Rhizobacter

Folia Microbiol (Praha), 2003, 48(4), 510 - 20
Polyphasic taxonomy of symbiotic rhizobia from wild leguminous plants growing in Egypt; Zahran HH et al.; About 20 strains of rhizobia from wild legumes were characterized based on numerical analysis of phenotypic characteristics, nodulating ability, fatty acid methyl esters (FAME) and SDS-PAGE profiles of whole cell proteins . FAME analysis revealed that palmitic (16:0), stearic (18:0) and arachidonic (20:0) were detected in most of wild-legume rhizobia, the latter being uncommon in fatty acid profiles of Rhizobium and Sinorhizobium . Numerical analysis of FAME classified strains of wild-legume rhizobia into 9 clusters and one heterogeneous group . There was both agreement and disagreement with the clustering data based on phenotypic analysis and FAME analysis . Four strains were grouped together in the same cluster based on both methods . However, 4 another strains, which were placed in one cluster of phenotypic analysis, were distributed in several clusters after FAME analysis . SDS-PAGE of whole-cell proteins revealed that the rhizobial strains exhibited protein profiles with peptide bands ranging from 5-19 band per profile and showed molar mass of 110-183 kDa . As in the case of FAME analysis, numerical analysis of protein bands was compared with clustering of phenotypic analysis . Agreement of the two methods was obvious when clustering some strains but conflicted in the classification of some other strains . However, integration of the three methods could be the basis of a polyphasic taxonomy . The twenty strains of wild-legume rhizobia were finally classified as follows: 12 strains related to Rhizobium leguminosarum, 5 strains related to Sinorhizobium meliloti and 3 strains to Rhizobium spp . Rhizobia nodulating wild herb legumes are among indigenous strains nodulating crop legumes in cultivated as well as noncultivated lands.

Syst Appl Microbiol, 2003 Sep, 26(3), 453 - 65
A catalogue of molecular, physiological and symbiotic properties of soybean-nodulating rhizobial strains from different soybean cropping areas of China; Thomas-Oates J et al.; We have analysed 198 fast-growing soybean-nodulating rhizobial strains from four different regions of China for the following characteristics: generation time; number of plasmids; lipopolysaccharide (LPS), nodulation factors (LCOs) and PCR profiles; acidification of growth medium; capacity to grow at acid, neutral, and alkaline pH; growth on LC medium; growth at 28 and 37 degrees C; melanin production capacity; Congo red absorption and symbiotic characteristics . These unbiased analyses of a total subset of strains isolated from specific soybean-cropping areas (an approach which could be called "strainomics") can be used to answer various biological questions . We illustrate this by a comparison of the molecular characteristics of five strains with interesting symbiotic properties . From this comparison we conclude, for instance, that differences in the efficiency of nitrogen fixation or competitiveness for nodulation of these strains are not apparently related to differences in Nod factor structure.

Microbiol Res, 2003, 158(3), 243 - 8
Growth promoting influence of siderophore-producing Pseudomonas strains GRP3A and PRS9 in maize (Zea mays L.) under iron limiting conditions; Sharma A et al.; Maize seeds were bacterized with siderophore-producing pseudomonads with the goal to develop a system suitable for better iron uptake under iron-stressed conditions . Siderophore production was compared in fluorescent Pseudomonas spp . GRP3A, PRS9 and P . chlororaphis ATCC 9446 in standard succinate (SSM) and citrate (SCM) media . Succinate was better suited for siderophore production, however, deferration of media resulted in increased siderophore production in all the strains . Maximum siderophore level (216.23 microg/ml) was observed in strain PRS9 in deferrated SSM after 72 h of incubation . Strains GRP3A and PRS9 were used for plant growth promotion experiments . Strains GRP3A and PRS9 were also antagonistic against the phytopathogens, Colletotrichum dematium, Rhizoctonia solani and Sclerotium rolfsii . Bacterization of maize seeds with strains GRP3A and PRS9 showed significant increase in germination percentage and plant growth . Maximum shoot and root length and dry weight were observed with 10 microM Fe3+ along with bacterial inoculants suggesting application of siderophore producing plant growth promoting rhizobacterial strains in crop productivity in calcareous soil system.

Biotechnol Lett, 2003 Aug, 25(15), 1267 - 70
Anions effects on biosorption of Mn(II) by extracellular polymeric substance (EPS) from Rhizobium etli; Pulsawat W et al.; Microbial extracellular polymeric substances (EPS) are potential biosorbents for metal remediation and recovery . The Langmuir and Freundlich kinetics of Mn(II) binding by the EPS from a novel Mn(II) oxidising strain of Rhizobium etli were determined . Maximum manganese specific adsorptions (q(max)) decreased in the sequence: sulphate (62 mg Mn per g EPS) > nitrate (53 mg g(-1)) > chloride (21 mg g(-1)) . Consideration of the anion during kinetic studies is usually neglected but is important in providing more practical and comparable data between different biosorbent systems.

Biotechnol Lett, 2003 Sep, 25(17), 1407 - 13
Screening, identification and kinetic characterization of a bacterium for Mn(II) uptake and oxidation; Moy YP et al.; Following sample collection and screening at a number of Mn-associated mine sites in Northern Australia, a microbial strain was selected for its enhanced rate of Mn uptake . The strain was identified by phylogenetic analysis as a Rhizobium sp . Kinetic studies of Mn(II) uptake and oxidation by this strain in glucose-based media established that the uptake of Mn(II) was much greater than the conversion of Mn(II) to Mn oxide . Chemical analysis and scanning electron microscopy confirmed the production of significant amounts of polysaccharides by this strain . These polysaccharides may play a role both in enhancing Mn(II) accumulation and in minimizing Mn oxide production.

Environ Microbiol, 2003 Oct, 5(10), 916 - 24
Effect of thorium on the growth and capsule morphology of Bradyrhizobium; Santamaria M et al.; The thorium effect on Bradyrhizobium growth was assayed in liquid media . Th4+ inhibited the growth of Bradyrhizobium (Chamaecytisus) BGA-1, but this effect decreased in the presence of suspensions of live or dead bacterial cells . Th4+ induced the formation of a gel-like precipitate when added to a dense suspension of B . (Chamaecytisus) BGA-1 cells . Viable Bradyrhizobium cells remained in suspension after precipitate formation . Thorium was recovered in the precipitate, in which polysaccharide, lipopolysaccharide and proteins were also found . After Th4+ addition, the morphology of B . (Chamaecytisus) BGA-1 or Bradyrhizobium japonicum USDA 110 sedimented cells studied by scanning electron microscopy changed from an entangled network of capsulated bacteria to uncapsulated individual cells and an amorphous precipitate . Energy-dispersive X-ray spectroscopy showed that thorium was mainly in the amorphous fraction . Precipitate was also formed between B . (Chamaecytisus) BGA-1 and Al3+, which was also toxic to this bacterium . Precipitate induced by Th4+ or Al3+ was found in all Bradyrhizobium and Sinorhizobium strains tested, but not in Rhizobium, Salmonella typhimurium, Aerobacter aerogenes or Escherichia coli . These results suggest a specific defence mechanism based on metal precipitation by extracellular polymers.

Environ Microbiol, 2003 Oct, 5(10), 888 - 95
Temporal change in culturable phenanthrene degraders in response to long-term exposure to phenanthrene in a soil column system; Bodour AA et al.; Widespread environmental contamination by polycyclic aromatic hydrocarbons (PAH) has led to increased interest in the use of natural attenuation as a clean-up strategy . However, few bioremediation studies have investigated the behaviour of the indigenous PAH-degrading community after long-term exposure to a PAH . In this study, a column packed with sandy loam soil was exposed to a solution saturated with phenanthrene ( approximately 1.2 mg l-1) for a 6-month period to examine the temporal response of the indigenous phenanthrene-degrading community . Initial soil, effluent, and final soil samples were collected and analysed for phenanthrene concentration and culturable phenanthrene degraders . Phenanthrene-degrading isolates were grouped by colony morphology . For each unique group, 16S rDNA polymerase chain reaction was performed, and then sequencing analysis was used to identify the isolate at the genus level . Twenty-five phenanthrene-degrading isolates, potentially representing 19 genera, were obtained from this analysis . Of these, eight genera have not been reported previously to degrade phenanthrene, including Afipia, Janthinobacterium, Leptothrix, Massilia, Methylobacterium, Rhizobium, Sinorhizobium and Thiobacillus . Results indicate that the dominant phenanthrene-degrading population changed over the course of this 6-month experiment . Specifically, the isolates obtained initially from the soil were not subsequently found in either effluent samples or the soil at the end of the experiment . Furthermore, several isolates that were found in the soil at the end of the experiment were not observed in the soil initially or in the effluent samples . This study confirms earlier findings indicating that a diverse community participates in phenanthrene degradation in the environment, and also suggests that the composition of this community is temporally variable.

Planta, 2003 Nov, 218(1), 42 - 9 Epub 2003 Sep 24.
Expression of ENOD40 during tomato plant development; Vleghels I et al.; In legumes, ENOD40 expression is increased upon interaction of plants with rhizobia . Little is known of the expression pattern of ENOD40 during other stages of the plant life cycle . Studies of ENOD40 expression in non-legume development may give an indication of the function of the gene . To investigate the ENOD40 expression pattern during plant development, a fusion between the beta-glucuronidase (GUS) reporter gene and 150 bp of the 5' untranslated region plus 3,000 bp of 5' untranscribed tomato ENOD40 sequence was constructed and introduced into Lycopersicon esculentum Miller . Based on the observed GUS expression patterns in transgenic tomato we speculate that ENOD40 in tomato has a role in counteracting ethylene-provoked responses.

Curr Microbiol, 2003 Aug, 47(2), 134 - 7
nodD alleles of Sinorhizobium fredii USDA191 differentially influence soybean nodulation, nodC expression, and production of exopolysaccharides; Machado D et al.; All Rhizobium strains examined to date have one or multiple alleles of nodD . At least one copy of nodD and the presence of flavonoid exudates are required for nod gene induction and nodulation . Sinorhizobium fredii USDA191 has two copies of nodD . In this study, we demonstrate that inactivation of either copy of nodD caused a reduction in basal levels of expression of nodC . Extra copies of nodD1 had no effect on the expression of nodC when compared with the wild type, but extra copies of nodD2 abolished the inducer requirement, thereby rendering nodC constitutive . A nodD1 mutant was unable to nodulate soybean cultivars 'Peking' and 'McCall' . Inactivation of nodD2 or addition of extra copies of nodD1 or nodD2 caused delayed nodulation on Peking, and reduced the number of nodules on McCall . Both nodD alleles of S . fredii USDA191 appear to be involved in regulation of exopolysaccharide production; however, nodD2 appears to be more important in this respect than nodD1.

Microb Ecol, 2003 Oct, 46(3), 302 - 11 Epub 2003 Sep 17.
Microbial processes associated with roots of bulbous rush coated with iron plaques; Kusel K et al.; Bulbous rush (Juncus bulbosus) is a pioneer species in acidic, iron-rich, coal mining lakes in the eastern part of Germany . Juncus roots are coated with iron plaques, and it has been suggested that microbial processes under the iron plaques might be supportive for Juncus plant growth . The objectives of this work were to enumerate the microbes involved in the turnover of iron and organic root exudates in the rhizoplane, to investigate the effect of oxygen and pH on the utilization of these exudates by the rhizobacteria, and to study the ability of the root-colonizing microbiota to reduce sulfate . Enumeration studies done at pH 3 demonstrated that 10(6) Fe(III) reducers and 10(7) Fe(II) oxidizers g (fresh wt root)(-1) were associated with Juncus roots . When roots were incubated in goethite-containing medium without and with supplemental glucose, Fe(II) was formed at rates approximating 1.1 mmol g (fresh wt root) (-1) d(-1) and 3.6 mmol g (fresh wt root)(-1) d(-1) under anoxic conditions, respectively . These results suggest that a rapid microbially mediated cycling of iron occurs in the rhizosphere of Juncus roots under changing redox conditions . Most-probable-number estimates of aerobes and anaerobes capable of consuming root exudates at pH 3 were similar in the rhizosphere sediment and in Juncus roots, but numbers of aerobes were significantly higher than those of anaerobes . At pH 3, supplemental organic exudates were primarily subject to aerobic oxidation to CO2 and not subject to fermentation . However, at pH 4.5, root exudates were also rapidly utilized under anoxic conditions . Root-associated sulfate reduction was not observed at pH 3 to 4.5 but was observed at pH 4.9 . The pH increased during all root-incubation studies both under oxic and anoxic conditions . Thus, as result of the microbial turnover of organic root exudates, pH and CO2 levels might be elevated at the root surface and favor Juncus plants to colonize acidic habitats.

Int J Parasitol, 2003 Sep 30, 33(11), 1269 - 76
Interactions between bacteria and plant-parasitic nematodes: now and then; Bird DM et al.; Based on genome-to-genome analyses of gene sequences obtained from plant-parasitic, root-knot nematodes (Meloidogyne spp.), it seems likely that certain genes have been derived from bacteria by horizontal gene transfer . Strikingly, a common theme underpinning the function of these genes is their apparent direct relationship to the nematodes' parasitic lifestyle . Phylogenetic analyses implicate rhizobacteria as the predominant group of 'gene donor' bacteria . Root-knot nematodes and rhizobia occupy similar niches in the soil and in roots, and thus the opportunity for genetic exchange may be omnipresent . Further, both organisms establish intimate developmental interactions with host plants, and mounting evidence suggests that the mechanisms for these interactions are shared too . We propose that the origin of parasitism in Meloidogyne may have been facilitated by acquisition of genetic material from soil bacteria through horizontal transfer, and that such events represented key steps in speciation of plant-parasitic nematodes . To further understand the mechanisms of horizontal gene transfer, and also to provide experimental tools to manipulate this promising bio-control agent, we have initiated a genomic sequence of the bacterial hyper-parasite of plant parasitic nematodes, Pasteuria penetrans . Initial data have established that P . penetrans is closely related to Bacillus spp., to the extent that considerable genome synteny is apparent . Hence, Bacillus serves as a model for Pasteuria, and vice versa.

Indian J Environ Health, 2002 Oct, 44(4), 282 - 9
Mechanical, physico-chemical and microbial analysis of oil refinery waste receiving agricultural soil; Ashok BT et al.; The mechanical, physico-chemical characteristics of soil and the activity of indigenous microflora were studied in the agricultural soil steadily receiving petroleum refinery effluent at Mathura, U.P, India The data on the soil grain size and texture revealed that the soil in the test region was basically loam or silty loam . Physic-chemical analysis showed considerable variability in the soil pH, temperature, moisture content and water holding capacity (WHC) . A substantially higher microbial activity was noticed at the test sites as evident from the total variable (10(5) to 10(9) CFU g-1 soil) bacterial population . In addition, a significant population of proteolytic and cellulolytic bacteria, rhizobium and actinomycetes was detected . Oligotrophs were isolated and characterized into four types (I-IV) . A fraction of oligotrophic bacteria, particularly those belonging to type II and type IV exhibited appreciable in distilled water . Invariably higher microbial biomass ranging from 366 to 1604 mg CO2 . 100 g-1 soil, clearly implied that the soil in the test region was very well nourished and the refinery waste was providing enough nitrites to support the growth of soil microflora.

