Induction of H(2)O(2) synthesis by beta-glucan elicitors in soybean is independent of cytosolic calcium transients.

Soybean cell suspension cultures have been used to investigate the role of the elevation of the cytosolic Ca(2+) concentration in beta-glucan elicitors-induced defence responses, such as H(2)O(2) and phytoalexin production. The intracellular Ca(2+) concentration was monitored in transgenic cells expressing the Ca(2+)-sensing aequorin. Two lines of evidence showed that a transient increase of the cytosolic Ca(2+) concentration is not necessarily involved in the induction of H(2)O(2) generation: (i) a Bradyrhizobium japonicum cyclic beta-glucan induced the H(2)O(2) burst without increasing the cytosolic Ca(2+) concentration; (ii) two ion channel blockers (anthracene-9-carboxylate, A9C; 5-nitro-2-(3-phenylpropylamino)-benzoate, NPPB) could not prevent a Phytophthora soja beta-glucan elicitor-induced H(2)O(2) synthesis but did prevent a cytosolic Ca(2+) concentration increase. Moreover, A9C and NPPB inhibited P. sojae beta-glucan-elicited defence-related gene inductions as well as the inducible accumulation of phytoalexins, suggesting that the P. sojae beta-glucan-induced transient cytosolic Ca(2+) increase is not necessary for the elicitation of H(2)O(2) production but is very likely required for phytoalexin synthesis.

Bradyrhizobium japonicum mutants defective in cyclic beta-glucan synthesis show enhanced sensitivity to plant defense responses.

Susceptibility of the nitrogen-fixing soybean symbiont Bradyrhizobium japonicum to inducible plant defense metabolites such as phytoalexin and H2O2, was investigated. On the wild-type strain USDA 110 the soybean phytoalexin, glyceollin, showed bacteriostatic activity. Viable bacteria isolated from intact nodules were adapted to glyceollin. H2O2 in physiological concentrations did not affect wild-type bacteria. B. japonicum mutants defective in the biosynthesis of cyclic beta-(1-->3)-(1-->6)-glucans showed higher susceptibility to both phytoalexin and H2O2.

Further studies of the role of cyclic beta-glucans in symbiosis. An NdvC mutant of Bradyrhizobium japonicum synthesizes cyclodecakis-(1-->3)-beta-glucosyl.

The cyclic beta-(1-->3),beta-(1-->6)-D-glucan synthesis locus of Bradyrhizobium japonicum is composed of at least two genes, ndvB and ndvC. Mutation in either gene affects glucan synthesis, as well as the ability of the bacterium to establish a successful symbiotic interaction with the legume host soybean (Glycine max). B. japonicum strain AB-14 (ndvB::Tn5) does not synthesize beta-glucans, and strain AB-1 (ndvC::Tn5) synthesizes a cyclic beta-glucan lacking beta-(1-->6)-glycosidic bonds. We determined that the structure of the glucan synthesized by strain AB-1 is cyclodecakis-(1-->3)-beta-D-glucosyl, a cyclic beta-(1-->3)-linked decasaccharide in which one of the residues is substituted in the 6 position with beta-laminaribiose. Cyclodecakis-(1-->3)-beta-D-glucosyl did not suppress the fungal beta-glucan-induced plant defense response in soybean cotyledons and had much lower affinity for the putative membrane receptor protein than cyclic beta-(1-->3),beta-(1-->6)-glucans produced by wild-type B. japonicum. This is consistent with the hypothesis presented previously that the wild-type cyclic beta-glucans may function as suppressors of a host defense response.

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.

Cyclolaminarinose. A new biologically active beta-(1-->3) cyclic glucan.

