda 137, and ak13 1c of bradyrhizobium japonicum induced hup activity when growing heterotrophically in medium with carbon substrate and nh4cl in the presence of 2% h2 and 2% o2. hup activity was induced during heterotrophic growth in the presence of carbon substrates, which were assimilated during the time of h2 oxidation. strains i-110 ars and sr grown heterotrophically or chemoautotrophically for 3 days had similar rates of h2 oxidation. similar rates of hup ...19873115959
analysis of lectin binding by bradyrhizobium japonicum strains grown on nitrocellulose filters using peroxidase-labeled lectin.a procedure was developed to assess the ability of wild-type and mutant strains of bradyrhizobium japonicum to bind soybean lectin. the lectin-binding ability of bacteria grown on nitrocellulose filters was determined using peroxidase-labeled soybean lectin. the assay produced clear differences between strains known to be unable to bind soybean lectin and those which can. the assay gave results identical to those of the fluorescein isothiocyanate-soybean lectin-binding assay of t. v. bhuvaneswar ...19873118739
characterization of the rhizobium leguminosarum genes nodlmn involved in efficient host-specific nodulation.three nodulation genes, nodl, nodm and nodn, were isolated from rhizobium leguminosarum and their dna sequences were determined. the three genes are in the same orientation as the previously described nodfe genes and the predicted molecular weights of their products are 20,105 (nodl), 65,795 (nodm) and 18,031 (nodn). analysis of gene regulation using operon fusions showed that nodl, nodm and nodn are induced in response to flavanone molecules and that this induction is nodd-dependent. in additio ...19883132583
regulation of nod gene expression in bradyrhizobium japonicum.the best inducers of nod::lacz translational fusions in bradyrhizobium japonicum are isoflavones, primarily genistein and daidzein. upstream of the nodabc genes in b. japonicum is a novel gene, nody, which is coregulated with nodabc. measurements of the activity of lacz fusions to the nodd gene of b. japonicum show that this gene is inducible by soybean seed extract and selected flavonoid chemicals. the induction of the nody abc and nodd operons appears to require a functional nodd gene, indicat ...19883146016
characterization of adenosine triphosphatase from rhizobium japonicum. 19853161817
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 ...19883192511
in vitro and in vivo antibacterial activities of me1207, a new oral cephalosporin.me1207 (pivaloyloxymethyl ester of me1206) is a new oral cephalosporin. me1206 is (6r,7r)-7-[(z)-2-(2-aminothiazol-4-yl)-2-(methoxyimino)- acetamido]-3-[(z)-2-(4-methylthiazol-5-yl)-ethyl]-cephem-4-carboxy lic acid. the susceptibilities of about 1,600 clinical isolates to me1206 were determined by the agar dilution method. me1206 showed a broad spectrum of activity against gram-positive and gram-negative bacteria. me1206 was more active than cefaclor, t-2525, and cefixime against staphylococcus ...19883264132
the symbiotic nitrogen fixation regulatory operon (fixrnifa) of bradyrhizobium japonicum is expressed aerobically and is subject to a novel, nifa-independent type of activation.the bradyrhizobium japonicum n2 fixation regulatory gene, nifa, was sequenced and its transcription start site determined. between the start of transcription and the nifa gene an open reading frame of 278 codons was found and named fixr. a deletion in fixr which allowed transcription into nifa resulted in a 50% reduced fix activity. the fixrnifa operon was expressed in soybean root nodules, in cultures grown anaerobically with nitrate as terminal electron acceptor, in microaerobic cultures, and ...19873313281
effect of ph on tritium exchange and hydrogen production and uptake in free-living cells and in bacteroids of bradyrhizobium japonicum.soybean nodule bacteroids and bradyrhizobium japonicum free-living cells induced for h2-uptake hydrogenase, actively catalyze the evolution of h2 in a reaction highly dependent on the ph. the optimal phs for the evolution and uptake reactions were 4.0 and 7.5-8.0, respectively. no differences were found between free-living cells and bacteroids with respect to hydrogen acceptor specificity, although absolute rates of h2 uptake were higher for free-living cells. both types of cells were able to ev ...19873322198