Int J Syst Evol Microbiol, 2003 Sep, 53(Pt 5), 1575 - 83
Characterization of rhizobia isolated from legume species within the genera Astragalus and Lespedeza grown in the Loess Plateau of China and description of Rhizobium loessense sp . nov; Wei GH et al.; Twenty-nine rhizobial isolates from root nodules of Astragalus and Lespedeza spp . growing in the Loess Plateau of China were characterized by numerical taxonomy, RFLP and sequencing of PCR-amplified 16S rRNA genes, measurement of DNA G+C content, DNA-DNA relatedness and cross-nodulation with selected legume species . Based on the results of numerical taxonomy, the isolates formed two clusters (1 and 2) with some single isolates at a similarity level of 82 % . Cluster 1 contained six isolates from Astragalus and Lespedeza spp . Cluster 2 consisted of nine isolates from Astragalus spp . DNA relatedness was greater than 80 % among isolates within cluster 2 . Phylogenetic analysis based on 16S rRNA gene sequences showed that CCBAU 7190B(T), representing cluster 2, was closely related to Rhizobium galegae and Rhizobium huautlense . DNA-DNA relatedness between CCBAU 7190B(T) and reference strains of R . galegae, R . huautlense and other related species ranged from 0 to 48.6 % . The cluster 2 isolates could also be differentiated phenotypically from related species . Based on these data, a novel species, Rhizobium loessense sp . nov., is proposed for cluster 2, with the type strain CCBAU 7190B(T) (=AS1.3401(T)=LMG 21975(T)).

Mol Cell Biol, 2003 Oct, 23(19), 6780 - 9
Targeted disruption of the mouse PAS domain serine/threonine kinase PASKIN; Katschinski DM et al.; PASKIN is a novel mammalian serine/threonine kinase containing two PAS (Per-Arnt-Sim) domains . PASKIN is related to the Rhizobium oxygen sensor protein FixL and to AMP-regulated kinases . Like FixL, the sensory PAS domain of PASKIN controls the kinase activity by autophosphorylation in a (unknown) ligand-dependent manner . In Saccharomyces cerevisiae, the two PASKIN orthologues PSK1 and PSK2 phosphorylate three translation factors and two enzymes involved in glycogen synthesis, thereby coordinately regulating protein synthesis and glycolytic flux . To elucidate the function of mammalian PASKIN, we inactivated the mouse Paskin gene by homologous recombination in embryonic stem cells . Paskin(-/-) mice showed normal development, growth, and reproduction . The targeted integration of a lacZ reporter gene allowed the identification of the cell types expressing mouse PASKIN . Surprisingly, PASKIN expression is strongly upregulated in postmeiotic germ cells during spermatogenesis . However, fertility and sperm production and motility were not affected by the PASKIN knockout . The Ppp1r7 gene encoding Sds22, a regulatory subunit of protein phosphatase 1, shares the promoter region with the Paskin gene, pointing towards a common transcriptional regulation . Indeed, Sds22 colocalized with the cell types expressing PASKIN in vivo, suggesting a functional role of protein phosphatase-1 in the regulation of PASKIN autophosphorylation.

Mol Plant Microbe Interact, 2003 Sep, 16(9), 796 - 807
Distinct patterns of symbiosis-related gene expression in actinorhizal nodules from different plant families; Pawlowski K et al.; Phylogenetic analyses suggest that, among the members of the Eurosid I clade, nitrogen-fixing root nodule symbioses developed multiple times independently, four times with rhizobia and four times with the genus Frankia . In order to understand the degree of similarity between symbiotic systems of different phylogenetic subgroups, gene expression patterns were analyzed in root nodules of Datisca glomerata and compared with those in nodules of another actinorhizal plant, Alnus glutinosa, and with the expression patterns of homologous genes in legumes . In parallel, the phylogeny of actinorhizal plants was examined more closely . The results suggest that, although relationships between major groups are difficult to resolve using molecular phylogenetic analysis, the comparison of gene expression patterns can be used to inform evolutionary relationships . In this case, stronger similarities were found between legumes and intracellularly infected actinorhizal plants (Alnus) than between actinorhizal plants of two different phylogenetic subgroups (Alnus/Datisca).

Mol Plant Microbe Interact, 2003 Sep, 16(9), 743 - 51
Characterization of Nops, nodulation outer proteins, secreted via the type III secretion system of NGR234; Marie C et al.; The nitrogen-fixing symbiotic bacterium Rhizobium species NGR234 secretes, via a type III secretion system (TTSS), proteins called Nops (nodulation outer proteins) . Abolition of TTSS-dependent protein secretion has either no effect or leads to a change in the number of nodules on selected plants . More dramatically, Nops impair nodule development on Crotalaria juncea roots, resulting in the formation of nonfixing pseudonodules . A double mutation of nopX and nopL, which code for two previously identified secreted proteins, leads to a phenotype on Pachyrhizus tuberosus differing from that of a mutant in which the TTSS is not functional . Use of antibodies and a modification of the purification protocol revealed that NGR234 secretes additional proteins in a TTSS-dependent manner . One of them was identified as NopA, a small 7-kDa protein . Single mutations in nopX and nopL were also generated to assess the involvement of each Nop in protein secretion and nodule formation . Mutation of nopX had little effect on NopL and NopA secretion but greatly affected the interaction of NGR234 with many plant hosts tested . NopL was not necessary for the secretion of any Nops but was required for efficient nodulation of some plant species . NopL may thus act as an effector protein whose recognition is dependent upon the hosts' genetic background.

J Appl Microbiol, 2003, 95(4), 832 - 8
Effect of Azospirillum-mediated plant growth promotion on the development of bacterial diseases on fresh-market and cherry tomato; Romero AM et al.; AIMS: Plant growth-promoting (PGP) activity of two Azospirillum strains and their effects on foliar and vascular bacterial diseases were evaluated on fresh market and cherry tomato . METHODS AND RESULTS: Tomato seeds were inoculated with A . brasilense Sp7 or Azospirillum sp . BNM-65 . Four-week-old plants were challenge-inoculated with Clavibacter michiganensis subsp . michiganensis (bacterial canker) or with Xanthomonas campestris pv . vesicatoria (bacterial spot) . Azospirillum-induced PGP was greater on cherry than on fresh-market tomato . Cherry tomato was more resistant to bacterial canker but more susceptible to bacterial spot than the fresh-market tomato . Canker severity was not affected by Azospirillum seed treatments . However, leaf- and plant-death were delayed on Azospirillum-treated plants compared with nontreated controls . Azospirillum increased the bacterial spot severity on cherry but not on fresh-market tomato . CONCLUSIONS: PGP was observed on both tomato genotypes, although growth effects were larger on cherry tomato . Also, Azospirillum treatments may alter tomato susceptibility to bacterial diseases . SIGNIFICANCE AND IMPACT OF THE STUDY: The interaction between PGP rhizobacteria like Azospirillum spp., not known to induce systemic resistance, with plant pathogens distantly located is frequently overlooked . This work demonstrates the importance of this kind of evaluation.

Environ Manage . 2003 Sep 11; {Epub ahead of print}
Comparison of Four Sesbania Species to Remediate Pb/Zn and Cu Mine Tailings; Chan GY et al.; A 6-month greenhouse pot trial was performed, aimed at screening appropriate Sesbania species for remediation of Pb/Zn and Cu mine tailings . Performances of young seedlings of four Sesbania species ( S . cannabina, S . grandiflora, S . rostrata, and S . sesban) were compared with and without inoculation of rhizobia . Seedlings were planted in two types of tailings amended with garden soil or garden soil mixed with river sediment . The results indicated that inoculated plants generally produced a higher biomass than samples without inoculation . Pb/Zn mine tailings containing rather high concentrations of total and water-soluble Cu, Pb, and Zn were toxic to plant growth compared with Cu mine tailings, according to the growth performance of the four species . Sesbania sesban and S . rostrata showed superior growth performance, compared to the other two species . Thus, they can serve as pioneer species to modify the barren environment, by providing organic matter and essential nutrients such as nitrogen, upon decomposition, in a relatively short period of time . This is especially true for S . rostrata, which is an annual plant that forms both stem and root nodules . However, a longer-term field trial should be conducted to investigate if superior species can beneficially modify the habitat for the growth of subsequent plant communities.

J Biol Chem, 2003 Nov 28, 278(48), 47915 - 21 Epub 2003 Sep 08.
Temperature-controlled structural alterations of an RNA thermometer; Chowdhury S et al.; Thermoresponsive structures in the 5'-untranslated region of mRNA are known to control translation of heat shock and virulence genes . Expression of many rhizobial heat shock genes is regulated by a conserved sequence element called ROSE for repression of heat shock gene expression . This cis-acting, untranslated mRNA is thought to prevent ribosome access at low temperature through an extended secondary structure, which partially melts when the temperature rises . We show here by a series of in vivo and in vitro approaches that ROSE is a sensitive thermometer responding in the physiologically relevant temperature range between 30 and 40 degrees C . Point mutations predicted to disrupt base pairing enhanced expression at 30 degrees C . Compensatory mutations restored repression, emphasizing the importance of secondary structures in the sensory RNA . Only moderate inducibility of a 5'-truncated ROSE variant suggests that interactions between individual stem loops coordinate temperature sensing . In the presence of a complementary oligonucleotide, the functionally important stem loop of ROSE was rendered susceptible to RNase H treatment at heat shock temperatures . Since major structural rearrangements were not observed during UV and CD spectroscopy, subtle structural changes involving the Shine-Dalgarno sequence are proposed to mediate translational control . Temperature perception by the sensory RNA is an ordered process that most likely occurs without the aid of accessory factors.

Nature, 2003 Sep 4, 425(6953), 78 - 81
Host sanctions and the legume-rhizobium mutualism; Kiers ET et al.; Explaining mutualistic cooperation between species remains one of the greatest problems for evolutionary biology . Why do symbionts provide costly services to a host, indirectly benefiting competitors sharing the same individual host? Host monitoring of symbiont performance and the imposition of sanctions on 'cheats' could stabilize mutualism . Here we show that soybeans penalize rhizobia that fail to fix N(2) inside their root nodules . We prevented a normally mutualistic rhizobium strain from cooperating (fixing N(2)) by replacing air with an N(2)-free atmosphere (Ar:O(2)) . A series of experiments at three spatial scales (whole plants, half root systems and individual nodules) demonstrated that forcing non-cooperation (analogous to cheating) decreased the reproductive success of rhizobia by about 50% . Non-invasive monitoring implicated decreased O(2) supply as a possible mechanism for sanctions against cheating rhizobia . More generally, such sanctions by one or both partners may be important in stabilizing a wide range of mutualistic symbioses.

FEMS Microbiol Lett, 2003 Aug 29, 225(2), 227 - 33
Nematode-enhanced microbial colonization of the wheat rhizosphere; Knox OG et al.; The mechanisms by which seed-applied bacteria colonize the rhizosphere in the absence of percolating water are poorly understood . Without mass flow, transport of bacteria by growing roots or soil animals, particularly nematodes may be important . We used a sand-based microcosm system to investigate the ability of three species of nematodes (Caenorhabditis elegans, Acrobeloides thornei and a Cruznema sp.) to promote rhizosphere colonization by four strains of beneficial rhizobacteria . In nearly all cases, rhizosphere colonization was substantially increased by the presence of nematodes, irrespective of bacterial or nematode species . Our results suggest that nematodes are important vectors for bacteria rhizosphere colonization in the absence of percolating water.

Science, 2003 Oct 24, 302(5645), 630 - 3 Epub 2003 Aug 28.
LysM domain receptor kinases regulating rhizobial Nod factor-induced infection; Limpens E et al.; The rhizobial infection of legumes has the most stringent demand toward Nod factor structure of all host responses, and therefore a specific Nod factor entry receptor has been proposed . The SYM2 gene identified in certain ecotypes of pea (Pisum sativum) is a good candidate for such an entry receptor . We exploited the close phylogenetic relationship of pea and the model legume Medicago truncatula to identify genes specifically involved in rhizobial infection . The SYM2 orthologous region of M . truncatula contains 15 putative receptor-like genes, of which 7 are LysM domain-containing receptor-like kinases (LYKs) . Using reverse genetics in M . truncatula, we show that two LYK genes are specifically involved in infection thread formation . This, as well as the properties of the LysM domains, strongly suggests that they are Nod factor entry receptors.

Biophys J, 2003 Sep, 85(3), 1345 - 57
Bacterial flagellar microhydrodynamics: Laminar flow over complex flagellar filaments, analog archimedean screws and cylinders, and its perturbations; Trachtenberg S et al.; The flagellar filament, the bacterial organelle of motility, is the smallest rotary propeller known . It consists of 1), a basal body (part of which is the proton driven rotary motor), 2), a hook (universal joint-allowing for off-axial transmission of rotary motion), and 3), a filament (propeller-a long, rigid, supercoiled helical assembly allowing for the conversion of rotary motion into linear thrust) . Helically perturbed (so-called "complex") filaments have a coarse surface composed of deep grooves and ridges following the three-start helical lines . These surface structures, reminiscent of a turbine or Archimedean screw, originate from symmetry reduction along the six-start helical lines due to dimerization of the flagellin monomers from which the filament self assembles . Using high-resolution electron microscopy and helical image reconstruction methods, we calculated three-dimensional density maps of the complex filament of Rhizobium lupini H13-3 and determined its surface pattern and boundaries . The helical symmetry of the filament allows viewing it as a stack of identical slices spaced axially and rotated by constant increments . Here we use the closed outlines of these slices to explore, in two dimensions, the hydrodynamic effect of the turbine-like boundaries of the flagellar filament . In particular, we try to determine if, and under what conditions, transitions from laminar to turbulent flow (or perturbations of the laminar flow) may occur on or near the surface of the bacterial propeller . To address these questions, we apply the boundary element method in a manner allowing the handling of convoluted boundaries . We tested the method on several simple, well-characterized cylindrical structures before applying it to real, highly convoluted biological surfaces and to simplified mechanical analogs . Our results indicate that under extreme structural and functional conditions, and at low Reynolds numbers, a deviation from laminar flow might occur on the flagellar surface . These transitions, and the conditions enabling them, may affect flagellar polymorphism and the formation and dispersion of flagellar bundles-factors important in the chemotactic response.

Adv Biochem Eng Biotechnol, 2003, 84, 49 - 89
Rhizobacterial diversity in India and its influence on soil and plant health; Johri BN et al.; The rhizosphere or the zone of influence around roots harbors a multitude of microorganisms that are affected by both abiotic and biotic stresses . Among these are the dominant rhizobacteria that prefer living in close vicinity to the root or on its surface and play a crucial role in soil health and plant growth . Both free-living and symbiotic bacteria are involved in such specific ecological niches and help in plant matter degradation, nutrient mobilization and biocontrol of plant disease . While the rhizosphere as a domain of fierce microbial activity has been studied for over a century, the availability of modern tools in microbial ecology has now permitted the study of microbial communities associated with plant growth and development, in situ localization of important forms, as well as the monitoring of introduced bacteria as they spread in the soil and root environment . This interest is linked to environmental concerns for reduced use of chemicals for disease control as well as an appreciation for utilization of biologicals and organics in agriculture . Indian researchers have studied the diversity of rhizobacteria in a variety of plants, cereals, legumes and others along with assessment of their functionality based on the release of enzymes (soil dehydrogenase, phosphatase, nitrogenase, etc.), metabolites (siderophores, antifungals, HCN, etc.), growth promoters (IAA, ethylene) and as inducers of systemic disease resistance (ISR) . Based on such primary screening protocols, effective rhizobacteria have been field tested with success stories from various agroecological zones of the country, as reflected in the control of root- and soil-borne diseases, improved soil health and increased crop yields . Several commercial formulations, mostly based on dry powder (charcoal, lignite, farmyard manure, etc.) have been prepared and field tested, however, problems of appropriate shelf-life and cell viability are still to be solved . Also, inherent in such low cost technologies are the problems of variability in field performance and successful establishment of introduced inoculants in the root zone . In addition, most products available in the market are not properly monitored for quality before they reach the farmer . As a consequence, the acceptance of rhizobacterial formulations in the country is limited . However, several laboratories have now developed protocols for the rapid characterization of effective isolates based on molecular fingerprinting and other similar tools . Also, the use of molecular markers (gus, lux, gfp, etc.) makes it easy to monitor introduced inoculants in situ in soil and rhizosphere environments . The government initiative in integrated nutrient management and pest management systems has provided additional incentives to relate rhizobacterial science to other ongoing activities so that the benefit of this research leads to technologies that are environmentally and socially acceptable.