A unique glucan has been isolated from a recombinant strain of a Rhizobium meliloti TY7, a cyclic beta-(1-->2) glucan mutant carrying a locus specifying beta-(1-->3; 1-->6) glucan synthesis from Bradyrhizobium japonicum USDA110. This compound, which appears to have considerable hydrophobic affinity, was separated from a perchloric acid cell extract by adsorption to a C-18 silica column. Unlike those cyclic glucans previously isolated from Rhizobium meliloti or Bradyrhizobium japonicum, this molecule contains neither phosphoglycerol nor phosphocholine substituents, respectively. 2D NMR, FAB mass spectrometric analysis and high-performance anion exchange chromatography with pulsed amperometric detection (HPAEC-PAD) confirmed that this glucan is a single, cyclic decasaccharide (cyclolaminarinose) in which one of the residues is substituted in its 6-position with beta-laminarabiose. This structural assignment was confirmed by mass spectral and NMR analyses of the product obtained from two consecutive Smith degradations. Unlike the complex 13C spectrum of the unoxidized material, the spectrum of this product consisted of only six resonances due to rapid time averaging of its symmetrical structure on the relatively slow NMR timescale. Synthesis of this newly described cyclic beta-glucan in the R. meliloti ndvB mutant restored the symbiotic and hypoosmotic adaptation characteristics of the R. meliloti wild type strain.

Nodule development induced by mutants of Bradyrhizobium japonicum defective in cyclic B-glucan synthesis.

The soybean response to inoculation with B. japonicum mutants defective in cyclic beta-(1-->3), beta-(1-->6) glucan synthesis was examined by electron microscopy and by monitoring the expression of early and late nodulin genes. Two mutants were examined. Strain AB-14 is an ndvB mutant and is unable to synthesize beta-glucans. Strain AB-1 is an ndvC mutant and produces cyclic glucans containing 95 to 100% beta-(1-->3) glycosidic linkages. Nodules formed by either mutant were defective in nitrogen fixation activity. Soybean plants inoculated with strain AB-14 formed nodules roughly at the same rate as the wild-type strain USDA110, but nodulation by strain AB-1 was significantly delayed. Microscopy of nodules formed by strain AB-14 showed an overall ultrastructure similar to nodules formed by the wild type. However, in some nodules bacteroids were limited to only one part of the infected cells and in others the few bacteroids present showed signs of degradation. Nodulelike structures were formed by strain AB-1 that showed some signs of cellular differentiation. For example, clear parenchyma and sclerenchyma tissue could be seen. However, no infection threads or bacteria were evident in these structures. The expression of early (e.g., ENOD2 and ENOD55) and late (e.g., NOD26 and leghemoglobin) nodulins was examined. Nodules formed by strain AB-14 expressed all of the nodulins tested but at a reduced level. Expression of late nodulins was delayed in strain AB-14-induced nodules. Nodules formed by strain AB-1 were more strongly affected in nodulin expression. Although leghemoglobin synthesis was not detected, infection-specific nodulin transcripts of GmN93 and ENOD55 were detected. However, expression of the early nodulins ENOD2 and ENOD55 was considerably delayed and only apparent when assayed 21 days postinoculation. A low level of expression of intermediate nodulin GmN70 and late nodulin NOD26 could also be detected by 21 days postinoculation. The microscopy data show that tissue differentiation occurs in these nodules even in the absence of active bacterial invasion. These results demonstrate the importance of cyclic beta-(1-->3), beta-(1-->6) glucan synthesis to symbiotic development in soybean.

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.

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).

Identification and cloning of Bradyrhizobium japonicum genes expressed strain selectively in soil and rhizosphere.

The growth of Bradyrhizobium japonicum USDA 110 and USDA 438 in soil extract-supplemented medium led to transcription of a large amount of DNA not expressed in basal medium. Strain USDA 438 was more competitive for the nodulation of soybean than strain USDA 110. To identify and isolate DNA regions which were expressed specifically in strain USDA 438 but not in strain USDA 110 in response to soil extract or soybean root exudate, we developed a subtractive RNA hybridization procedure. Several cosmid clones which showed strain-specific gene expression were isolated from a USDA 438 gene library. Two clones enhanced competitive nodulation when mobilized to USDA 110. The method described may be useful for identifying genes expressed in response to environmental stimuli or genes expressed differently in related microbial strains.

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.

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.

Hemoproteins of Bradyrhizobium japonicum Cultured Cells and Bacteroids.

