Polyamines in Rhizobium, Bradyrhizobium, Azorhizobium and Argobacterium

Abstract Eighteen strains of Rhizobium including four species, R. leguminosarum, R. meliloti, R. loti and R. fredii , nine strains of Bradyrhizobium japonicum and three strains of Azorhizobium caulinodans contained putrescine and honospermidine as major polyamines. All these nodulating N₂-fixing rhizobia lack spermidine. Spermidine and cadaverine were present only in a limited number of R. meliloti and B. japonicum . Polymanine-synthetic activity was not affected by the differences in ability to produce phytoxine (rhizobitoxine and dihydrorhizobitoxine) H₂-uptake-hydrogenation in the organisms. Putrescine and homospermidine were major polyamined in a strain of Agrobacterium rhizogenes . All the eight strains of Agrobacterium tumefaciens as well as A. rubi, A. radiobacter and two other strains of A. rhizogenes contained putrescine and spermidine as major polyamines and homospermidine and spermine (and thermospermine) as minor polyamines.     

Location of nif genes on large plasmids in Rhizobium strains isolated from legume tree root nodules

Abstract The presence of plasmids in Rhizobium strains isolated from legume tree root nodules has been studied. Bacteria isolated from Acacia melanoxylon, A. cyanophylla, Prosopis chilensis and Sophora microphylla harbour 2 to 5 plasmids of an M _r higher than 115 · 10⁶. Hybridization experiments have shown DNA homology between plasmid pID1 ( R. meliloti 41 nifH and D genes) and one of the plasmids in each of the bacteria studied.     

Characterization of Rhizobium etli and other Rhizobium spp. that nodulate Phaseolus vulgaris L. in an Austrian soil

Phaseolus vulgaris L. (common bean) is nodulated by rhizobia present in the fields around the Seibersdorf laboratory despite the fact that common bean has not been grown for a long time. Using PCR analysis with repetitive primers, plasmid profiles, nifH profiles, PCR-RFLP analysis of the 16S rRNA gene and of the 16S rRNA-23S rRNA intergenic spacer and the nodulation phenotype, two well-differentiating groups could be distinguished. One group showed high similarity to Rhizobium sp. R602sp, isolated from common bean in France, while the other showed the same characteristics as R. etli . We detected little variation in the symbiotic regions but found higher diversity when using approaches targeting the whole genome. Many isolates obtained in this study might have diverged from a limited number of strains, therefore the Austrian isolates showed high saprophytic and nodulation competence in that particular soil.     

Differentiating Indigenous Soybean Bradyrhizobium and Rhizobium spp. of Indian Soils

Soybean is extensively cultivated worldwide and is the largest source of biologically fixed nitrogen among legumes. It is nodulated by both slow and fast growing rhizobia. Indigenous soybean rhizobia in Vertisols of central India were assessed for utilization of 35 carbon sources and intrinsic resistance to 19 antibiotics. There was greater utilization of trehalose and raffinose by fast growers (87 and 73 % by fast vs. 35 and 30 % by slow growers); but slow growers had higher ability to utilize glucosamine (75 % by slow vs. 33 % by fast growers). A larger proportion of slow growers were resistant to vancomycin, polymyxin-B and rifampicin (70, 65 and 55 %) compared to fast growers (13, 7 and 7 % each). Among the two 16S rRNA sequence types in the slow growers, those belonging to Bradyrhizobium spp. utilized glucosamine while those belonging to Rhizobium radiobacter did not. All the fast growers had 16S rRNA homology to R. radiobacter and majority could not utilize glucosamine. It is suggested that during initial isolations and screening of rhizobia in strain selection programmes, using carbon sources like glucosamine and antibiotics like vancomycin, polymyxin-B and rifampicin in the media may provide a simple way of distinguishing Bradyrhizobium strains from R. radiobacter among the slow growers.     

Composted sawdust as a carrierfor Bradyrhizobium,Rhizobium and Azospirillum in crop inoculation

Sawdust was composted by inoculation with a cellulose-decomposing fungus (Cephalosporium sp.) and an N₂-fixing bacterium (Azospirillum brasilense). The product was investigated as a possible carrier for Bradyrhizobium, Rhizobium and Azospirillum. The simple technology and composition of the carrier supported good growth and survival of the investigated strains. Yield increases following crop inoculation with the carrier containing the Bradyrhizobium/Rhizobium/Azospirillum mixture were observed with soybean (34–62%), groundnuts (4–39%), lucerne (24–82%) and a grass mixture of bird's foot trefoil and ryegrass (20–21%).     