Nucleic Acids Res, 2003 Sep 1, 31(17), 5003 - 15
Structural motifs in the RNA encoded by the early nodulation gene enod40 of soybean; Girard G et al.; The plant gene enod40 is highly conserved among legumes and also present in various non-legume species . It is presumed to play a central regulatory role in the Rhizobium-legume interaction, being expressed well before the initiation of cortical cell divisions resulting in nodule formation . Two small peptides encoded by enod40 mRNA as well as its secondary structure have been shown to be key elements in the signalling processes underlying nodule organogenesis . Here results concerning the secondary structure of mRNA of enod40 in soybean are presented . This study combined a theoretical approach, involving structure prediction and comparison, as well as structure probing . Our study indicates five conserved domains in enod40 mRNA among numerous leguminous species . Structure comparison suggests that some domains are also conserved in non-leguminous species and that an additional domain exists that was found only in leguminous species developing indeterminate nodules . Enzymatic and chemical probing data support the structure for three of the domains, and partially for the remaining two . The rest of the molecule appears to be less structured . Some of the domains include motifs, such as U-containing internal loops and bulges, which seem to be conserved . Therefore, they might be involved in the regulatory role of enod40 RNA.

J Mol Biol, 2003 Aug 29, 331(5), 1093 - 108
The axial alpha-helices and radial spokes in the core of the cryo-negatively stained complex flagellar filament of Pseudomonas rhodos: recovering high-resolution details from a flexible helical assembly; Cohen-Krausz S et al.; Of the two known "complex" flagellar filaments, those of Pseudomonas are far more flexible than those of Rhizobium . Their diameter is larger and their outer three-start ridges and grooves are more prominent . Although the symmetry of both complex filaments is similar, the polymer's linear mass density and the flagellin molecular mass of the latter are lower . A recent comparison of a three-dimensional reconstruction of the filament of Pseudomonas rhodos to that of Rhizobium lupini indicates that the outer flagellin domain (D3) is missing in R.lupini . Here, we concentrate on the structure of the inner core of the filament of P.rhodos using field emission cryo-negative staining electron microscopy and a hybrid helical/single particle reconstruction technique . Averaging 158 filaments caused the density band corresponding to the radial spokes to nearly average out due to their variability and inferred flexibility . Treating the Z=0 cross-sections through the aligned individual three-dimensional density maps as images, classifying them by correspondence analysis (using a mask containing the radial spokes domain) and re-averaging the subclasses (using helical reconstruction techniques) allowed a recovery of the radial spokes and resolved the alpha-helices in domain D0 and the triple alpha-helical bundles in domain D1 at a resolution of 1/7A(-1) . Although the perturbed components of the helical lattice are present along the entire filament's radius, the interior of the complex filament is similar to that of the plain one, whereas it's exterior is altered . Reconstructions of vitrified and cryo-negatively stained plain, right-handed filaments of Salmonella typhimurium SJW1655 prepared and imaged under conditions identical with those used for P.rhodos confirm the similarity of their inner cores and that the secondary structures in the interior of the flagellar filament can, under critical conditions of image recording and correction, be resolved in negative stain.

Plant Physiol, 2003 Aug, 132(4), 1982 - 8
Nod factor-induced root hair curling: continuous polar growth towards the point of nod factor application; Esseling JJ et al.; A critical step in establishing a successful nitrogen-fixing symbiosis between rhizobia and legume plants is the entrapment of the bacteria between root hair cell walls, usually in characteristic 180 degrees to 360 degrees curls, shepherd's crooks, which are formed by the host's root hairs . Purified bacterial signal molecules, the nodulation factors (NFs), which are lipochitooligosaccharides, induce root hair deformation in the appropriate host legume and have been proposed to be a key player in eliciting root hair curling . However, for curling to occur, the presence of intact bacteria is thought to be essential . Here, we show that, when spot applied to one side of the growing Medicago truncatula root hair tip, purified NF alone is sufficient to induce reorientation of the root hair growth direction, or a full curl . Using wild-type M . truncatula containing the pMtENOD11::GUS construct, we demonstrate that MtENOD11::GUS is expressed after spot application . The data have been incorporated into a cell biological model, which explains the formation of shepherd's crook curls around NF-secreting rhizobia by continuous tip growth reorientation.

J Appl Microbiol, 2003, 95(3), 484 - 91
Effect of trehalose on survival of Bradyrhizobium japonicum during desiccation; Streeter JG; AIMS: A major reason for the ineffectiveness of legume inoculants in the field is the rapid death of rhizobia because of desiccation . The major purpose of this study was to identify conditions under which alpha,alpha-trehalose would improve survival of Bradyrhizobium japonicum during desiccation . METHODS AND RESULTS: Trehalose was added to cultures just prior to desiccation or was supplied to bacteria during the 6-day growth period . A wide variety of trehalose concentrations was tested . Trehalose added to cultures at the time of desiccation improved survival slightly, but trehalose loading during growth was much more effective in protection against desiccation . Growth of bacteria with 3 mmol l-1 trehalose increased trehalose concentration in cells by about threefold and increased survival of cells placed on soya bean {Glycine max (L.) Merr.} seeds by two- to four-fold after 2 or 24 h . Average of overall results indicate that growth of bacteria with trehalose in the medium resulted in a 294% increase in survival after 24 h of desiccation . The concentration of trehalose in cells was very highly correlated with survival of bacteria . When trehalose-loaded cells were suspended in buffer or water, 60-85% of cellular trehalose was lost in about 1 h and, in spite of these losses, survival during desiccation was not reduced . CONCLUSIONS: Accumulation of trehalose in the cytoplasm is critical to the survival of B . japonicum during desiccation . Increasing the periplasmic concentration of trehalose is also beneficial but is not so critical as the concentration of trehalose in the cytoplasm . Because B . japonicum cannot utilize trehalose as a carbon source, cells can be loaded with trehalose by providing the disaccharide during the growth period . SIGNIFICANCE AND IMPACT OF THE STUDY: Although it may not be practical to use trehalose as a carbon source in inoculant production, it may be possible to engineer greater trehalose accumulation in rhizobia . Trehalose concentration in cells should be a useful predictor of survival during desiccation.

Appl Environ Microbiol, 2003 Aug, 69(8), 4396 - 402
Rhizobium leguminosarum biovar viciae 1-aminocyclopropane-1-carboxylate deaminase promotes nodulation of pea plants; Ma W et al.; Ethylene inhibits nodulation in various legumes . In order to investigate strategies employed by Rhizobium to regulate nodulation, the 1-aminocyclopropane-1-carboxylate (ACC) deaminase gene was isolated and characterized from one of the ACC deaminase-producing rhizobia, Rhizobium leguminosarum bv . viciae 128C53K . ACC deaminase degrades ACC, the immediate precursor of ethylene in higher plants . Through the action of this enzyme, ACC deaminase-containing bacteria can reduce ethylene biosynthesis in plants . Insertion mutants with mutations in the rhizobial ACC deaminase gene (acdS) and its regulatory gene, a leucine-responsive regulatory protein-like gene (lrpL), were constructed and tested to determine their abilities to nodulate Pisum sativum L . cv . Sparkle (pea) . Both mutants, neither of which synthesized ACC deaminase, showed decreased nodulation efficiency compared to that of the parental strain . Our results suggest that ACC deaminase in R . leguminosarum bv . viciae 128C53K enhances the nodulation of P . sativum L . cv . Sparkle, likely by modulating ethylene levels in the plant roots during the early stages of nodule development . ACC deaminase might be the second described strategy utilized by Rhizobium to promote nodulation by adjusting ethylene levels in legumes.

Arch Microbiol, 2003 Nov, 180(5), 309 - 18 Epub 2003 Aug 01.
Poly beta-hydroxybutyrate depolymerase (PhaZ) in Azospirillum brasilense and characterization of a phaZ mutant; Kadouri D et al.; Like many other prokaryotes, rhizobacteria of the genus Azospirillum produce high levels of poly-beta-hydroxybutyrate (PHB) under sub-optimal growth conditions . Utilization of PHB by bacteria under stress has been proposed as a mechanism that favors their compatible establishment in competitive environments . PHB depolymerase (PhaZ) is an essential enzyme in PHB degradation . The phaZ gene was identified in Azospirillum brasilense, cloned, sequenced, and shown to be located on the chromosome . Insertion of a kanamycin-resistant cassette within phaZ of A . brasilense resulted in a phaZ mutant that was unable to degrade PHB; however, carbon source utilization was similar in both the wild-type and the mutant strain . The ability of the wild-type to endure starvation conditions, ultraviolet irradiation, heat, and osmotic shock, and to grow in the presence of hydrogen peroxide was higher than that of the mutant strain . By contrast, the ability of the phaZ mutant strain to endure desiccation was higher than that of the wild-type strain . No differences between the strains were seen in their ability to endure sonication, or to survive in carrier materials used for soil inoculants . In addition, motility was the same between the two strains, whereas cell aggregation and exopolysaccharide production were higher in the wild-type than in the phaZ mutant strain.

Can J Microbiol, 2003 Apr, 49(4), 237 - 43
Comparative analysis of the Bradyrhizobium japonicum sucA region; Green LS et al.; To study the adjustments made to the tricarboxylic acid cycle during symbiosis of nitrogen-fixing rhizobia with their host legumes, we have characterized the genes encoding the alpha-ketoglutarate dehydrogenase enzyme complex in Bradyrhizobium japonicum . The genes were arranged in the order sucA-sucB-scdA-lpdA, where scdArepresents a short-chain dehydrogenase gene (GenBank accession No . AY049030) . All four genes appeared to be co-transcribed, an arrangement that is so far unique to B . japonicum . The mdh gene, encoding malate dehydrogenase, was located upstream of the sucA operon, and its primary transcript appeared to be monocistronic . Primer extension indicated that the sucA operon and mdh were transcribed from typical housekeeping promoters.

Res Microbiol, 2003 Jul-Aug, 154(6), 433 - 42
Exopolysaccharide synthesis in Rhizobium leguminosarum bv . trifolii is related to various metabolic pathways; Janczarek M et al.; Rhizobium leguminosarum bv . trifolii synthesizes extracellular polysaccharide (EPS) that is postulated to be a biologically active signalling molecule in clover symbiosis . A group of seven exopolysaccharide-deficient (Exo), non-nitrogen-fixing mutants of R . leguminosarum bv . trifolii strain 24.1 isolated by transposon mutagenesis were complemented to mucoid phenotype by a low-copy plasmid carrying the pssA gene encoding the first glucosyl-IP-transferase . Some of these mutants were not corrected in their symbiotic defect by the pssA gene . Precise localization of Tn5 insertion sites by subcloning and sequencing the adjacent genomic DNA in the Exo mutants identified the disrupted genes and their possible functions . Only one mutant (Rt74) was mutated in pssA gene; others were mutated in diverse genes that were not directly involved in EPS biosynthesis . The suppression of EPS deficiency in these mutants by additional copies of pssA indicated a possible connection between exopolysaccharide biosynthesis and various metabolic pathways.

Int J Syst Evol Microbiol, 2003 Jul, 53(Pt 4), 1207 - 17
Description of new Ensifer strains from nodules and proposal to transfer Ensifer adhaerens Casida 1982 to Sinorhizobium as Sinorhizobium adhaerens comb . nov . Request for an opinion; Willems A et al.; A group of four diverse rhizobial isolates and two soil isolates that are highly related to Ensifer adhaerens were characterized by a polyphasic approach . On the basis of DNA-DNA hybridizations and phenotypic features, these strains cannot be distinguished clearly form Ensifer adhaerens, a soil bacterium that was described in 1982, mainly on the basis of phenotypic characteristics . Phylogenetically, Ensifer and Sinorhizobium form a single group in the 16S rDNA dendrogram of the alpha-Proteobacteria, as well as in an analysis of partial recA gene sequences . They may therefore be regarded as a single genus . Because Sinorhizobium was proposed in 1988, according to the Bacteriological Code (1990 Revision) the older name, Ensifer, has priority . However, there are several reasons why a change from Sinorhizobium to Ensifer may not be the best solution and making an exception to Rule 38 may be more appropriate . We therefore propose the species Sinorhizobium adhaerens comb . nov . and put forward a Request for an Opinion to the Judicial Commission regarding the conservation of Sinorhizobium adhaerens over Ensifer adhaerens.

Biotechnol Lett, 2003 Jan, 25(2), 115 - 9
Enhanced biosynthesis of polyhydroxyalkanoates in a mutant strain of Rhizobium meliloti; Lakshman K et al.; Strains of Rhizobium spp . isolated from leguminous plants and standard strains accumulated 27% to 57% polyhydroxyalkanoate (PHA) of their cell biomass . Among these cultures, one strain of Rhizobium meliloti synthesized 10-30% more PHA than others and contained 3% hydroxyvalerate (HV) when grown on sucrose as carbon substrate . The occurrence of hydroxybutyrate (HB) and HV was confirmed by GC and 1H NMR analysis . Treatment of the culture with 4'-N-piperidinobutyl-2-chlorophenoxazine resulted in a mutant which synthesized up to 69%, PHA of the cell biomass with an improved yield of 11 to 47% under different carbon and nitrogen ratios, compared to the parent strain.

Ann Bot (Lond), 2003 Aug, 92(2), 247 - 58
Morphological compatibility of white clover and perennial ryegrass cultivars grown under two nitrate levels in flowing solution culture; Collins RP et al.; The effects of nitrate (NO3-) supply on shoot morphology, vertical distribution of shoot and root biomass and total nitrogen (N) acquisition by two perennial ryegrass (Lolium perenne L.) cultivars (AberElan and Preference) and two white clover (Trifolium repens L.) cultivars (Grasslands Huia and AberHerald) were studied in flowing nutrient culture . Cultivars were grown from seed as monocultures and the clovers inoculated with Rhizobium . The 6-week measurement period began on day 34 (grasses) and day 56 (clovers) when the NO3- supply was adjusted to either 2 mmol m-3 (low nitrogen, LN) or 50 mmol m-3 (high nitrogen, HN) . These treatments were subsequently maintained automatically . Plants were harvested at intervals to measure their morphology and N content . Cultivars of both species differed significantly in several aspects of their response to NO3- supply . In the grasses, the LN treatment increased the root : shoot ratio of AberElan but did not affect the distribution of root length in the root profile . In contrast, this treatment changed the root distribution of Preference compared with HN, resulting in a larger proportion of root length being distributed further down the root profile . The morphology of white clover Grasslands Huia was for the most part unaffected by the level of NO3- supply . In contrast, AberHerald exhibited different growth strategies, with LN plants increasing their stolon weight per unit length at the expense of leaf production, leaf area and stolon length, whereas HN plants showed reduced stolon thickness, greater leaf area production and stolon length per plant . Cultivars with different morphological/physiological strategies in response to NO3- supply may be of value in the construction of 'compatible mixtures' aimed at reducing oscillations in sward clover content by extending the range of conditions that allow balanced coexistence of species to occur.