The hemoprotein content of 17 strains of Bradyrhizobium japonicum bacteroids from field-grown plants and the corresponding strains of cultured cells was determined spectrally. The major terminal oxidases, cytochromes (cyt) aa(3) and o, were present in all strains of cultured cells. cyt aa(3) was present in significant amounts in bacteroids only in strains of DNA homology group II. cyt o appeared to be present in bacteroids of all strains, and the average level was the same as in cultured cells. cyt b and c in the membrane fractions were higher in bacteroids of all strains compared with cultured cells. cyt P-450 was present in both the membrane and soluble fractions of bacteroids of most strains. The total P-450 content varied sixfold among strains. A CO-reactive hemoprotein, P-422, was present in the soluble fraction of all strains of cultured cells. P-422 may be a hemoglobinlike protein, and it was present in significant amounts in bacteroids only in DNA homology group I strains.

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.

Cytochrome mutants of bradyrhizobium induced by transposon tn5.

Transposon Tn5 was used to mutate Bradyrhizobium japonicum USDA 61N. From over 5000 clones containing Tn5, 12 were selected and purified using a chemical reaction to identify oxidase-deficient clones. Four classes of mutants were identified based on the alterations in cytochromes. Most of the mutants had alterations in more than one cytochrome. Southern hybridization analysis of restricted genomic DNA of a representative strain of each class demonstrated that each mutant had a single Tn5 insert. Thus a single Tn5 insert produced pleiotropic effects on cytochromes. One class, which was totally deficient in cytochromes aa(3) and c, produced ineffective nodules on soybeans. Most of the strains representing the other classes produced effective nodules but exceptions were observed in each class. Bacteroids of the wild-type strain contained cytochrome aa(3). Bacteroids from one class of mutants were totally devoid of cytochrome aa(3). Several of these strains produced effective symbioses indicating that cytochrome aa(3) is not required for an effective symbiosis in this DNA homology group II strain which normally has this terminal oxidase in bacteroids.

Induction of Symbiotically Defective Auxotrophic Mutants of Rhizobium fredii HH303 by Transposon Mutagenesis.

Symbiotically defective auxotrophic mutants were isolated by transposon Tn5 mutagenesis of Rhizobium fredii HH303, a fast-growing microsymbiont of North American commercial soybean cultivars such as Glycine max cv. Williams. Three different Tn5-carrying suicide vectors, pBLK1-2, pSUP1011, and pGS9, were used for mutagenesis with transposition frequencies of 4 x 10, 3 x 10, and 1 x 10, respectively, while the frequency of background mutation resistant to 500 mug of kanamycin per ml was 1 x 10. From 2,600 Tn5-induced mutants, 14 auxotrophic mutants were isolated and classified in seven groups including adenosine (four), aspartate (two), cysteine or methionine (two), isoleucine and valine (two), nicotinic acid (one), pantothenic acid (one), and uracil (two). All the auxotrophs induced nodulation on soybean, but the symbiotic effectiveness of each mutant was different. Three auxotrophs (two cysteine or methionine and one pantothenic acid) formed effective nodules similar to those of the wild type. Three auxotrophs (one nicotinic acid and two aspartate) produced mature nodules like those of the wild type, but the nodules lacked the characteristic pink color inside and were unable to fix nitrogen. Four auxotrophs (two adenosine and two uracil) induced pseudonodules unable to fix nitrogen. The other four auxotrophs repeatedly induced both effective and ineffective nodules, but bacteroids isolated from the effective nodules were prototrophic revertants. The symbiotic phenotype and the degree of effectiveness of the auxotrophic mutants varied with the type of mutation.

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 species. The kinetics of growth inhibition are multiphasic, with no apparent effect for several hours after addition, followed by a period of total inhibition. Subsequently, growth resumed at a slower rate. Resumption of growth was not due to destruction of the nucleotide. Studies of the intracellular cGMP concentration did not reveal significant changes in cells grown under aerobic or microaerobic conditions. No effect of cGMP on the derepression of respiratory nitrate reductase was observed.