Hox genes in sea spiders (Pycnogonida) and the homology of arthropod head segments

The pycnogonids (or sea spiders) are an enigmatic group of arthropods, classified in recent phylogenies as a sister-group of either euchelicerates (horseshoe crabs and arachnids), or all other extant arthropods. Because of their bizarre morpho-anatomy, homologies with other arthropod taxa have been difficult to assess. We review the main morphology-based hypotheses of correspondence between anterior segments of pycnogonids, arachnids and mandibulates. In an attempt to provide new relevant data to these controversial issues, we performed a PCR survey of Hox genes in two pycnogonid species, Endeis spinosa and Nymphon gracile, from which we could recover nine and six Hox genes, respectively. Phylogenetic analyses allowed to identify their orthology relationships. The Deformed gene from E. spinosa and the abdominal-A gene from N. gracile exhibit unusual sequence divergence in their homeodomains, which, in the latter case, may be correlated with the extreme reduction of the posterior region in pycnogonids. Expression patterns of two Hox genes (labial and Deformed) in the E. spinosa protonymphon larva are discussed. The anterior boundaries of their expression domains favour homology between sea spider chelifores, euchelicerates chelicerae and mandibulate (first) antennae, in contradistinction with previously proposed alternative schemes such as the protocerebral identity of sea spider chelifores or the absence of a deutocerebrum in chelicerates. In addition, while anatomical and embryological evidences suggest the possibility that the ovigers of sea spiders could be a duplicated pair of pedipalps, the Hox data support them as modified anterior walking legs, consistent with the classical views.Supplementary material is available for this article at and is accessible for authorized users.Guest editors Jean Deutsch and Gerhard Scholtz     

Root nodule Bradyrhizobium spp. harbor tfdAalpha and cadA, homologous with genes encoding 2,4-dichlorophenoxyacetic acid-degrading proteins

The distribution of tfdAalpha and cadA, genes encoding 2,4-dichlorophenoxyacetate (2,4-D)-degrading proteins which are characteristic of the 2,4-D-degrading Bradyrhizobium sp. isolated from pristine environments, was examined by PCR and Southern hybridization in several Bradyrhizobium strains including type strains of Bradyrhizobium japonicum USDA110 and Bradyrhizobium elkanii USDA94, in phylogenetically closely related Agromonas oligotrophica and Rhodopseudomonas palustris, and in 2,4-D-degrading Sphingomonas strains. All strains showed positive signals for tfdAalpha, and its phylogenetic tree was congruent with that of 16S rRNA genes in alpha-Proteobacteria, indicating evolution of tfdAalpha without horizontal gene transfer. The nucleotide sequence identities between tfdAalpha and canonical tfdA in beta- and gamma-Proteobacteria were 46 to 57%, and the deduced amino acid sequence of TfdAalpha revealed conserved residues characteristic of the active site of alpha-ketoglutarate-dependent dioxygenases. On the other hand, cadA showed limited distribution in 2,4-D-degrading Bradyrhizobium sp. and Sphingomonas sp. and some strains of non-2,4-D-degrading B. elkanii. The cadA genes were phylogenetically separated between 2,4-D-degrading and nondegrading strains, and the cadA genes of 2,4-D degrading strains were further separated between Bradyrhizobium sp. and Sphingomonas sp., indicating the incongruency of cadA with 16S rRNA genes. The nucleotide sequence identities between cadA and tftA of 2,4,5-trichlorophenoxyacetate-degrading Burkholderia cepacia AC1100 were 46 to 53%. Although all root nodule Bradyrhizobium strains were unable to degrade 2,4-D, three strains carrying cadA homologs degraded 4-chlorophenoxyacetate with the accumulation of 4-chlorophenol as an intermediate, suggesting the involvement of cadA homologs in the cleavage of the aryl ether linkage. Based on codon usage patterns and GC content, it was suggested that the cadA genes of 2,4-D-degrading and nondegrading Bradyrhizobium spp. have different origins and that the genes would be obtained in the former through horizontal gene transfer.     