J Biol Chem, 2003 Oct 10, 278(41), 39269 - 79 Epub 2003 Jul 16.
Expression cloning and biochemical characterization of a Rhizobium leguminosarum lipid A 1-phosphatase; Karbarz MJ et al.; Lipid A of Rhizobium leguminosarum, a nitrogen-fixing plant endosymbiont, displays several significant structural differences when compared with Escherichia coli . An especially striking feature of R . leguminosarum lipid A is that it lacks both the 1- and 4'-phosphate groups . Distinct lipid A phosphatases that attack either the 1 or the 4' positions have previously been identified in extracts of R . leguminosarum and Rhizobium etli but not Sinorhizobium meliloti or E . coli . Here we describe the identification of a hybrid cosmid (pMJK-1) containing a 25-kb R . leguminosarum 3841 DNA insert that directs the overexpression of the lipid A 1-phosphatase . Transfer of pMJK-1 into S . meliloti 1021 results in heterologous expression of 1-phosphatase activity, which is normally absent in extracts of strain 1021, and confers resistance to polymyxin . Sequencing of a 7-kb DNA fragment derived from the insert of pMJK-1 revealed the presence of a lipid phosphatase ortholog (designated LpxE) . Expression of lpxE in E . coli behind the T7lac promoter results in the appearance of robust 1-phosphatase activity, which is normally absent in E . coli membranes . Matrix-assisted laser-desorption/time of flight and radiochemical analysis of the product generated in vitro from the model substrate lipid IVA confirms the selective removal of the 1-phosphate group . These findings show that lpxE is the structural gene for the 1-phosphatase . The availability of lpxE may facilitate the re-engineering of lipid A structures in diverse Gram-negative bacteria and allow assessment of the role of the 1-phosphatase in R . leguminosarum symbiosis with plants . Possible orthologs of LpxE are present in some intracellular human pathogens, including Francisella tularensis, Brucella melitensis, and Legionella pneumophila.

J Bacteriol, 2003 Aug, 185(15), 4382 - 92
immX immunity region of rhizobium phage 16-3: two overlapping cistrons of repressor function; Csiszovszki Z et al.; 16-3 is a temperate phage of the symbiotic nitrogen-fixing bacterium Rhizobium meliloti 41 . Its prophage state and immunity against superinfection by homoimmune phages are governed by a complex set of controls: the immC and immX repressor systems and the avirT element are all located in well-separated, distinct regions which span 25 kb on the bacteriophage chromosome . The anatomy and function of the immC region are well documented; however, fewer analyses have addressed the immX and avirT regions . We focused in this paper on the immX region and dissected it into two major parts: X(U/L) and X(V) . The X(U/L) part (0.6 kb) contained two overlapping cistrons, X(U) and X(L), coding for proteins pXU and pXL, respectively . Inactivation of either gene inactivated the repressor function of the immX region . Loss-of-function mutants of X(U) and X(L) complemented each other in trans in double lysogens . The X(V) part (1 kb) contained a target for X(U/L) repressor action . Mutations at three sites in X(V) led to various degree of ImmX insensitivity in a hierarchic manner . Two sites (X(V1) and X(V3)) exhibited the inverted-repeat structures characteristic of many repressor binding sites . However, X(V1) could also be folded into a transcription terminator . Of the two immunity regions of 16-3, immX seems to be unique both in its complex genetic anatomy and in its sequence . To date, no DNA or peptide sequence homologous to that of ImmX has been found in the data banks . In contrast, immC shares properties of a number of immunity systems commonly found in temperate phages.

Plant Mol Biol, 2003 May, 52(2), 303 - 16
The Lotus japonicus ndx gene family is involved in nodule function and maintenance; Gronlund M et al.; To elucidate the function of the ndx homeobox genes during the Rhizobium-legume symbiosis, two Lotus japonicus ndr genes were expressed in the antisense orientation under the control of the nodule-expressed promoter Psenod12 in transgenic Lotus japonicus plants . Many of the transformants obtained segregated into plants that failed to sustain proper development and maintenance of root nodules concomitant with down-regulation of the two ndx genes . The root nodules were actively fixing nitrogen 3 weeks after inoculation, but the plants exhibited a stunted growth phenotype . The nodules on such antisense plants had under-developed vasculature and lenticels when grown on medium lacking nitrogen sources . These nodules furthermore entered senescence earlier than the wild-type nodules . Normal plant growth was resumed upon external addition of nitrogen . This suggests that assimilated nitrogen is not properly supplied to the plants in which the two ndx genes are down-regulated . The results presented here, indicate that the ndx genes play a role in the development of structural nodule features, required for proper gas diffusion into the nodule and/or transport of the assimilated nitrogen to the plant.

Mol Plant Microbe Interact, 2003 Jul, 16(7), 617 - 25
Extracellular proteins involved in soybean cultivar-specific nodulation are associated with pilus-like surface appendages and exported by a type III protein secretion system in Sinorhizobium fredii USDA257; Krishnan HB et al.; Several gram-negative plant and animal pathogenic bacteria have evolved a type III secretion system (TTSS) to deliver effector proteins directly into the host cell cytosol . Sinorhizobium fredii USDA257, a symbiont of soybean and many other legumes, secretes proteins called Nops (nodulation outer proteins) into the extracellular environment upon flavonoid induction . Mutation analysis and the nucleotide sequence of a 31.2-kb symbiosis (sym) plasmid DNA region of USDA257 revealed the existence of a TTSS locus in this symbiotic bacterium . This locus includes rhc (rhizobia conserved) genes that encode components of a TTSS and proteins that are secreted into the environment (Nops) . The genomic organization of the TTSS locus of USDA257 is remarkably similar to that of another broad-host range symbiont, Rhizobium sp . strain NGR234 . Flavonoids that activate the transcription of the nod genes of USDA257 also stimulate the production of novel filamentous appendages known as pili . Electron microscope examination of isolated pili reveals needle-like filaments of 6 to 8 nm in diameter . The production of the pili is dependent on a functional nodD1 and the presence of a nod gene-inducing compound . Mutations in several of the TTSS genes negate the ability of USDA257 to elaborate pili . Western blot analysis using antibodies raised against purified NopX, Nop38, and Nop7 reveals that these proteins were associated with the pili . Mutations in rhcN, rhcJ, rhcC, and ttsI alter the ability of USDA257 to form nodules on Glycine max and Macroptilium atropurpureum.

Pest Manag Sci, 2003 Jun-Jul, 59(6-7), 665 - 70
United States Department of Agriculture-Agricultural Research Service research programs on microbes for management of plant-parasitic nematodes; Meyer SL; Restrictions on the use of conventional nematicides have increased the need for new methods of managing plant-parasitic nematodes . Consequently, nematode-antagonistic microbes, and active compounds produced by such organisms, are being explored as potential additions to management practices . Programs in this area at the USDA Agricultural Research Service investigate applied biocontrol agents, naturally occurring beneficial soil microbes and natural compounds . Specific research topics include use of plant growth-promoting rhizobacteria and cultural practices for management of root-knot and ring nematodes, determination of management strategies that enhance activity of naturally occurring Pasteuria species (bacterial obligate parasites of nematodes), studies on interactions between biocontrol bacteria and bacterial-feeding nematodes, and screening of microbes for compounds active against plant-parasitic nematodes . Some studies involve biocontrol agents that are active against nematodes and soil-borne plant-pathogenic fungi, or combinations of beneficial bacteria and fungi, to manage a spectrum of plant diseases or to increase efficacy over a broader range of environmental conditions . Effective methods or agents identified in the research programs are investigated as additions to existing management systems for plant-parasitic nematodes.

Biosci Biotechnol Biochem, 2003 May, 67(5), 1144 - 8
Rhizoremediation of dioxin-like compounds by a recombinant Rhizobium tropici strain expressing carbazole 1,9a-dioxygenase constitutively; Saiki Y et al.; A recombinant Rhizobium strain, PBK3-IS, that constitutively expressed the oxygenase component of carbazole 1,9a-dioxygenase from Sphingomonas sp . strain KA1, was constructed . In the water-cultured siratro rhizospheres inoculated with strain PBK3-IS, 48% of the dibenzofuran was removed within 3 days (initial substrate, 25 microg) . Similar results were obtained in soil-cultured siratro rhizospheres using sterile vermiculite . When non-sterile field soils were used instead of sterile vermiculite, the inoculated recombinant strain could grow on the siratro root in all soils tested, except for wet paddy field.

Proteomics, 2003 Jun, 3(6), 1077 - 85
Proteome analysis of aerobic and fermentative metabolism in Rhizobium etli CE3; Encarnacion S et al.; Rhizobium etli undergoes a transition from an aerobic to a fermentative metabolism during successive subcultures in minimal medium . This metabolic transition does not occur in cells subcultured in rich medium, or in minimal medium containing either biotin or thiamine . In this report, we characterize the aerobic and fermentative metabolism of R . etli using proteome analysis . According to their synthesis patterns in response to aerobic (rich medium, minimal medium with biotin or minimal medium with thiamine) or fermentative (minimal medium without supplements) growth conditions, proteins were assigned to five different classes: (i) proteins produced only in aerobic conditions (e.g., catalase-peroxidase KatG and the E2 component of pyruvate dehydrogenase); (ii) protein produced under both conditions but strongly induced in aerobic metabolism (e.g., malate dehydrogenase and the succinyl-CoA synthetase beta subunit); (iii) proteins that were induced equally under all conditions tested (e.g., AniA, DnaK, and GroEL); (iv) proteins downregulated during aerobic metabolism, and (v) proteins specific to only one of the conditions analyzed . Northern blotting studies of katG expression confirmed the proteome data for this protein . The negative regulation of carbon metabolism proteins observed in fermentative metabolism is consistent with the drastic physiological changes which occur during this process.

FEMS Microbiol Lett, 2003 Jun 27, 223(2), 239 - 44
The ECF sigma factor RpoI of R . leguminosarum initiates transcription of the vbsGSO and vbsADL siderophore biosynthetic genes in vitro; Yeoman KH et al.; When complexed with Escherichia coli RNA polymerase core enzyme, purified RpoI protein of Rhizobium leguminosarum initiated transcription in vitro from promoters of the vbsADL and vbsGSO operons, which are needed to synthesise the siderophore vicibactin . There is a single transcription initiation site for rpoI, regardless of whether the cells are grown in Fe-replete or Fe-depleted media, but levels of rpoI mRNA were reduced, though not abolished, in the presence of Fe . Unlike PvdS, a similar Pseudomonas sigma factor needed to transcribe genes involved in pyoverdine synthesis, RpoI transcribes vbsADL and vbsGSO in the absence of the cognate siderophore . The RpoI sigma factor is not required for transcription of rpoI.

Ying Yong Sheng Tai Xue Bao, 2003 Feb, 14(2), 187 - 90
{Role of Rhizobium in wheat-astragalus mixed cropping system}; Zhong Z et al.; Inoculation experiments were carried out in wheat-astragalus mixed cropping system to study the changes of plant biomass, nitrogen content in plants and soil, and enzyme activity of Rhizobium . The results showed that in the mixed cropping system inoculated with Rhizobium, the growth of plants was stimulated, total nitrogen of plants and soil was increased significantly, and the increment of enzyme activity of Rhizobium was also found.

Arch Microbiol, 2003 Aug, 180(2), 118 - 26 Epub 2003 Jun 19.
IS Rm31, a new insertion sequence of the IS 66 family in Sinorhizobium meliloti; Biondi EG et al.; Sinorhizobium meliloti natural populations show a high level of genetic polymorphism possibly due to the presence of mobile genetic elements such as insertion sequences (IS), transposons, and bacterial mobile introns . The analysis of the DNA sequence polymorphism of the nod region of S . meliloti p SymA megaplasmid in an Italian isolate led to the discovery of a new insertion sequence, IS Rm31 . IS Rm31 is 2,803 bp long and has 22-bp-long terminal inverted repeat sequences, 8-bp direct repeat sequences generated by transposition, and three ORFs (A, B, C) coding for proteins of 124, 115, and 541 amino acids, respectively . ORF A and ORF C are significantly similar to members of the transposase family . Amino acid and nucleotide sequences indicate that IS Rm31 is a member of the IS 66 family . IS Rm31 sequences were found in 30.5% of the Italian strains analyzed, and were also present in several collection strains of the Rhizobiaceae family, including S . meliloti strain 1021 . Alignment of targets sites in the genome of strains carrying IS Rm31 suggested that IS Rm31 inserts randomly into S . meliloti genomes . Moreover, analysis of IS Rm31 insertion sites revealed DNA sequences not present in the recently sequenced S . meliloti strain 1021 genome . In fact, IS Rm31 was in some cases linked to DNA fragments homologous to sequences found in other rhizobia species.

ScientificWorldJournal, 2001 Nov 10, 1 Suppl 2, 17 - 21
Symbiotic performance of herbaceous legumes in tropical cover cropping systems; Ibewiro B et al.; Increasing use of herbaceous legumes such as mucuna ( Mucuna pruriens var . utilis {Wright} Bruck) and lablab ( Lablab purpureus {L.} Sweet) in the derived savannas of West Africa can be attributed to their potential to fix atmospheric nitrogen (N2) . The effects of management practices on N2 fixation in mucuna and lablab were examined using 15N isotope dilution technique . Dry matter yield of both legumes at 12 weeks was two to five times more in in situ mulch (IM) than live mulch (LM) systems . Land Equivalent Ratios, however, showed 8 to 30% more efficient utilization of resources required for biomass production under LM than IM systems . Live mulching reduced nodule numbers in the legumes by one third compared to values in the IM systems . Similarly, nodule mass was reduced by 34 to 58% under LM compared to the IM systems . The proportion of fixed N2 in the legumes was 18% higher in LM than IM systems . Except for inoculated mucuna, the amounts of N fixed by both legumes were greater in IM than LM systems . Rhizobia inoculation of the legumes did not significantly increase N2 fixation compared to uninoculated plots . Application of N fertilizer reduced N2 fixed in the legumes by 36 to 51% compared to inoculated or uninoculated systems . The implications of cover cropping, N fertilization, and rhizobia inoculation on N contributions of legumes into tropical low-input systems were discussed.

ScientificWorldJournal, 2001 Dec 11, 1 Suppl 2, 722 - 7
Economy of fertilizer nitrogen through organic sources in rain-fed rice-legume cropping systems in West Bengal, India; Puste AM et al.; Field experiments were conducted at a farmers" plot adjacent to the Regional Research Station, red and laterite zone, Sub-center Sekhampur (Birbhum district) of West Bengal, India, situated 23 degrees 24' N latitude, 87 degrees 24' E longitude, to study the effect of different bio- and organic sources of nutrients instead of total fertilizer N in terms of crop productivity in the sequence and building up of soil fertility . During the wet seasons of 1997 and 1998, 12 combinations of bio- and organic sources (crop residues, well decomposed cow dung, dhanicha as green manure) were substituted for 25-50% of N fertilizer applied on transplanted rice (Cv . IR 36) . Subsequently, during the winters of 1997-1998 and 1998-1999, leguminous pulse crops like lentil (Lens culinaris {L.} Medic.), gram ( Cicer arietinum L.) and lathyrus (Lathyrus sativus L.) were grown with and without inoculation of Rhizobium . Results revealed that the application of inorganic N in combination with organic sources exhibited a significant increase in rice yield (3.60-3.84 t ha(-1) ) compared to the yield from sole application of N (3.19-3.26 t ha(-1) ) . The study showed that about 25% of total applied N was saved without significant yield reduction with simultaneous improvement of soil physical properties (pH, organic matter, available N, P, K, and CEC) . Seed yield of pulses (lentil, gram, and lathyrus) were more pronounced in the treatment inoculated with Rhizobium, with a saving of 42.6-48.4 kg N ha(-1) . Therefore, the results suggest that the combined application of inorganic and organic N sources in a 75:25 ratio is a superior N-management practice with regards to crop yields as well as improvement of soil fertility.