regulation of the fixa gene and fixbc operon in bradyrhizobium japonicum.the transcriptional start site of the bradyrhizobium japonicum fixbc operon was identified by nuclease s1 mapping. it was located approximately 700 base pairs upstream of fixb and was preceded by a promoter sequence that showed strong homology to the b. japonicum fixa promoter and thus to the general nif consensus promoter sequence. further transcript mapping experiments revealed that fixa and fixbc transcription in b. japonicum strictly depended on the presence of the regulatory gene nifa and o ...19883343218
lack of carbon substrate repression of uptake hydrogenase activity in bradyrhizobium japonicum sr.the expression of ex planta uptake hydrogenase (hup) activity in bradyrhizobium japonicum sr induced in the absence or presence of carbon substrates was compared. hup activity was influenced by ph, indicating that acidification of induction medium with low buffering capacity resulting from carbon substrate metabolism inhibited hup activity. cell suspensions in medium with adequate buffering capacity and carbon substrate were limited in o2; increasing o2 availability to cells during induction sti ...19883350794
identification of a new bradyrhizobium japonicum gene (frxa) encoding a ferredoxinlike protein.an open reading frame of 74 codons was identified downstream of the nifb gene of bradyrhizobium japonicum 110. the predicted amino acid sequence shared 63% similarity with the rhodopseudomonas palustris ferredoxin i sequence. we propose to name the gene frxa. the frxa gene was found to be cotranscribed with the nifb gene. an insertion mutation within frxa hardly affected nitrogen fixation activity.19883350797
essential and non-essential domains in the bradyrhizobium japonicum nifa protein: identification of indispensable cysteine residues potentially involved in redox reactivity and/or metal binding.the amino acid sequence of the bradyrhizobium japonicum nitrogen fixation regulatory protein nifa, as derived from the nucleotide sequence of the nifa gene, was aligned to the corresponding protein sequences from klebsiella pneumoniae, rhizobium meliloti and rhizobium leguminosarum biovar viciae. high conservation was found in the central domain and in the cooh-terminal, putative dna binding domain, whereas very little homology was present within the first 250 amino acids from the nh2-terminus. ...19883357773
quantitative assay for binding of bradyrhizobium japonicum to cultured soybean cells.incubation of bradyrhizobium japonicum with the cultured soybean cell line sb-1 resulted in the adhesion of the bacteria to the plant cells. an antiserum was raised against b. japonicum, and the 125i-labeled immunoglobulin fraction was used to quantitate the number of bacteria bound to the soybean cells. the measurement of 125i-labeled antibody binding correlated well with parallel assays by microscopic observation. using this quantitation, we have optimized the parameters of the assay in terms ...19883410819
dicarboxylic acid transport in bradyrhizobium japonicum: use of rhizobium meliloti dct gene(s) to enhance nitrogen fixation.a recombinant plasmid encoding rhizobium meliloti sequences involved in dicarboxylic acid transport (plasmid prk290:4:46) (e. bolton, b. higgisson, a. harrington, and f. o'gara, arch. microbiol. 144:142-146, 1986) was used to study the relationship between dicarboxylic acid transport and nitrogen fixation in bradyrhizobium japonicum. the expression of the dct sequences on plasmid prk290:4:46 in b. japonicum cj1 resulted in increased growth rates in media containing dicarboxylic acids as the sole ...19883422072
laboratory and clinical evaluation of isolation media for campylobacter jejuni.six selective isolation media were evaluated for their ability to support the growth of campylobacter jejuni. colony counts of 70 isolated strains of c. jejuni and recovery studies on these strains in simulated positive feces samples demonstrated that bolton and hutchinson' charcoal, cefoperazone, deoxycholate agar and karmali's charcoal-based selective medium produced the highest recovery rates with the greatest suppression of other fecal flora. c. jejuni colonies were more easily recognized on ...19873429621