Influence of Glycine spp. on Competitiveness of Bradyrhizobium japonicum and Rhizobium fredii

The displacement of indigenous Bradyrhizobium japonicum in soybean nodules with more effective strains offers the possibility of enhanced N₂ fixation in soybean (Glycine max (L.) Merr.). Our objective was to determine whether the wild soybean (G. soja Sieb. & Zucc.) genotype PI 468397 would cause reduced competitiveness of important indigenous B. japonicum strains USDA 31, 76, and 123 and thereby permit nodulation by Rhizobium fredii, the fast-growing microsymbiont of soybean. In an initial experiment, PI 468397 nodulated and fixed moderate amounts of N₂ with USDA 31 and 76 but, despite the formation of nodules, fixed essentially no N₂ with USDA 123. In contrast, PI 468397 formed a highly effective symbiosis with R. fredii strain USDA 193. In two subsequent experiments, Williams soybean and PI 468397 were grown in a pasteurized soil mixture or in soybean rhizobium-free soil and inoculated with both USDA 123 and USDA 193. In each experiment, more than 90% of the nodules of Williams contained USDA 123, while only a maximum of 2% were occupied with USDA 193. In contrast, in the two experiments, 16 and 11%, respectively, of the nodules produced on PI 468397 were occupied by USDA 123, while in both experiments 87% contained USDA 193. Thus, in relation to the cultivar Williams, which is commonly grown and used as a parent in soybean breeding programs in the United States, PI 468397 substantially reduced the competitive ability of B. japonicum strain USDA 123 in relation to R. fredii strain USDA 193.     

Expression and regulation of the lactose transposon Tn951 in Rhizobium spp.

Abstract The expression of β-galactosidase by the lac transposon Tn 951 , in Escherichia coli , was found to be cAMP-dependent. This finding provided the basis for an investigation of the effect of cAMP on Tn 951 lac expression in Rhizobium , with the ultimate aim of using the Tn 951 system as a specific probe for cAMP mediated catabolite repression. When introduced into Rhizobium , Tn 951 directed the synthesis of β-galactosidase, which was inducible by isopropyl-β- d -thiogalactopyranoside (IPTG). Marked quantitative and qualitative differences in β-galactosidase expression were found between R. meliloti and R. japonicum during the growth cycle, with expression being higher in the former. β-Galactosidase levels were, however, unaffected by exogenous cAMP under catabolite repressing conditions.     

Behaviour of bacteriophage Mu-based IncP suicide vector plasmids in Rhizobium spp.

Abstract The ability of P-type plasmids, containing bacteriophage Mu, to survive in Rhizobia was examined under conditions in which the plasmids were not directly selected. The plasmid pRP4::Mu::Tn7 survived in all tested strains of Rhizobia and Agrobacteria . The surviving plasmids were analysed for expression of plasmid coded properties and for presence of both bacteriophage Mu and Tn7 DNA. A large variation in plasmid size and coding capacity was observed. In the parent plasmid Tn7 is inserted into the Mu DNA. The majority of surviving plasmids had lost Mu DNA but the Tn7 DNA was retained. Analysis of surviving plasmids which contained Mu DNA indicated that a small deletion of Mu is sufficient to allow plasmid survival. Deleted derivatives of pJB4J1 were also isolated in Rhizobium under similar conditions. The potential of these suicide vectors as potential vectors for transposon mutagenesis in Rhizobium is discussed.     

两株高效相思根瘤菌的抗药性研究及其质粒组成分析

目的:研究相思根瘤菌质粒与其抗药性之间的关系。方法:研究了相思根瘤菌MZ和AJ018在含不同浓度的抗生素的固体培养基和液体培养基的生长情况,并用碱裂解方法对其质粒组成进行检测。结果:两菌株对链霉素和卡那霉素均无抗性,而对其它抗生素都有不同程度的抗性。MZ菌株对实验中的氯霉素、氨苄青霉素、四环素三种抗生素的耐受范围与最大耐受值都比AJ018强,当平板培养基中氨苄青霉素、四环素、氯霉素的终浓度分别为250μg/ml、75μg/ml、150μg/ml时,AJ018在平板上无菌落生长,当三种抗生素终浓度分别为800μg/ml1、50μg/ml、400μg/ml时无菌落生长。两菌株都含有一个大约50kb的质粒。     