Arch Microbiol, 2003 Jul, 180(1), 45 - 52 Epub 2003 Jun 07.
Genetic diversity of fast-growing rhizobia that nodulate soybean ( Glycine max L . Merr); Saldana G et al.; The fast-growing Rhizobium sp . strain NGR234, isolated from Papua New Guinea, and 13 strains of Sinorhizobium fredii, isolated from China and Vietnam, were fingerprinted by means of RAPD, REP, ERIC and ARDRA . ERIC, REP and RAPD markers revealed a considerable genetic diversity among fast-growing rhizobia . Chinese isolates showed higher levels of diversity than those strains isolated from Vietnam . ARDRA analysis revealed three different genotypes among fast-growing rhizobia that nodulate soybean, even though all belonged to a subcluster that included Sinorhizobium saheli and Sinorhizobium meliloti . Among S . fredii rhizobia, two strains, SMH13 and HH303, might be representatives of other species of nitrogen-fixing organisms . Although restriction analysis of the nifD- nifK intergenic DNA fragment confirmed the unique nature of Rhizobium sp . strain NGR234, several similarities between Rhizobium sp . strain NGR234 and S . fredii USDA257, the ARDRA analysis and the full sequence of the 16S rDNA confirmed that NGR234 is a S . fredii strain . In addition, ARDRA analysis and the full sequence of the 16S rDNA suggested that two strains of rhizobia might be representatives of other species of rhizobia.

Genome Biol . 2003;4(6):R39 . Epub 2003 May 19.
Horizontally transferred genes in plant-parasitic nematodes: a high-throughput genomic approach; Scholl EH et al.; BACKGROUND: Published accounts of horizontally acquired genes in plant-parasitic nematodes have not been the result of a specific search for gene transfer per se, but rather have emerged from characterization of individual genes . We present a method for a high-throughput genome screen for horizontally acquired genes, illustrated using expressed sequence tag (EST) data from three species of root-knot nematode, Meloidogyne species . RESULTS: Our approach identified the previously postulated horizontally transferred genes and revealed six new candidates . Screening was partially dependent on sequence quality, with more candidates identified from clustered sequences than from raw EST data . Computational and experimental methods verified the horizontal gene transfer candidates as bona fide nematode genes . Phylogenetic analysis implicated rhizobial ancestors as donors of horizontally acquired genes in Meloidogyne . CONCLUSIONS: High-throughput genomic screening is an effective way to identify horizontal gene transfer candidates . Transferred genes that have undergone amelioration of nucleotide composition and codon bias have been identified using this approach . Analysis of these horizontally transferred gene candidates suggests a link between horizontally transferred genes in Meloidogyne and parasitism.

Genome Biol . 2003;4(6):R36 . Epub 2003 May 13.
The mosaic structure of the symbiotic plasmid of Rhizobium etli CFN42 and its relation to other symbiotic genome compartments; Gonzalez V et al.; BACKGROUND: Symbiotic bacteria known as rhizobia interact with the roots of legumes and induce the formation of nitrogen-fixing nodules . In rhizobia, essential genes for symbiosis are compartmentalized either in symbiotic plasmids or in chromosomal symbiotic islands . To understand the structure and evolution of the symbiotic genome compartments (SGCs), it is necessary to analyze their common genetic content and organization as well as to study their differences . To date, five SGCs belonging to distinct species of rhizobia have been entirely sequenced . We report the complete sequence of the symbiotic plasmid of Rhizobium etli CFN42, a microsymbiont of beans, and a comparison with other SGC sequences available . RESULTS: The symbiotic plasmid is a circular molecule of 371,255 base-pairs containing 359 coding sequences . Nodulation and nitrogen-fixation genes common to other rhizobia are clustered in a region of 125 kilobases . Numerous sequences related to mobile elements are scattered throughout . In some cases the mobile elements flank blocks of functionally related sequences, thereby suggesting a role in transposition . The plasmid contains 12 reiterated DNA families that are likely to participate in genomic rearrangements . Comparisons between this plasmid and complete rhizobial genomes and symbiotic compartments already sequenced show a general lack of synteny and colinearity, with the exception of some transcriptional units . There are only 20 symbiotic genes that are shared by all SGCs . CONCLUSIONS: Our data support the notion that the symbiotic compartments of rhizobia genomes are mosaic structures that have been frequently tailored by recombination, horizontal transfer and transposition.

Nucleic Acids Res, 2003 Jun 15, 31(12), 3143 - 56
Inactivation of the nod box distal half-site allows tetrameric NodD to activate nodA transcription in an inducer-independent manner; Feng J et al.; In Rhizobium leguminosarum, NodD can activate nodA transcription in response to inducer flavonoids . Here, we show that the inducible nodA promoter contains an intrinsic part through which NodD can activate nodA transcription in an inducer-independent manner . Evidence was provided that NodD binds to target DNA through anchoring the two half-sites of the nod box as a tetramer . An imperfect inverted repeat AT-N10-GAT was found in each half-site and is critical for NodD binding . Mutation of the inverted repeat of the nod box distal half-site allowed NodD to activate nodA transcription in an inducer-independent manner in vivo, and to modulate the DNA bending of the NodD-nod box complex in the absence of inducer in vitro.

Biotechnol Prog, 2003 May-Jun, 19(3), 714 - 9
Metabolite profiles and growth characteristics of Rhizobium meliloti cultivated at different specific growth rates; Ong LC et al.; Rhizobium meliloti (ATCC 55340) was grown at different specific growth rates in a chemostat apparatus . Metabolic products, relating to the Embden-Meyerhof-Parnas (EMP) pathway and the tricarboxylic acid (TCA) cycle, were measured and quantified to probe the influence of specific growth rate on the distribution of important metabolites . The detection of propionate in the fermentation broth implies that the imbalance of reducing equivalents of FADH(2) and NADH + H(+) resulted in a partially reductive operation of the TCA cycle . Additionally, experimental results show that the specific growth rate plays an essential role in modulating the biomass concentration, the specific substrate uptake rate, the cell length, the specific exopolysaccharide (EPS) production rate, the distribution of EPS molecular weight, and the profiles of carbohydrate and organic acid . The specific EPS production rate (varying from 13.3 to 111 mg EPS/g-DW/h) follows a growth-associated pattern at the specific growth rate ranging from 0.06 to 0.20 h(-1) and switches into non-growth-associated mode when the specific growth rate is over 0.20 h(-1).

Appl Environ Microbiol, 2003 Jun, 69(6), 3561 - 8
Citrate synthase mutants of Sinorhizobium fredii USDA257 form ineffective nodules with aberrant ultrastructure; Krishnan HB et al.; The tricarboxylic acid (TCA) cycle plays an important role in generating the energy required by bacteroids to fix atmospheric nitrogen . Citrate synthase is the first enzyme that controls the entry of carbon into the TCA cycle . We cloned and determined the nucleotide sequence of the gltA gene that encodes citrate synthase in Sinorhizobium fredii USDA257, a symbiont of soybeans (Glycine max {L.} Merr.) and several other legumes . The deduced citrate synthase protein has a molecular weight of 48,198 and exhibits sequence similarity to citrate synthases from several bacterial species, including Sinorhizobium meliloti and Rhizobium tropici . Southern blot analysis revealed that the fast-growing S . fredii strains and Rhizobium sp . strain NGR234 contained a single copy of the gene located in the bacterial chromosome . S . fredii USDA257 gltA mutant HBK-CS1, which had no detectable citrate synthase activity, had diminished nodulation capacity and produced ineffective nodules on soybean . Light and electron microscopy observations revealed that the nodules initiated by HBK-CS1 contained very few bacteroids . The infected cells contained large vacuoles and prominent starch grains . Within the vacuoles, membrane structures that appeared to be reminiscent of disintegrating bacteroids were detected . The citrate synthase mutant had altered cell surface characteristics and produced three times more exopolysaccarides than the wild type produced . A plasmid carrying the USDA257 gltA gene, when introduced into HBK-CS1, was able to restore all of the defects mentioned above . Our results demonstrate that a functional citrate synthase gene of S . fredii USDA257 is essential for efficient soybean nodulation and nitrogen fixation.

Appl Environ Microbiol, 2003 Jun, 69(6), 3244 - 50
Involvement of the reserve material poly-beta-hydroxybutyrate in Azospirillum brasilense stress endurance and root colonization; Kadouri D et al.; When grown under suboptimal conditions, rhizobacteria of the genus Azospirillum produce high levels of poly-beta-hydroxybutyrate (PHB) . Azospirillum brasilense strain Sp7 and a phbC (PHB synthase) mutant strain in which PHB production is impaired were evaluated for metabolic versatility, for the ability to endure various stress conditions, for survival in soil inoculants, and for the potential to promote plant growth . The carbon source utilization data were similar for the wild-type and mutant strains, but the generation time of the wild-type strain was shorter than that of the mutant strain with all carbon sources tested . The ability of the wild type to endure UV irradiation, heat, osmotic pressure, osmotic shock, and desiccation and to grow in the presence of hydrogen peroxide was greater than that of the mutant strain . The motility and cell aggregation of the mutant strain were greater than the motility and cell aggregation of the wild type . However, the wild type exhibited greater chemotactic responses towards attractants than the mutant strain exhibited . The wild-type strain exhibited better survival than the mutant strain in carrier materials used for soil inoculants, but no difference in the ability to promote plant growth was detected between the strains . In soil, the two strains colonized roots to the same extent . It appears that synthesis and utilization of PHB as a carbon and energy source by A . brasilense under stress conditions favor establishment of this bacterium and its survival in competitive environments . However, in A . brasilense, PHB production does not seem to provide an advantage in root colonization under the conditions tested.

Mol Microbiol, 2003 Jun, 48(5), 1305 - 16
The structure of the helically perturbed flagellar filament of Pseudomonas rhodos: implications for the absence of the outer domain in other complex flagellins and for the flexibility of the radial spokes; Cohen-Krausz S et al.; Bacterial flagella, the organelles of motility, are commonly divided into two classes: 'plain' and 'complex' . The complex filaments are pairwise, helically perturbed forms of the plain filaments and have been reported to occur only in Rhizobium and Pseudomonas . Previously, we reconstructed and analysed the structure of the complex filaments of Rhizobium lupini H13-3 and determined their unique symmetry and origin of the perturbations (Trachtenberg et al., 1986, J Mol Biol 190: 569-576; 1987, 195: 603-620; 1998, 276: 759-773; Cohen-Krausz and Trachtenberg, 1998, J Struct Biol 122: 267-282) . Here, we analyse the structure of the flagellar filament of the other known complex filament, that of Pseudomonas rhodos, as reconstructed from electron microscope images . Compared with the filament of R . lupini, the filament of P . rhodos is more flexible, as implied from high-intensity darkfield light microscopy and, although constructed from flagellins of higher molecular weights (59 versus 41 kDa), has similar symmetry . Using cryonegative stained specimens and low-dose, field emission electron microscopy, we reconstructed and averaged 158 filaments each containing 170 statistically significant layer lines . The three-dimensional density maps of P . rhodos clearly suggest, when compared with those of R . lupini and the right-handed Salmonella typhimurium SJW1655, that R . lupini is missing the outer flagellin domain (D3), that the interior of the complex filament is rather similar to that of the plain filament and that the radial spokes (connecting domains D0 and D1), present in individual density maps, average out because of their variability and implied flexibility . Extending the three-start grooves and ridges on the propeller's surface, in the form of an Archimedean screw, may further improve the motility of the cell in viscous environments.

Mol Microbiol, 2003 Jun, 48(5), 1195 - 207
The twin-arginine translocation (Tat) system is essential for Rhizobium-legume symbiosis; Meloni S et al.; The Tat (twin-arginine translocation) system mediates export of periplasmic proteins in folded conformation . Proteins transported via Tat contain a characteristic twin-arginine motif in their signal peptide . Genetic determinants (tatABC genes) of the Tat system from Rhizobium leguminosarum bv . viciae were cloned and characterized, and a tatBC deletion mutant was constructed . The mutant lacked the ability for membrane targeting of hydrogenase, a known Tat substrate, and was impaired in hydrogenase activity . Interestingly, in the absence of a functional Tat system, only small, white nodules unable to fix nitrogen were induced in symbiosis with pea plants . Analysis of nodule structure and location of green fluorescent protein (GFP)-tagged bacteria within nodules indicated that the symbiotic process was blocked in the tat mutant at a stage previous to bacteria release into cortical cells . The R . leguminosarum Tat-deficient mutant lacked a functional cytochrome bc1 complex . This was consistent with the fact that R . leguminosarum Rieske protein, a key component of the symbiosis-essential cytochrome bc1 complex, contained a typical twin-arginine signal peptide . However, comparative analyses of nodule structure indicated that nodule development in the tat mutant was arrested at an earlier step than in a cytochrome bc1 mutant . These data indicate that the Tat pathway is also critical for proteins relevant to the initial stages of the symbiotic process.

J Am Chem Soc, 2003 May 21, 125(20), 6103 - 12
Synthesis and biological evaluation of Rhizobium sin-1 lipid A derivatives; Demchenko AV et al.; A highly convergent strategy for the synthesis of several derivatives of the lipid A of Rhizobium sin-1 has been developed . The approach employed the advanced intermediate 3-O-acetyl-6-O-(3-O-acetyl-4,6-O-benzylidene-2-deoxy-2-phthalimido-beta-d-glucopyrano-syl)-2-azido-4-O-benzyl-2-deoxy-1-thio-alpha-d-glucopyranoside (5), which is protected in such a way that the anomeric center, the C-2 and C-2' amino groups, and the C-3 and C-3' hydroxyls can be selectively functionalized . The synthetic strategy was used for the preparation of 2-deoxy-6-O-{2-deoxy-3-O-{(R)-3-hydroxy-hexadecanoyl}-2-{(R)-3-octacosanoyloxy-hexadecan}amido-beta-d-glucopyranosyl}-2-{(R)-3-hydroxy-hexadecan}amido-3-O-{(R)-3-hydroxy-hexadecanoyl}-alpha-d-glucopyranose (11) and 2-deoxy-6-O-{2-deoxy-3-O-{(R)-3-hydroxy-hexadecanoyl}-2-{(R)-3-octacosanoyloxy-hexadecan}amido-beta-d-glucopyranosyl}-2-{(R)-3-hydroxy-hexadecan}amido-3-O-{(R)-3-hydroxy-hexadecanoyl}-d-glucono-1,5-lactone (13), which contain an unusual octacosanoic acid moiety and differ in the oxidation state of the anomeric center . The results of biological studies indicate that 11 and 13 lack the proinflammatory effects of Escherichia coli lipopolysaccharides (LPS) . Furthermore, 13 emulated the ability of heterogeneous R . sin-1 LPS to antagonize enteric LPS, providing evidence for the critical role of the gluconolactone moiety of R . sin-1 LPS in mediating this antagonistic effect . Compound 13 is the first example of a lipid A derivative that is devoid of phosphate but possesses antagonistic properties, making it an attractive lead compound for development of a drug to use in the treatment of Gram-negative septicemia.

Antonie Van Leeuwenhoek, 2003, 83(3), 285 - 91
Prevalence of 1-aminocyclopropane-1-carboxylate deaminase in Rhizobium spp; Ma W et al.; This is the first report documenting the presence of 1-aminocyclopropane-1-carboxylate (ACC) deaminase in Rhizobium . This enzyme, previously found in free-living bacteria, yeast and fungi, degrades ACC, the immediate precursor of ethylene in higher plants . Thirteen different rhizobial strains were examined by Southern hybridization, Western blots and ACC deaminase enzyme assay . Five of them tested positive for ACC deaminase . Induction of the expression of ACC deaminase was examined in one of the positively tested strains, Rhizobium leguminosarum bv . viciae 128C53K . This rhizobial ACC deaminase had a trace basal level of expression without ACC, but could be induced by a concentration of ACC as low as 1 microM . The more ACC added to this Rhizobium the higher the expression level of the ACC deaminase.