organization and characterization of genes essential for symbiotic nitrogen fixation from bradyrhizobium japonicum i110.a total of 96 independent tn5 insertions within a 39-kilobase-pair (kbp) segment of chromosomal dna containing the three structural genes for nitrogenase (nifh, nifd, and nifk) from bradyhizobium japonicum i110 were obtained in escherichia coli and transferred to the wild-type strain by marker exchange. individual transconjugants containing a tn5 insertion were inoculated onto glycine max cv. wilkin (soybeans) and analyzed for their effect on symbiotic nitrogen fixation. in addition to the three ...19863462181
nitrogenase promoter-lacz fusion studies of essential nitrogen fixation genes in bradyrhizobium japonicum i110.dna fragments containing either the nifd or nifh promoter and 5' structural gene sequences from bradyrhizobium japonicum i110 were fused in frame to the lacz gene. stable integration of these nif promoter-lacz fusions by homologous double reciprocal crossover into a symbiotically nonessential region of the b. japonicum chromosome provided an easy assay for the effects of potential nif regulatory mutants. the level of beta-galactosidase activity expressed from these two nif promoter-lacz fusions ...19863462182
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 ...19873473063
variability in molybdenum uptake activity in bradyrhizobium japonicum strains.twenty naturally occurring strains of bradyrhizobium japonicum in 11 serogroups were screened for the ability to take up mo as bacteroids from soybean root nodules. the strains varied greatly in their ability to take up mo in a 1-min period. the best strain was usda 136, which had an mo uptake activity of almost 3.0 pmol/min per mg of bacteroid (dry weight). in contrast, the poorest strain, usda 62, had an mo uptake activity of 0.35 pmol of mo per min per mg of bacteroid. there were similarities ...19873473064
bacterial heme synthesis is required for expression of the leghemoglobin holoprotein but not the apoprotein in soybean root nodules.in bradyrhizobium japonicum/soybean symbiosis, the leghemoglobin (legume hemoglobin) apoprotein is a plant product, but the origin of the heme prosthetic group is not known. b. japonicum strain lo505 is a transposon tn5-induced cytochrome-deficient mutant; it excreted the oxidized heme precursor coproporphyrin iii into the growth medium. mutant strain lo505 was specifically deficient in protoporphyrinogen oxidase (protoporphyrinogen-ix:oxygen oxidoreductase, ec 1.3.3.4) activity, and thus it cou ...19873479799
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 ...19863511036
expression and regulation of the escherichia coli glutamate dehydrogenase gene (gdh) in rhizobium japonicum.the glutamate dehydrogenase (gdh) gene of escherichia coli was transferred into an ammonium assimilation deficient mutant (asm-) of rhizobium japonicum (cj9) using plasmid prp301, a broad host range derivative of rp4. exconjugants capable of growth on ammonia as sole n-source occurred at a frequency of 6.8 x 10(-6). assimilatory gdh (nadp+) activity was detected in the strain carrying the e. coli gdh gene and the pattern of ammonia assimilation via gdh was similar to that of the asm+ wild type s ...19863516109
aerobic purification of hydrogenase from rhizobium japonicum by affinity chromatography.we purified active hydrogenase from free-living rhizobium japonicum by affinity chromatography. the uptake hydrogenase of r. japonicum has been treated previously as an oxygen-sensitive protein. in this purification, however, reducing agents were not added nor was there any attempt to exclude oxygen. in fact, the addition of sodium dithionite to aerobically purified protein resulted in the rapid loss of activity. purified hydrogenase was more stable when stored under o2 than when stored under ar ...19863519580
reversible inactivation of the o2-labile hydrogenases from azotobacter vinelandii and rhizobium japonicum.hydrogenases catalyze the reversible activation of dihydrogen. the hydrogenases from the aerobic, n2-fixing microorganisms azotobacter vinelandii and rhizobium japonicum are nickel- and iron-containing dimers that belong to the group of o2-labile enzymes. exposure of these hydrogenases to o2 results in an irreversible inactivation; therefore, these enzymes are purified anaerobically in a fully active state. we describe in this paper an electron acceptor-requiring and ph-dependent, reversible ina ...19863525552