氮源对紫云英根瘤菌nod基因表达的作用

高浓度的硫酸铵阻碍了紫云英根瘤菌诱导紫云英根毛发生典型的根毛变形并明显抑制了紫云英结瘤能力。通过对融合子的β-半乳精苷酶活性的测定进一步表明高浓度的硫酸铵对紫云英的结瘤调节基因nodDZ、共同结瘤基因nodA及nodBC的表达有抑制作用而对结瘤调节基因nodD1的表达无抑制作用。     

Root nodule development: origin, function and regulation of nodulin genes

The symbiotic root nodule, an organ formed on leguminous plants, is a product of successful interactions between the host plant and the soil bacteria, Rhizobium spp. Plant hormones play an important role in the genesis of this organ. The hormonal balance appears to be modulated by the signals produced by bacteria. Many host genes induced during nodule organogenesis and the symbiotic state have been identified and characterized from several legumes. These genes encode nodule-specific proteins (nodulins) which perform diverse functions in root nodule development and metabolism. Formation of a subcellular compartment housing the bacteria is essential to sustain the symbiotic state, and several nodulins are involved in maintaining the integrity and function of this compartment. The bacteroid enclosed in the perbacteroid membrane behaves as an 'organelle,'completely dependent on the host for all its requirements for carbon, nitrogen and other essential elements. Thus it seems likely that the nodulins in the peribacteroid membrane perform specific transport functions. While the function of a few other nodulins is known (e.g. nodulin-100, nodulin-35), a group of uncharacterized nodulins exists in soybean root nodules. These nodulins share structural similarities and seem to have been derived from a common ancestor. Induction of nodulin genes occurs prior to and independent of nitrogen fixation, and thus is a prelude to symbiosis. Although some of the early nodulin genes are induced prior to or during infection, induction of late nodulins requires endocytotic release of bacteria.     

Utilization of poly-β-hydroxybutyrate in free-living cultures of Rhizobium ORS571

Abstract The effect of carbon starvation on growth and poly-β-hydroxybutyrate (PHB) utilization in oxygen-limited chemostat cultures of Rhizobium ORS571 was studied. Under oxygen-limited growth conditions PHB was not degraded. When in a nitrogen-fixing oxygen-limited culture, after stopping the medium supply, the dissolved oxygen concentration was maintained at 10 μM, a slow breakdown of PHB was observed. Addition of ammonia and air to a nitrogen-fixing oxygen-limited culture after the medium supply had been stopped, resulted in the simultaneous utilization of PHB and succinate. The possible use of the energy derived from PHB degradation in Rhizobia bacteria and bacteroids is discussed.     

The formation of R-prime deletion mutants and the identification of the symbiotic genes in Rhizobium fredii strain USDA191

Summary R-prime plasmids were formed between the plasmid of Rhizobium fredii strain USDA191 containing nodulation and nitrogen-fixation genes, pRjaUSDA191c, and pRL180, and RP1 derivative. R. fredii USDA191 contains four HindIII fragments that hybridize with an 8.7 kb EcoRI fragment that contains nodulation genes from R. meliloti. These four fragments are on pRjaUSDA191c and are 15.5 kb, 12.5 kb, 6.8 kb, and 5.2 kb in size. A series of R-primes generated in E. coli of pRjaUSDA191c were transferred into a Nod^- Nif^- derivative of strain USDA191 to determine which nodulation region is necessary for nodule formation. Transconjugants containing the 12.5 kb and the 6.8 kb HindIII fragments on segments of pRjaUSDA191c produced nodules on soybean plants. However, transconjugants containing the 12.5 kb HindIII fragment alone were unable to form nodules, suggesting that the 6.8 kb HindIII fragment or the 6.8 kb and the 12.5 kb HindIII fragments together were needed for nodule formation. The 6.8 kb HindIII fragment was subcloned into the vector pVK102 and transferred into transconjugants containing no sequences homologous to R. meliloti nodulation DNA or to transconjugants containing only the 12.5 kb HindIII fragment. Nodules were formed on soybeans only when both the 12.5 kb and the 6.8 kb HindIII fragments were present in R. frediistrain USDA191.     

Competition between Rhizobium strains in nodule formation: Interaction between nodulating and non-nodulating strains

Summary Nodulation of pea cv. Afghanistan and cv. Iran by a nodulating Rhizobium strain is suppressed by the presence of a non-nodulating strain. The degree of suppression varies, dependent on the Rhizobium strains used. There is a great variation in the competitive ability of the Rhizobium strains and this is not related to the ability to form root nodules. The critical period of competition is restricted to ca. 24 hours after inoculation.