J Exp Bot, 2003 Jul, 54(388), 1691 - 700 Epub 2003 May 28.
Sugar uptake and proton release by protoplasts from the infected zone of Vicia faba L . nodules: evidence against apoplastic sugar supply of infected cells; Peiter E et al.; Symbiotic dinitrogen fixation of legume nodules is fuelled by phloem-imported carbohydrates . These have to pass several cell layers to reach cells infected with Rhizobium bacteroids . It is unclear whether apoplastic steps are involved in carbohyd-rate translocation within the nodule . Protoplasts were isolated from the infected and uninfected cells of the central tissue of Vicia faba nodules using a recently developed protocol . These protoplasts were used to elucidate pathways for sugar transport in this tissue . Both types of protoplasts released protons into the medium . Acidification was inhibited by vanadate and erythrosin B . However, it was stimulated by fusicoccin only in uninfected cells . A symport of sugars with protons can therefore be energized in both cell types . Uptake of 14C-labelled sugars was determined using a phthalate centrifugation technique . Uninfected protoplasts accumulated glucose through high-affinity H+/glucose-symport that was not competitively inhibited by fructose or sucrose . Uninfected protoplasts also absorbed sucrose with biphasic kinetics . At 0.1, 1, and 10 mM sucrose, uptake was inhibited by CCCP . Fusicoccin did not stimulate the linear phase of sucrose uptake . Glucose inhibited sucrose uptake nearly completely . This was not related to sucrose cleavage in the medium because sucrose was absorbed at a much higher rate than glucose, and glucose concentration did not increase in sucrose-containing protoplast suspensions . By contrast with uninfected protoplasts, infected cells did not show transporter-mediated glucose or sucrose uptake . The findings underline a role of uninfected cells in sugar translocation . Infected cells are not apoplastically supplied with sugars and possibly depend on uninfected cells for carbon supply.

Plant J, 2003 May, 34(4), 495 - 506
The NFP locus of Medicago truncatula controls an early step of Nod factor signal transduction upstream of a rapid calcium flux and root hair deformation; Amor BB et al.; Establishment of the Rhizobium-legume symbiosis depends on a molecular dialogue, in which rhizobial nodulation (Nod) factors act as symbiotic signals, playing a key role in the control of specificity of infection and nodule formation . Using nodulation-defective (Nod-) mutants of Medicago truncatula to study the mechanisms controlling Nod factor perception and signalling, we have previously identified five genes that control components of a Nod factor-activated signal transduction pathway . Characterisation of a new M . truncatula Nod- mutant led to the identification of the Nod Factor Perception (NFP) locus . The nfp mutant has a novel phenotype among Nod- mutants of M . truncatula, as it does not respond to Nod factors by any of the responses tested . The nfp mutant thus shows no rapid calcium flux, the earliest detectable Nod factor response of wild-type plants, and no root hair deformation . The nfp mutant is also deficient in Nod factor-induced calcium spiking and early nodulin gene expression . While certain genes controlling Nod factor signal transduction also control the establishment of an arbuscular mycorrhizal symbiosis, the nfp mutant shows a wild-type mycorrhizal phenotype . These data indicate that the NFP locus controls an early step of Nod factor signal transduction, upstream of previously identified genes and specific to nodulation.

Lett Appl Microbiol, 2003, 36(6), 349 - 53
Isolation and characterization of phorate degrading soil bacteria of environmental and agronomic significance; Bano N et al.; Phorate {O,O-diethyl-S-(ethylthio)methyl phosphoradiothioate} degrading bacteria were isolated from agricultural soil and characterized based on their morphological and biochemical characteristics . The selected isolates PS-1, PS-2 and PS-3 were presumptively identified as Rhizobium, Pseudomonas and Proteous species, respectively . The HPLC analysis of phorate in bioaugmented soil revealed its complete disappearance within 40 days . The degradation isotherms of the isolates PS-1, PS-2 and PS-3 suggested time-dependent disappearance of phorate following the first order rate kinetics at the corresponding rate constants of 0.04, 0.05 and 0.04 days-1 . Besides, the isolates concurrently exhibited substantial phosphate solubilization, indole acetic acid, and siderophore production . The isolate PS-3 also showed anti-fungal activity against a phytopathogen Fusarium oxysporum . As a result of the multifarious biological properties, the isolates have been suggested to be important bioresource for efficient bioinoculant development.

Syst Appl Microbiol, 2003 Mar, 26(1), 47 - 53
Description of Devosia neptuniae sp . nov . that nodulates and fixes nitrogen in symbiosis with Neptunia natans, an aquatic legume from India; Rivas R et al.; Neptunia natans is a unique aquatic legume indigenous to tropical and sub-tropical regions and is nodulated symbiotically by rhizobia using an unusual infection process unlike any previously described . Previously, isolates of neptunia-nodulating rhizobia from Senegal were characterized as Allorhizobium undicola . Here we report on a different group of neptunia-nodulating rhizobia isolated from India . Sequencing of the 16S rDNA gene from two of these Indian isolates (strains J1T and J2) show that they belong in the genus Devosia rather than Allorhizobium . Currently, the only described Devosia species is D . riboflavina (family Hyphomicrobiaceae, order Rhizobiales) . The complete 16S rDNA sequences of strains J1T and J2 are 95.9% homologous to the type strain, D . riboflavina LMG 2277T, suggesting that these neptunia-nodulating strains from India belong to a new Devosia species . This hypothesis was confirmed by further studies of polyphasic taxonomy (DNA-DNA hybridisation, TP-RAPD patterns, SDS-PAGE of cellular proteins, 16S rDNA RFLP patterns, carbon source utilisation, cellular fatty acid analysis and other phenotypic characterisations), all of which support the proposal that these neptunia-nodulating strains constitute a new Devosia species, which we name Devosia neptuniae sp . nov . These gram negative, strictly aerobic short rods are motile by a subpolar flagellum, positive for catalase, oxidase, urease and beta-galactosidase, can utilise several carbohydrates (but not organic acids) as carbon sources and contain C18:0 3-OH, cis-7 C18:1 11-methyl and cis-7 C18:1 as their major cellular fatty acids . Unlike D . riboflavina, the longer-chain C24:1 3-OH and C26:1 3-OH hydroxy fatty acids are not detected . The type strain of D . neptuniae is LMG 21357T (CECT 5650T) . Assignment of this new taxon represents the fourth example in the literature of a non-rhizobial genus of bacteria capable of forming a bonafide dinitrogen-fixing root-nodule symbiosis with legume plants.

Plant Physiol, 2003 May, 132(1), 311 - 7
Nod factor and elicitors activate different phospholipid signaling pathways in suspension-cultured alfalfa cells; den Hartog M et al.; Lipo-chitooligosaccharides (Nod factors) are produced by symbiotic Rhizobium sp . bacteria to elicit Nod responses on their legume hosts . One of the earliest responses is the formation of phosphatidic acid (PA), a novel second messenger in plant cells . Remarkably, pathogens have also been reported to trigger the formation of PA in nonlegume plants . To investigate how host plants can distinguish between symbionts and pathogens, the effects of Nod factor and elicitors (chitotetraose and xylanase) on the formation of PA were investigated in suspension-cultured alfalfa (Medicago sativa) cells . Theoretically, PA can be synthesized via two signaling pathways, i.e . via phospholipase D (PLD) and via phospholipase C in combination with diacylglycerol (DAG) kinase . Therefore, a strategy involving differential radiolabeling with {(32)P}orthophosphate was used to determine the contribution of each pathway to PA formation . In support, PLD activity was specifically measured by using the ability of the enzyme to transfer the phosphatidyl group of its substrate to a primary alcohol . In practice, Nod factor, chitotetraose, and xylanase induced the formation of PA and its phosphorylated product DAG pyrophosphate within 2 min of treatment . However, whereas phospholipase C and DAG kinase were activated during treatment with all three different compounds, PLD was only activated by Nod factor . No evidence was obtained for the activation of phospholipase A(2).

Mol Plant Microbe Interact, 2003 May, 16(5), 371 - 5
Nitrogen comes down to earth: report from the 5th European Nitrogen Fixation Conference; De Hoff P et al.; For four days and four nights, with almost 50 presentations and more than 175 posters, the 5th European Nitrogen Fixation Conference continued a tradition of excellence, bringing scientists from diverse fields such as microbiology, biochemistry, computational genomics, and plant physiology together to address the complex problems associated with biological nitrogen fixation (BNF) . The conference was hosted by the John Innes Center and the University of East Anglia in Norwich, England and took place from September 6 through 10, 2002 . A diverse range of topics was presented, from the evolution of rhizobial genomes to the plant genes involved in bacterial and fungal symbiosis, to the structure of nitrogenase, and to the means by which nitrogen is shuttled between the symbiotic bacteria and the plant . Additionally, sessions involving broader issues, such as nitrogen fertilizer use and work being done in developing countries, brought home the importance of the research being carried out in BNF around the world.

Mol Plant Microbe Interact, 2003 Apr, 16(4), 335 - 41
Rhizobium-lnduced calcium spiking in Lotus japonicus; Harris JM et al.; Legumes and rhizobium bacteria form a symbiosis that results in the development of nitrogen-fixing nodules on the root of the host plant . The earliest plant developmental changes are triggered by bacterially produced nodulation (Nod) factors . Within minutes of exposure to Nod factors, sharp oscillations in cytoplasmic calcium levels (calcium spiking) occur in epidermal cells of several closely related legumes . We found that Lotus japonicus, a legume that follows an alternate developmental pathway, responds to both its bacterial partner and to the purified bacterial signal with calcium spiking . Thus, calcium spiking is not restricted to a particular pathway of nodule development and may be a general component of the response of host legumes to their bacterial partner . Using Nod factor-induced calcium spiking as a tool to identify mutants blocked early in the response to Nod factor, we show that the L . japonicus Ljsym22-1 mutant but not the Ljsym30 mutant fails to respond to Nod factor with calcium spiking.

Mol Plant Microbe Interact, 2003 Apr, 16(4), 326 - 34
The role of nod factor substituents in actin cytoskeleton rearrangements in Phaseolus vulgaris; Cardenas L et al.; In order to define the symbiotic role of some of the chemical substituents in the Rhizobium etli Nod factors (NFs), we purified Nod metabolites secreted by the SM25 strain, which carries most of the nodulation genes, and SM17 with an insertion in nodS . These NFs were analyzed for their capabilities to induce root hair curling and cytoskeletal rearrangements . The NFs secreted by strain SM17 lack the carbamoyl and methyl substituents on the nonreducing terminal residue and an acetyl moiety on the fucosyl residue on the reducing-terminal residue as determined by mass spectrometry . We have reported previously that the root hair cell actin cytoskeleton from bean responds with a rapid fragmentation of the actin bundles within 5 min of NF exposure, and also is accompanied by increases in the apical influxes and intracellular calcium levels . In this article, we report that methyl-bearing NFs are more active in inducing root hair curling and actin cytoskeleton rearrangements than nonmethylated NFs . However, the carbamoyl residue on the nonreducing terminal residue and the acetyl group at the fucosyl residue on the reducing terminal residue do not seem to have any effect on root hair curling induction or in actin cytoskeleton rearrangement.

Curr Opin Biotechnol, 2003 Apr, 14(2), 200 - 5
Genomics insights into symbiotic nitrogen fixation; Weidner S et al.; Following an interaction with rhizobial soil bacteria, legume plants are able to form a novel organ, termed the root nodule . This organ houses the rhizobial microsymbionts, which perform the biological nitrogen fixation process resulting in the incorporation of ammonia into plant organic molecules . Recent advances in genomics have opened exciting new perspectives in this field by providing the complete gene inventory of two rhizobial microsymbionts . The complete genome sequences of Mesorhizobium loti, the symbiont of several Lotus species, and Sinorhizobium meliloti, the symbiont of alfalfa, were determined and annotated in detail . For legume macrosymbionts, expressed sequence tag projects and expression analyses using DNA arrays in conjunction with proteomics approaches have identified numerous genes involved in root nodule formation and nitrogen fixation . The isolation of legume genes by tagging or positional cloning recently allowed the identification of genes that control the very early steps of root nodule organogenesis.

J Bacteriol, 2003 May, 185(10), 2988 - 98
Discordant phylogenies within the rrn loci of Rhizobia; van Berkum P et al.; It is evident from complete genome sequencing results that lateral gene transfer and recombination are essential components in the evolutionary process of bacterial genomes . Since this has important implications for bacterial systematics, the primary objective of this study was to compare estimated evolutionary relationships among a representative set of alpha-Proteobacteria by sequencing analysis of three loci within their rrn operons . Tree topologies generated with 16S rRNA gene sequences were significantly different from corresponding trees assembled with 23S rRNA gene and internally transcribed space region sequences . Besides the incongruence in tree topologies, evidence that distinct segments along the 16S rRNA gene sequences of bacteria currently classified within the genera Bradyrhizobium, Mesorhizobium and Sinorhizobium have a reticulate evolutionary history was also obtained . Our data have important implications for bacterial taxonomy, because currently most taxonomic decisions are based on comparative 16S rRNA gene sequence analysis . Since phylogenetic placement based on 16S rRNA gene sequence divergence perhaps is questionable, we suggest that the proposals of bacterial nomenclature or changes in their taxonomy that have been made may not necessarily be warranted . Accordingly, a more conservative approach should be taken in the future, in which taxonomic decisions are based on the analysis of a wider variety of loci and comparative analytical methods are used to estimate phylogenetic relationships among the genomes under consideration.

Microbiology, 2003 May, 149(Pt 5), 1357 - 65
Fur is not the global regulator of iron uptake genes in Rhizobium leguminosarum; Wexler M et al.; Rhizobium leguminosarum fur mutants were unaffected in Fe-dependent regulation of several operons that specify different Fe uptake systems, yet cloned R . leguminosarum fur partially corrected an Escherichia coli fur mutant and R . leguminosarum Fur protein bound to canonical fur boxes . The lack of a phenotype in fur mutants is not due to functional redundancy with Irr, another member of the Fur superfamily found in the rhizobia, since irr fur double mutants are also unaffected in Fe-responsive regulation of several operons involved in Fe uptake . Neither Irr nor Fur is needed for symbiotic N(2) fixation on peas . As in Bradyrhizobium japonicum, irr mutants accumulated protoporphyrin IX . R . leguminosarum irr is not regulated by Fur and its Irr protein lacks the motif needed for haem-dependent post-translational modification that occurs in B . japonicum Irr . The similarities and differences in the Fur superfamily in the rhizobia and other Gram-negative bacteria are discussed.

Microbiology, 2003 May, 149(Pt 5), 1165 - 76
Only one catalase, katG, is detectable in Rhizobium etli, and is encoded along with the regulator OxyR on a plasmid replicon; Vargas Mdel C et al.; The plasmid-borne Rhizobium etli katG gene encodes a dual-function catalase-peroxidase (KatG) (EC 1.11.1.7) that is inducible and heat-labile . In contrast to other rhizobia, katG was shown to be solely responsible for catalase and peroxidase activity in R . etli . An R . etli mutant that did not express catalase activity exhibited increased sensitivity to hydrogen peroxide (H(2)O(2)) . Pre-exposure to a sublethal concentration of H(2)O(2) allowed R . etli to adapt and survive subsequent exposure to higher concentrations of H(2)O(2) . Based on a multiple sequence alignment with other catalase-peroxidases, it was found that the catalytic domains of the R . etli KatG protein had three large insertions, two of which were typical of KatG proteins . Like the katG gene of Escherichia coli, the R . etli katG gene was induced by H(2)O(2) and was important in sustaining the exponential growth rate . In R . etli, KatG catalase-peroxidase activity is induced eightfold in minimal medium during stationary phase . It was shown that KatG catalase-peroxidase is not essential for nodulation and nitrogen fixation in symbiosis with Phaseolus vulgaris, although bacteroid proteome analysis indicated an alternative compensatory mechanism for the oxidative protection of R . etli in symbiosis . Next to, and divergently transcribed from the catalase promoter, an ORF encoding the regulator OxyR was found; this is the first plasmid-encoded oxyR gene described so far . Additionally, the katG promoter region contained sequence motifs characteristic of OxyR binding sites, suggesting a possible regulatory mechanism for katG expression.