endogenous lectins from cultured soybean cells: isolation of a protein immunologically cross-reactive with seed soybean agglutinin and analysis of its role in binding of rhizobium japonicum.incubation of rhizobium japonicum with the cultured soybean cell line sb-1, originally derived from the roots of glycine max, resulted in specific adhesion of the bacteria to the plant cells. this binding interaction appears to be mediated via carbohydrate recognition, since galactose can inhibit the heterotypic adhesion but glucose cannot. affinity chromatography, on a sepharose column derivatized with n-caproyl-galactosamine, of the supernatant fraction of a sb-1 cell suspension after enzymati ...19863528167
cloning and expression of bradyrhizobium japonicum uptake hydrogenase structural genes in escherichia coli.to identify the structural genes for the components of bradyrhizobium japonicum uptake hydrogenase (mr 60,000 and 30,000), we have expressed these genes in escherichia coli and shown that the products cross-react with antibodies to the respective hydrogenase subunits. we constructed subclones of overlapping dna fragments from an uptake hydrogenase-complementing cosmid, phu52 [lambert, g. r., cantrell, m. a., hanus, f. j., russell, s. a., haddad, k. r. & evans, h. j. (1985) proc. natl. acad. sci. ...19863532119
nucleotide sequence of the gene encoding the nitrogenase iron protein of thiobacillus ferrooxidans.the dna sequence was determined for the cloned thiobacillus ferrooxidans nifh and part of the nifd genes. a putative t. ferrooxidans nifh promoter was identified whose sequences showed perfect consensus with those of the klebsiella pneumoniae nif promoter. two putative consensus upstream activator sequences were also identified. the amino acid sequence was deduced from the dna sequence. in a comparison of nifh dna sequences from t. ferrooxidans and eight other nitrogen-fixing microbes, a rhizobi ...19873539923
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 expressio ...19873558318
a locus encoding host range is linked to the common nodulation genes of bradyrhizobium japonicum.by using cloned rhizobium meliloti, rhizobium leguminosarum, and rhizobium sp. strain mpik3030 nodulation (nod) genes as hybridization probes, homologous regions were detected in the slow-growing soybean symbiont bradyrhizobium japonicum usda 110. these regions were found to cluster within a 25-kilobase (kb) region. specific nod probes from r. meliloti were used to identify noda-, nodb-, nodc-, and nodd-like sequences clustered on two adjacent hindiii restriction fragments of 3.9 and 5.6 kb. a 7 ...19873584066
structure of the bradyrhizobium japonicum gene hema encoding 5-aminolevulinic acid synthase.the nucleotide (nt) sequence of the hema gene, which encodes 5-aminolevulinic acid synthase (alas) from the bacterium bradyrhizobium japonicum, is presented. this sequence predicts a protein of 408 amino acids (aa) with an mr of 44,599. this predicted amino acid sequence is highly homologous to that of the chicken embryonic liver alas, exhibiting a 48.8% identical amino acid sequence over the entire length of the bacterial protein. a single mrna start point was demonstrated by s1 protection anal ...19873609750
mapping and nucleotide sequence of the nifs promoter of bradyrhizobium japonicum. 19873684606
effect of lectin on nodulation by wild-type bradyrhizobium japonicum and a nodulation-defective mutant.the nodulation characteristics of wild-type bradyrhizobium japonicum usda 110 and mutant strain hs111 were examined. mutant strain hs111 exhibits a delayed-nodulation phenotype, a result of its inability to initiate successful nodulation promptly following inoculation of the soybean root. previously, we showed that the defect in initiation of infection leading to subsequent nodulation which is found in hs111 can be phenotypically reversed by pretreatment with soybean root exudate or soybean seed ...19863707122
cell surface polysaccharides from bradyrhizobium japonicum and a nonnodulating mutant.the cell surface polysaccharides of wild-type bradyrhizobium japonicum usda 110 and a nonnodulating mutant, strain hs123, were analyzed. the capsular polysaccharide (cps) and exopolysaccharide (eps) of the wild type and the mutant strain do not differ in their sugar composition. cps and eps are composed of mannose, 4-o-methylgalactose/galactose, glucose, and galacturonic acid in a ratio of 1:1:2:1, respectively. h nuclear magnetic resonance spectra of the eps and cps of the wild type and mutant ...19873793715
nodulin-26, a peribacteroid membrane nodulin is expressed independently of the development of the peribacteroid compartment.the peribacteroid membrane (pbm) of root nodules is derived from the plant cell plasma membrane but contains in addition several nodule-specific host proteins (nodulins). antibodies raised against purified pbm of soybean were used to immunoprecipitate polysomes to isolate an rna fraction that served as a template for the synthesis of a cdna probe for screening a nodule-specific cdna library. clone p1b1 was found to encode a 26.5 kda polypeptide (nodulin-26) which is immunoprecipitable specifical ...19873822816
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 acti ...19853882669
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 ...19853900036
rhizobium japonicum hydrogenase: purification to homogeneity from soybean nodules, and molecular characterization.rhizobium japonicum hydrogenase was purified to homogeneity from soybean root nodules by four column chromatography steps after solubilization from membranes by treatment with a nonionic detergent. the specific activity was from 40 to 65 mumol h2 oxidized min-1 mg protein-1 and was increased 450-fold relative to that in bacteroids. the yield of activity was from 7 to 12%. the molecular weight of the native enzyme was 104,000 as determined by sucrose density gradient centrifugation. electrophores ...19853919648
nitric oxide-dependent proton translocation in various denitrifiers.respiration of no resulted in transient proton translocation in anaerobically grown cells of four physiologically diverse denitrifiers. paracoccus denitrificans, rhodopseudomonas sphaeroides subsp. denitrificans, "achromobacter cycloclastes," and rhizobium japonicum gave, respectively, h+/no ratios of 3.65, 4.96, 1.94, and 1.12. antimycin a completely inhibited no-dependent proton translocation in p. denitrificans and severely restricted translocation in the r. sphaeroides strain. proton uptake ...19853928599
production of monoclonal antibodies in culture.factors that affected the production of monoclonal antibodies by a mouse-mouse hybridoma cell line, propagated in vitro in stirred vessels, were investigated. the purpose of the research was to estimate the efficiency of this system for large scale production of monoclonal antibodies. the antibody produced by these hybridoma cells was an igg2a, specific for a surface antigen on rhizobium japonicum nr-7 cells. antibody content in the culture supernatant was determined by a radial-immunodiffusion ...19853930316
enzymes of the beta-ketoadipate pathway are inducible in rhizobium and agrobacterium spp. and constitutive in bradyrhizobium spp.protocatechuate is a universal growth substrate for members of the family rhizobiaceae, and these bacteria utilize the aromatic compound via the beta-ketoadipate pathway. this report describes transcriptional controls exercised by different subgroups of the rhizobiaceae over five enzymes that catalyze consecutive reactions in the pathway: protocatechuate oxygenase (ec 1.13.11.3), beta-carboxy-cis,cis-muconate lactonizing enzyme (ec 5.5.1.2), gamma-carboxymuconolactone decarboxylase (ec 4.1.1.44) ...19863941043
isolation and characterization of a soybean lectin having 4-o-methylglucuronic acid specificity.a new lectin from soybeans having specificity toward the extracellular 4-o-methyl-d-glucurono-l-rhamnans produced by certain strains of rhizobium japonicum has been purified and characterized. isolation was accomplished initially by isoelectric precipitation of contaminating globulins and subsequently by affinity chromatography on partially hydrolyzed glucuronorhamnan covalently coupled to amino-hexylagarose. residual globulins were removed by adsorption of the lectin on concanavalin a-agarose a ...19863964668