Ying Yong Sheng Tai Xue Bao, 2003 Jan, 14(1), 143 - 7
{Roles of rhizosphere in remediation of contaminated soils and its mechanisms}; Wei S et al.; Rhizosphere is a special 'ecological remediation unit' to treat contaminated soils, which contains a great quantity of microorganisms such as fungi and rhizobacteria living with plant roots . Thus, physiological and ecological roles of plant roots to remedy contaminated soils, to accumulate and to solidify heavy metals, to absorb and degrade organic pollutants in rhizosphere were illustrated, and the function of mycorrhizospheric fungi to absorb, barrier and chelate heavy metals, to degrade organic pollutants through their metabolism activities, the action of rhizobacteria to absorb and solidify heavy metals, to degrade organic pollutants in rhizosphere through their active living activities, and the combined remediation of fungi and bacteria to pollutants in rhizosphere and their relevant mechanisms were explained . It was suggested that the remediation role of rhizosphere was the main part of phytoremediation, and one of the main basic theories to remedy contaminated soils by the activity of green plants and other organisms . The use of hyperaccumulative plants in remedying soils contaminated by some heavy metals would be prospective . It would be one of the important approach to contaminated soils remediation by organic pollutants through the mechanism of screening some special plants whose roots had strong secreting ability to absorb and accumulate dissolvent organic pollutants on the basis of inoculating specific or non-specific fungi and bacteria from the rhizosphere . This will be a developing trend of research on the remediation of contaminated soils by organic pollutants.

J Hered, 2003 Mar-Apr, 94(2), 191 - 3
Allelic relationships of pea nodulation mutants; Novak K; Thirteen stable nonnodulating mutant lines of pea (Pisum sativum L.) originating from cv . Finale were tested for allelism in pairwise crosses . The F(1) plants were evaluated for the symbiotic phenotype under controlled growth conditions against the nodule bacterium Rhizobium leguminosarum bv . viciae strain 248 . All mutations were found to be recessive and the lines were classified into eight complementation groups comprising Risnod1-Risnod23, Risnod8, Risnod9-Risnod22, Risnod14, Risnod19-Risnod25, Risnod20, Risnod24-Risnod26, and Risnod32 . Position of Risnod21 was not firmly established, leaving the possibility of allelism both with Risnod19-Risnod25 and Risnod20 . The results were partially consistent with the previous reports on the allelism of these lines . Additional crosses confirmed the correspondence of Risnod14 with the locus sym7 and of Risnod19-Risnod25 with sym8 . The high number of eight complementation groups formed by 13 mutants provides an indication of additional nodulation loci in pea to those already reported and confirms the complexity of the genetic control of the early stages of nodulation.

J Exp Bot, 2003 May, 54(386), 1481 - 7
Suppression of arbuscular mycorrhizal colonization and nodulation in split-root systems of alfalfa after pre-inoculation and treatment with Nod factors; Catford JG et al.; Roots of legumes establish symbiosis with arbuscular mycorrhizal fungi (AMF) and nodule-inducing rhizobia . The existing nodules systemically suppress subsequent nodule formation in other parts of the root, a phenomenon termed autoregulation . Similarly, mycorrhizal roots reduce further AMF colonization on other parts of the root system . In this work, split- root systems of alfalfa (Medicago sativa) were used to study the autoregulation of symbiosis with Sinorhizobium meliloti and the mycorrhizal fungus Glomus mosseae . It is shown that nodulation systemically influences AMF root colonization and vice versa . Nodules on one half of the split-root system suppressed subsequent AMF colonization on the other half . Conversely, root systems pre-colonized on one side by AMF exhibited reduced nodule formation on the other side . An inhibition effect was also observed with Nod factors (lipo-chito-oligosaccharides) . NodSm-IV(C16:2, S) purified from S . meliloti systemically suppressed both nodule formation and AMF colonization . The application of Nod factors, however, did not influence the allocation of (14)C within the split-root system, excluding competition for carbohydrates as the regulatory mechanism . These results indicate a systemic regulatory mechanism in the rhizobial and the arbuscular mycorrhizal association, which is similar in both symbioses.

Carbohydr Res, 2003 May 1, 338(10), 1143 - 6
Enantioseparation using cyclosophoraoses as a novel chiral additive in capillary electrophoresis; Lee S et al.; Cyclosophoraoses, cyclic beta-(1-->2)-D-glucans produced by Rhizobium meliloti 2011, were used as a novel chiral additive for the separation of terbutaline, amethopterin, thyroxine and N-acetylphenylalanine enantiomers in aqueous capillary electrophoresis (CE) . Enantioseparation took place in the normal- or reversed-polarity mode when a high concentration of neutral (60 mM) or anionic (40 mM) cyclosophoraoses was added to the background electrolyte (BGE).

Biochem Biophys Res Commun, 2003 Apr 25, 304(1), 136 - 42
O2-specific regulation of the ferrous heme-based sensor kinase FixL from Sinorhizobium meliloti and its aberrant inactivation in the ferric form; Akimoto S et al.; FixL, a rhizobial heme-based O2-sensing histidine kinase, catalyzes autophosphorylation in the deoxy form at low O2 tension, while the kinase activity is inhibited in the case of the O2-bound form . The present study unambiguously shows that the binding of CO and NO does not significantly inhibit the kinase activity of dithiothreitol (DTT)-reduced ferrous FixL from Sinorhizobium meliloti, which is inconsistent with the spin state mechanism previously reported . Kinase inactivation is caused by aberrant disulfide (S-S) bond formation at Cys301 in the ferric homodimer, which explains these contradictory observations . The addition of DTT cleaved the S-S bond, leading to restoration of kinase activity in the ferric form as well as heme reduction, but, sodium hydrosulfite treatment produced the kinase-inactive deoxy form without S-S cleavage . On the basis of these experimental results, it can be concluded that ferrous FixL discriminates O2 from CO and NO, and signals the O2-bound state by downregulating the phosphoryl transfer reaction.

Genome Biol . 2003;4(4):R26 . Epub 2003 Mar 31.
Analysis and functional classification of transcripts from the nematode Meloidogyne incognita; McCarter JP et al.; BACKGROUND: Plant parasitic nematodes are major pathogens of most crops . Molecular characterization of these species as well as the development of new techniques for control can benefit from genomic approaches . As an entree to characterizing plant parasitic nematode genomes, we analyzed 5,700 expressed sequence tags (ESTs) from second-stage larvae (L2) of the root-knot nematode Meloidogyne incognita . RESULTS: From these, 1,625 EST clusters were formed and classified by function using the Gene Ontology (GO) hierarchy and the Kyoto KEGG database . L2 larvae, which represent the infective stage of the life cycle before plant invasion, express a diverse array of ligand-binding proteins and abundant cytoskeletal proteins . L2 are structurally similar to Caenorhabditis elegans dauer larva and the presence of transcripts encoding glyoxylate pathway enzymes in the M . incognita clusters suggests that root-knot nematode larvae metabolize lipid stores while in search of a host . Homology to other species was observed in 79% of translated cluster sequences, with the C . elegans genome providing more information than any other source . In addition to identifying putative nematode-specific and Tylenchida-specific genes, sequencing revealed previously uncharacterized horizontal gene transfer candidates in Meloidogyne with high identity to rhizobacterial genes including homologs of nodL acetyltransferase and novel cellulases . CONCLUSIONS: With sequencing from plant parasitic nematodes accelerating, the approaches to transcript characterization described here can be applied to more extensive datasets and also provide a foundation for more complex genome analyses.

Physiol Plant, 2003 May, 118(1), 10 - 15
Methods for isolating and characterizing ACC deaminase-containing plant growth-promoting rhizobacteria; Penrose DM et al.; One of the major mechanisms utilized by plant growth-promoting rhizobacteria (PGPR) to facilitate plant growth and development is the lowering of ethylene levels by deamination of 1-aminocyclopropane-1-carboxylic acid (ACC) the immediate precursor of ethylene in plants . The enzyme catalysing this reaction, ACC deaminase, hydrolyses ACC to alpha-ketobutyrate and ammonia . Several bacterial strains that can utilize ACC as a sole source of nitrogen have been isolated from rhizosphere soil samples . All of these strains are considered to be PGPR based on the ability to promote canola seedling root elongation under gnotobiotic conditions . The treatment of plant seeds or roots with these bacteria reduces the amount of ACC in plants, thereby lowering the concentration of ethylene . Here, a rapid procedure for the isolation of ACC deaminase-containing bacteria, a root elongation assay for evaluating the effects of selected bacteria on root growth, and a method of assessing bacterial ACC deaminase activity are described in detail . This should allow researchers to readily isolate new PGPR strains adapted to specific environments.

Meded Rijksuniv Gent Fak Landbouwkd Toegep Biol Wet, 2002, 67(2), 377 - 80
Biological control of soybean damping-off by antagonistic rhizobacteria; Sharifi Tehrani A et al.; Experiments were carried out with 133 bacterial isolates that were collected from soybean rhizosphere . These strains were used to investigate their biocontrol traits in vitro and their ability to suppress the soybean damping-off in vivo (soil and seed treatments) . Three highly effective isolates were selected from these antagonists for subsequent studies . According to the biochemical, physiological and morphological tests, these isolates (B-2, B-12 and B-80) were identified as Bacillus spp . In soil treatment, the isolate B-3 with 70.8%, B-12 with 66.7%, B-80 with 54.2% had the highest effect on reducing the soybean damping-off . In seed treatment, the isolates B-43 with 62.5%, B-12 with 58.4 and B-80 with 45.8%, had the greatest effect on reducing the disease . These isolates produced volatile metabolites that inhibited mycelial growth of Phytophthora sojae.

Meded Rijksuniv Gent Fak Landbouwkd Toegep Biol Wet, 2002, 67(2), 159 - 64
Disease control by means of induced resistance; Isebaert S et al.; The biological control of fungal diseases in agriculture and horticulture based on induced resistance can become an attractive alternative to traditional fungicides . The principle is that all plants have genetic information about resistance mechanisms against pathogens such as fungi, viruses, and bacteria . The purpose of our research is to activate these resistance factors that are latent present in the plant, by treating the plants with abiotic and biotic agents (elicitors) to activate defence responses . The resistance inducing products used in the experiments are based on non-pathogenic rhizobacteria, and metabolites of micro-organisms and plants . In our experiments the different products and organisms were tested against powdery mildew on tomato . Good results are obtained with Milsana, Elexa and Serenade, especially Milsana showed a strong reduction of the disease symptoms of O . lycopersicum . These products could contribute to an environmentally more acceptable crop protection and suits in the aim to durable production techniques in agriculture and horticulture.

Nature, 2003 Apr 17, 422(6933), 722 - 6
Amino-acid cycling drives nitrogen fixation in the legume-Rhizobium symbiosis; Lodwig EM et al.; The biological reduction of atmospheric N2 to ammonium (nitrogen fixation) provides about 65% of the biosphere's available nitrogen . Most of this ammonium is contributed by legume-rhizobia symbioses, which are initiated by the infection of legume hosts by bacteria (rhizobia), resulting in formation of root nodules . Within the nodules, rhizobia are found as bacteroids, which perform the nitrogen fixation: to do this, they obtain sources of carbon and energy from the plant, in the form of dicarboxylic acids . It has been thought that, in return, bacteroids simply provide the plant with ammonium . But here we show that a more complex amino-acid cycle is essential for symbiotic nitrogen fixation by Rhizobium in pea nodules . The plant provides amino acids to the bacteroids, enabling them to shut down their ammonium assimilation . In return, bacteroids act like plant organelles to cycle amino acids back to the plant for asparagine synthesis . The mutual dependence of this exchange prevents the symbiosis being dominated by the plant, and provides a selective pressure for the evolution of mutualism.

Mikrobiologiia, 2003 Jan-Feb, 72(1), 48 - 53
{Tomato root exudates and their effect on the growth and antifungal activity of Pseudomonas strains}; Kravchenko LV et al.; The study of the effect of the root exometabolites of tomato plants on the growth and antifungal activity of the plant growth-promoting Pseudomonas strains showed that the antifungal activity of plant growth-promoting rhizobacteria in the plant rhizosphere may depend on the sugar and organic acid composition of root exudates.

Mikrobiologiia, 2003 Jan-Feb, 72(1), 40 - 7
{The effect of the plant growth stimulant bactozole on Rhizobium leguminosarum bv . viciae 250a and its nitrogen-tolerant mutant M-71 under varied nitrogen supply}; Kosenko LV et al.; The effect of the plant growth stimulant bactozole on the growth of Rhizobium leguminosarum bv . viciae 250a and its nitrogen-tolerant mutant M-71 and the synthesis of extracellular carbohydrates was studied . At a low content of nitrate (6 mM) in the medium, all three bactozole concentrations tested (0.001, 0.01, and 0.1%) exerted similar stimulating effects on the growth of the parent strain 250a (about 1.5-fold) and the synthesis of extracellular carbohydrates (about 2-fold) . At a high content of nitrate (20 mM) in the medium, when the growth of the parent strain and the synthesis of extracellular carbohydrates were inhibited, bactozole at all three concentrations exerted only a growth-stimulating effect . At the same time, mutant M-71 showed better growth at higher concentrations of bactozole, whereas the ability of the mutant to synthesize extracellular carbohydrates decreased with increasing bactozole concentration . The cell biomass of the mutant accumulated at 20 mM nitrate was 1.8-2.5 times greater than it was at 6 mM nitrate . Bactozole enhanced the symbiosis of legume plants with both parent and mutant strains, raising the mass of plants and enhancing nodulation and the nitrogen-fixing activity of root nodules . The symbiotic parameters of mutant M-71 were better (irrespective of whether bactozole was present or not) when its inoculum was grown at a high nitrogen content (20 mM nitrate), whereas the respective parameters of the parent strain were better when it was grown at 6 mM nitrate . The inference is made that the better physiological characteristics of the mutant in the high-nitrate medium is due to its higher nitrate reductase activity (as compared with the parent strain) in both the free-living state and in legume nodules.

Meded Rijksuniv Gent Fak Landbouwkd Toegep Biol Wet, 2002, 67(3), 575 - 89
The role of some agricultural practices and fertilizer type on both the incidence of stem borers infestation and corn yield in Egypt; Mesbah HA et al.; Maize, Zea mays, L . is one of the most important field crops in Egypt . It is used mainly for human, animal and poultry feeding . Corn plants are usually attacked by several injourious insect pests at different stages of development . Out of them, the pink stem borer, Sesamia cretica (Led.), the purple lined borer, Chilo agamemnon (Bles.), and the European corn borer Ostrinia nubilalis (Hb.); which cause great damage and yield losses . It is profitable to adopt an effective and sustainable strategy for controlling these insect-pests . In this concern, sowing dates, planting spaces, foliar fertilizers (macro and micro-nutrients), mineral and/or biofertilization, were investigated to evaluate their role as tools in the so-called Integrated Pest Management (IPM) program of corn pests . In general, the used planting spaces of 60 and 70 cm apart between furrows insignificantly affected the level of stem borers infestation . It was clearly observed that the sowing dates have a role in the incidence of stem borers infestation throughout the corn growing seasons of 1994 and 1995 . Moreover, The biofertilized corn plants were more tolerant to the infestation by the stem borers than the minerally fertilized ones . Application of Polytrin significantly decreased the mean numbers of larvae . The tested nutrients preparations affected to less extent, the infestation levels . Concerning the interaction effect of applied nutrients preparations, used sowing dates and/or fertilizer type on the deduced means of larval numbers, it was revealed that: (i) the application of the nutrients preparations decreased to a great extent the effect of the studied sowing dates on the stem borers infestation; particularly in case of spraying ascorbic acid alone or in sequence with Polymex, coppersulphate & Potasin-F, (ii) the dressing of corn grains with the biofertilizers Phosphorin & Rhizobacterin before sowing, lowered to some extent the levels of infestation by Ch . agamemnon and O . nubilalis, in comparison to the minerally fertilized corn plants, especially in case of spraying Potasin-F, copper sulphate and scorbic acid followed by Polymex for Ch . agamemnon . Spraying Ascorbic acid alone or in sequence with Polymex; Potasin-F followed by Copper sulphate gave promising results for the control of O . nubilalis . In comparison to insecticide treatment, the used foliar nutrients & fertilizer type in both sowing dates gave positive interaction effects in decreasing levels of stem borers infestation and greatly improved the yield and yield characteristics of corn plants . Such agricultural practices enabled corn plants to tackle the going on infestation; thus crop loss due to the attack of the stem borers could be compensated.