expression of cytochrome o in hydrogen uptake constitutive mutants of rhizobium japonicum.mutant strains of rhizobium japonicum constitutive for h2 uptake activity (hupc) contained significantly more membrane-bound b-type cytochrome than did the wild type when grown heterotrophically. the hupc strains contained approximately three times more dithionite- and nadh-reducible co-reactive b-type cytochrome than did the wild type; the absorption features of the co spectra were characteristic of cytochrome o. this component, designated cytochrome b', was not reduced by nadh in the presence ...19853968033
role of ubiquinone in hydrogen-dependent electron transport in rhizobium japonicum.direct evidence for the involvement of ubiquinone in h2 oxidation by rhizobium japonicum was demonstrated; h2 reduced ca. 80% of the extractable ubiquinone. the inhibitor 2-n-heptyl-4-hydroxyquinoline-n-oxide blocked electron transport at a site between ubiquinone and the cytochromes. the results showed that no cytochrome component mediates electron flow from hydrogen to ubiquinone.19853968040
molecular and immunological comparison of membrane-bound, h2-oxidizing hydrogenases of bradyrhizobium japonicum, alcaligenes eutrophus, alcaligenes latus, and azotobacter vinelandii.the membrane-bound hydrogenases of bradyrhizobium japonicum, alcaligenes eutrophus, alcaligenes latus, and azotobacter vinelandii were purified extensively and compared. sodium dodecyl sulfate-polyacrylamide gel electrophoresis of each hydrogenase revealed two prominent protein bands, one near 60 kilodaltons and the other near 30 kilodaltons. the migration distances during nondenaturing polyacrylamide gel electrophoresis were similar for all except a. vinelandii hydrogenase, which migrated furth ...19854008438
conservation of symbiotic nitrogen fixation gene sequences in rhizobium japonicum and bradyrhizobium japonicum.southern hybridization with nif (nitrogen fixation) and nod (nodulation) dna probes from rhizobium meliloti against intact plasmid dna of rhizobium japonicum and bradyrhizobium japonicum strains indicated that both nif and nod sequences are on plasmid dna in most r. japonicum strains. an exception is found with r. japonicum strain usda194 and all b. japonicum strains where nif and nod sequences are on the chromosome. in r. japonicum strains, with the exception of strain usda205, both nif and nod ...19854008441
chemotaxis to aromatic and hydroaromatic acids: comparison of bradyrhizobium japonicum and rhizobium trifolii.rhizobia are bacteria well known for their ability to fix nitrogen in symbiosis with leguminous plants. members of diverse rhizobial species grow at the expense of hydroaromatic and aromatic compounds commonly found in plant cells and plant litter. using a quantitative capillary assay to measure chemotaxis, we tested the ability of hydroaromatic acids, selected aromatic acids, and their metabolites to serve as chemoattractants for two distantly related rhizobial species, bradyrhizobium japonicum ...19854019407
[the influence of acidic conditions on polar exopolysaccharides which bind rhizobium japonicum to soybean root hairs].the influence of acidic conditions on rhizobial exopolysaccharides (eps) and their symbiotic association in host plants, were investigated. light micrographs of soybean root hairs were taken with a fahraeus' slide assembly after the inocubation of rhizobial bacteria; curled and swollen root hairs, and the typical shepherd's crook with an infection thread were observed. the colonizations of soybean root hairs by rhizobia were different under acidic and neutral conditions. for the rhizobium japoni ...19854042755
accumulation of alpha,alpha-trehalose by rhizobium bacteria and bacteroids.four strains of rhizobium japonicum (61a76 and usda 110, 123, and 138) were grown in eight different defined media. regardless of the carbon or nitrogen source supplied, alpha, alpha-trehalose was the major carbohydrate (among mono- and disaccharides) accumulated by all four strains. after 7 to 9 days of growth, trehalose generally accounted for 90 to 100% of the mono- and disaccharides detected. none of the four strains would grow with trehalose as a carbon source, but the utilization of endoge ...19854044531
heterologous hybridization of bacterial dna to the endoglucanases a and b structural genes cela and celb of clostridium thermocellum.dna from various cellulolytic and non-cellulolytic bacteria was found to hybridize to clostridium thermocellum ncib10682 dna fragments carrying the structural genes cela and celb which code for endoglucanases a and b. homology to cela was detected in agrobacterium rhizogenes, azospirillum brasilense, bacillus subtilis, cellulomonas sp., clostridium stercorarium, erwinia chrysanthemi, pseudomonas solanacearum and streptomyces griseus. homology to celb was detected only in b. subtilis, c. stercora ...19854083831
isolation and partial characterization of the extracellular polysaccharides and lipopolysaccharides from fast-growing rhizobium japonicum usda 205 and its nod- mutant, hc205, which lacks the symbiotic plasmid.the extracellular polysaccharides and lipopolysaccharides (lpss) from two fast-growing rhizobium japonicum strains, usda 205 and hc205, were isolated and partially characterized. strain hc205 is a nod- mutant of usda 205 which lacks the symbiotic plasmid. the extracellular polysaccharides from both strains are very similar in composition, having galactose, glucose, glucuronic acid, and acyl groups. the extracellular polysaccharides do not contain detectable levels of pyruvate. methylation analys ...19854091554
fluorescent-antibody approach to study of rhizobia in soil.application of fluorescent-antibody (fa) techniques to the study of rhizobia as free-living soil bacteria was explored. antiserum to a particular strain of rhizobium japonicum proved specific in both agglutination and fa tests. within the r. japonicum group, 2 of 12 strains were stained by the conjugate and these fluoresced brightly; all others were entirely negative. fa tests were negative for 7 strains of r. meliloti, 9 strains of r. leguminosarum, 9 strains of r. trifolii, 6 strains of r. pha ...19684174666
competition between inoculum strains of rhizobium japonicum in the process of soybean nodulation during three planting periods. 19734201820
relationship between soybean cultivars and rhizobium japonicum serotypes with single- and multi-strain inoculants. i. greenhouse pot experiments. 19734205244
the phospholipid composition of rhizobium japonicum. 19714323346
electron paramagnetic resonance and temperature dependent spin state studies of ferric cytochrome p-450 from rhizobium japonicum. 19724339738
anaerobic-nitrate, symbiotic and aerobic growth of rhizobium japonicum: effects on cytochrome p 450 , other haemoproteins, nitrate and nitrite reductases. 19724341774
l-arabinose metabolism in rhizobium japonicum.l-arabinose was metabolized through an oxidative pathway by extracts of a strain of rhizobium japonicum. the findings showed that l-arabinose is converted into 2-keto-3-deoxy-l-arabonate, which is cleaved into glycoaldehyde and pyruvate.19744407017
streptomycin resistance in rhizobium japonicum. 19744441216
transformation of effectiveness in rhizobium japonicum. 19744477931
transmissible resistance to penicillin g, neomycin, and chloramphenicol in rhizobium japonicum.the genetic basis for resistance to a number of antibiotics was examined in rhizobium japonicum. resistance to penicillin g, neomycin, and chloramphenicol appears to be mediated by an extrachromosomal element similar to that found in the enterobacteriaceae. resistance to these antibiotics was eliminated from cells by treatment with acridine orange, and resistance to all three antibiotics could be transferred en bloc to agrobacterium tumefaciens under conditions excluding transformation or transd ...19734491197
deoxyribonucleate binding and transformation in rhizobium jpaonicum.rhizobium japonicum, capable of binding high-molecular-weight donor (32)p-labeled deoxyribonucleic acid (dna) during late log phase in a competence medium, was transformed for streptomycin resistance with a frequency of transformation ranging between 0.02 and 0.08%. eight to 10% of the homologous native (32)p-labeled input dna was bound irreversibly in a temperature-dependent manner. homologous denatured (32)p-labeled dna was incapable of binding to the recipient under similar conditions. cscl d ...19724538250
compatibility of different fungicides with rhizobium japonicum. 19724564552
a simple medium for quick growth of rhizobium japonicum. 19734580100
analysis of the intracellular amino acid pool and proteins from whole cells of rhizobium japonicum. 19734583873
survival of rhizobium japonicum in various carriers. 19734801277
relationship between soybean cultivars and rhizobium japonicum serotypes with single- and multi-strain inoculants. ii. field experiments. 19734801803
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