Meded Rijksuniv Gent Fak Landbouwkd Toegep Biol Wet, 2002, 67(3), 487 - 97
Effect of sequential applications of foliar nutrients, biofertilizers and sowing dates on the incidence of corn stem borers in Egypt; Mesbah HA et al.; In this study either early sown (May 1st) or lately sown (June 2nd) corn plants were treated with Phosphorin & Rhizobactrin as biofertilizers and sprayed with six selected foliar nutrients, i.e . Polymex; Greenzit SP100, Greenzit NPK, Potasin-F, Copper sulphate and Ascorbic acid; in mono-, bi-, and/or tri-sequential applications . Such practices were conducted to show their beneficial effects compared with the chemical treatment in checking the incidence of the stem borers and hence increasing the corn yield . The obtained results could be summarized in the following chief points: (a) the lately sown biofertilized plants showed somewhat higher levels of infestation than the early planted ones., (b) in general, spraying the biofertilized corn plants in both sowing dates with the tested foliar nutrients, significantly decreased the rate of the stem borers infestation than the untreated plants of control., (c) the foliar sprays of Greenzit NPK alone, bi- or tri-sequential applications of Potasin-F, Polymex, Ascorbic acid and Copper sulphate achieved considerable success in reducing larval numbers of the borers species . For example, in case of using the bi-sequential nutrients (Polymex/Ascorbic acid) the numbers were 1.2, 1.5 and 1.2 larvae/5 plants, whereas the numbers were 1.3, 1.0 and 0.7 larvae/5 plants as a result, of the tri-sequential applications (Potasin-F/Ascorbic acid/Polymex) for the pink stem borer, Sesamia cretica, (Led.), the purple lined borer, Chilo agamemnon, (Bels.), and the European corn borer Ostrinia nubilalis (Hb.), in respect, vs . 4.8, 4.5 and 2.9 larvae/5 plants for the same stem borers, respectively, in case of the untreated corn plants . In addition, the other trisequential applications (Polymex/ascorbic acid/Copper sulphate), (Potasin-F/Copper sulphate/ascorbic acid) and (Potasin-F/Copper sulphate/Polymex) reduced the stem borers infestation; (d) from the view point of the interaction effects of sowing dates and the tested foliar nutrients, it was found that the tri-sequential sprayings (Potasin-F/Copper sulphate/Polymex) and/or (Potasin-F/Copper sulphate/Ascorbic acid) have lowered the rate of the stem borers infestation to 3.3 and 3.3 and 5.7 and 4.3 larvae/5 plants for the tri-applications in the 1st and 2nd sowing dates, respectively . Such reductions in the levels of infestation led to an increase in the grain yield up to 6.9 and 7.2 and 5.4 and 5.8 ton/fed, for the early and lately sown corn plants, in respect, and (e) All the foliar nutrients, with no exception, proved to be efficient in managing the stem borers infestation as compared with the insecticide treatment using Polytrin . Although the chemical application had lowered the level of infestation to 2.3 and 5.7 larvae/5 plants in the 1st and 2nd sowing dates as compared with 9.7 and 14.7 larvae/5 untreated plants for the same sowing dates, lesser grain yield of 5.6 and 4.4 ton/fed . was obtained in the first and second dates of planting, successively, in comparison to the grain yield resulted from the tri-applications of Potasin-F/Copper sulphate with either Polymex or Ascorbic acid . The abovementioned results assured the profitable effects of using foliar nutrients as well as the biofertilizers for attaining healthy corn plants, which would be capable of tolerating the injury inflicted by the studied stem borers and compensating for the harmful effects of insects infestation, so high grain yields could be obtained than those of the untreated and/or the insecticide treated plants.

Indian J Exp Biol, 2002 Oct, 40(10), 1121 - 30
Symbiotic characteristics of cysteine and methionine auxotrophs of Sinorhizobium meliloti; Abbas BA et al.; Twenty one cysteine and 13 methionine auxotrophs of Sinorhizobium meliloti Rmd201 were obtained by random mutagenesis with transposon Tn5 . The cysteine auxotrophs were sulfite reductase mutants and each of these auxotrophs had a mutation in cysI/cysJ gene . The methionine auxotrophs were metA/metZ, metE and metF mutants . One hundred per cent co-transfer of Tn5-induced kanamycin resistance and auxotrophy from each Tn5-induced auxotrophic mutant indicated that each mutant cell most likely had a single Tn5 insertion . However, the presence of more than one Tn5 insertions in the auxotrophs used in our study cannot be ruled out . All cysteine and methionine auxotrophs induced nodules on alfalfa plants . The nodules induced by cysteine auxotrophs were fully effective like those of the parental strain-induced nodules, whereas the nodules induced by methionine auxotrophs were completely ineffective . The supplementation of methionine to the plant nutrient medium completely restored symbiotic effectiveness to the methionine auxotrophs . These results indicated that the alfalfa host provides cysteine but not methionine to rhizobia during symbiosis . Histological studies showed that the defective symbiosis of methionine auxotrophs with alfalfa plants was due to reduced number of infected nodule cells and incomplete transformation of bacteroids.

Yi Chuan Xue Bao, 2002 Dec, 29(12), 1118 - 25
{Genetic diversity and phylogeny of rhizobia isolated from peanut (Arachis hypogaea)}; Yang JK et al.; Forty three rhizobium strains isolated from peanut (Arachis hypogaea) and 15 reference strains from other genus and species were analyzed by the method of 16S rRNA RFLP, 16S rRNA sequencing and 16S-23S IGS PCR RFLP . The results of the 16S rRNA RFLP shown that 43 strains tested were all ascribed to the genus of Bradyrhizobium phylogenetically . Strains tested were adjacent to the B . japonicum and far from B . elkanii 16S rRNA genotype . The genotypes generated by the 4 restriction endonucleases, Mbo I, Dde I, Hae III and Msp I, were same as the representatives of B . japonicum . The dendrogram generated by 16S rRNA sequence and Neighbor-joining method shown that peanut rhizobia clustered into the subcluster represented by B . japonicum and B . liaoningense, were more close to B . liaoningense genetically, and the sequence difference between them was less than 1% . High sequence similarity was also determined between B . liaoningense and B . japonicum . JZ1, representative strain of peanut rhizobia were systematically far from the B . elkanii, and the sequence divergence about 2% . The results from IGS RFLP analysis indicated that although they were phylogenetically close to B . japonicum and B . elkanii, peanut rhizobia forming an independent group at the similarity of 71% could be further divided into four subgroups, A, B, C and D . Subgroup A consisted of strains from different region, subgroup B was composed of strains from Wuchang, Qianjiang and Jingzhou, subgroup C was mainly composed of strains from Jingzhou and starins of subgroup D mainly from Neijiang . Reference strains from B . japonicum and B . elkanii were independently clustered into the subgroup E at the similarity of 71% . The geographical factor effect on genetic diversity of rhizobia was found.

Mol Genet Genomics, 2003 Jun, 269(3), 312 - 20 Epub 2003 Mar 28.
The Lotus japonicus Sen1 gene controls rhizobial differentiation into nitrogen-fixing bacteroids in nodules; Suganuma N et al.; A Lotus japonicus mutant, Ljsym75, which forms ineffective symbiotic nodules and defines a new locus involved in the process of nitrogen fixation, was characterized in detail in order to identify the stage of developmental arrest of the nodules . No nitrogen-fixing activity was detectable in Ljsym75 nodules at any stage during plant development, and plant growth was markedly retarded . Ljsym75 plants formed twice as many nodules as the wild-type Gifu, and this phenotype was not influenced by the application of low concentrations of nitrate . Although the ineffective nodules formed on Ljsym75 were anatomically similar to effective Gifu nodules, Ljsym75 nodules senesced prematurely . Microscopic examination revealed that bacteria endocytosed into Ljsym75 nodules failed to differentiate into bacteroids . Moreover, the bacteria contained no nitrogenase proteins, whereas leghemoglobin was detected in the cytosol of the nodules . These results indicate that Ljsym75 is required for bacterial differentiation into nitrogen-fixing bacteroids in nodules, and thus the Ljsym75 gene was renamed sen1 (for stationary endosymbiont nodule) . Linkage analysis using DNA markers showed that Sen1 is located on chromosome 4.

Proc Natl Acad Sci U S A, 2003 Apr 15, 100(8), 4927 - 32 Epub 2003 Apr 08.
Bacterial volatiles promote growth in Arabidopsis; Ryu CM et al.; Several chemical changes in soil are associated with plant growth-promoting rhizobacteria (PGPR) . Some bacterial strains directly regulate plant physiology by mimicking synthesis of plant hormones, whereas others increase mineral and nitrogen availability in the soil as a way to augment growth . Identification of bacterial chemical messengers that trigger growth promotion has been limited in part by the understanding of how plants respond to external stimuli . With an increasing appreciation of how volatile organic compounds signal plants and serve in plant defense, investigations into the role of volatile components in plant-bacterial systems now can follow . Here, we present chemical and plant-growth data showing that some PGPR release a blend of volatile components that promote growth of Arabidopsis thaliana . In particular, the volatile components 2,3-butanediol and acetoin were released exclusively from two bacterial strains that trigger the greatest level of growth promotion . Furthermore, pharmacological applications of 2,3-butanediol enhanced plant growth whereas bacterial mutants blocked in 2,3-butanediol and acetoin synthesis were devoid in this growth-promotion capacity . The demonstration that PGPR strains release different volatile blends and that plant growth is stimulated by differences in these volatile blends establishes an additional function for volatile organic compounds as signaling molecules mediating plant-microbe interactions.

Appl Environ Microbiol, 2003 Apr, 69(4), 2276 - 83
Compatibility of rhizobial genotypes within natural populations of Rhizobium leguminosarum biovar viciae for nodulation of host legumes; Laguerre G et al.; Populations of Rhizobium leguminosarum biovar viciae were sampled from two bulk soils, rhizosphere, and nodules of host legumes, fava bean (Vicia faba) and pea (Pisum sativum) grown in the same soils . Additional populations nodulating peas, fava beans, and vetches (Vicia sativa) grown in other soils and fava bean-nodulating strains from various geographic sites were also analyzed . The rhizobia were characterized by repetitive extragenomic palindromic-PCR fingerprinting and/or PCR-restriction fragment length polymorphism (RFLP) of 16S-23S ribosomal DNA intergenic spacers as markers of the genomic background and PCR-RFLP of a nodulation gene region, nodD, as a marker of the symbiotic component of the genome . Pairwise comparisons showed differences among the genetic structures of the bulk soil, rhizosphere, and nodule populations and in the degree of host specificity within the Vicieae cross-inoculation group . With fava bean, the symbiotic genotype appeared to be the preponderant determinant of the success in nodule occupancy of rhizobial genotypes independently of the associated genomic background, the plant genotype, and the soil sampled . The interaction between one particular rhizobial symbiotic genotype and fava bean seems to be highly specific for nodulation and linked to the efficiency of nitrogen fixation . By contrast with bulk soil and fava bean-nodulating populations, the analysis of pea-nodulating populations showed preferential associations between genomic backgrounds and symbiotic genotypes . Both components of the rhizobial genome may influence competitiveness for nodulation of pea, and rhizosphere colonization may be a decisive step in competition for nodule occupancy.

Appl Environ Microbiol, 2003 Apr, 69(4), 2006 - 14
Identification of a third sulfate activation system in Sinorhizobium sp . strain BR816: the CysDN sulfate activation complex; Snoeck C et al.; Sinorhizobium sp . strain BR816 possesses two nodPQ copies, providing activated sulfate (3'-phosphoadenosine-5'-phosphosulfate {PAPS}) needed for the biosynthesis of sulfated Nod factors . It was previously shown that the Nod factors synthesized by a nodPQ double mutant are not structurally different from those of the wild-type strain . In this study, we describe the characterization of a third sulfate activation locus . Two open reading frames were fully characterized and displayed the highest similarity with the Sinorhizobium meliloti housekeeping ATP sulfurylase subunits, encoded by the cysDN genes . The growth characteristics as well as the levels of Nod factor sulfation of a cysD mutant (FAJ1600) and a nodP1 nodQ2 cysD triple mutant (FAJ1604) were determined . FAJ1600 shows a prolonged lag phase only with inorganic sulfate as the sole sulfur source, compared to the wild-type parent . On the other hand, FAJ1604 requires cysteine for growth and produces sulfate-free Nod factors . Apigenin-induced nod gene expression for Nod factor synthesis does not influence the growth characteristics of any of the strains studied in the presence of different sulfur sources . In this way, it could be demonstrated that the "household" CysDN sulfate activation complex of Sinorhizobium sp . strain BR816 can additionally ensure Nod factor sulfation, whereas the symbiotic PAPS pool, generated by the nodPQ sulfate activation loci, can be engaged for sulfation of amino acids . Finally, our results show that rhizobial growth defects are likely the reason for a decreased nitrogen fixation capacity of bean plants inoculated with cysD mutant strains, which can be restored by adding methionine to the plant nutrient solution.

Mol Microbiol, 2003 Apr, 48(2), 373 - 83
The uncoupling of oxygen sensing, phosphorylation signalling and transcriptional activation in oxygen sensor FixL and FixJ mutants; Saito K et al.; The rhizobial FixL/FixJ system, a member of the superfamily of bacterial two-component signal transducing systems, regulates the expression of nitrogen fixation-related genes by sensing environmental oxygen tension . Oxygen-free (deoxy) FixL is autophosphorylated at an invariant histidine residue with ATP, and the phosphoryl group is transferred to FixJ, leading to an enhancement in transcriptional activity at low oxygen tensions, but the histidine kinase activity of the oxygen-bound (oxy) form is inhibited . To investigate the mechanism of oxygen sensing, we established a FixL/FixJ-mediated PfixK-lacZ reporter system in Escherichia coli, and isolated FixL and FixJ mutations conferring an upregulation of lacZ gene expression on the reporter cells even under aerobic conditions . FixL mutant proteins, which contain single amino acid changes near the autophosphorylation site, showed elevated levels of autophosphorylation and a concomitant phosphoryl transfer to FixJ in the presence of oxygen, although their oxygen-binding affinities were unimpaired . These mutational analyses suggest that the autophosphorylation domain plays a crucial role in regulatory coupling between oxygen binding and kinase activity . FixJ mutants in helix alpha1 and strand beta5 of the N-terminal half exhibited the formation of a stable acyl phosphate bond . In contrast, those in helices alpha4 and alpha5 constitutively bound to the fixK promoter in a monomeric form, suggesting that the alpha4 and alpha5 helices may be involved in the post-phosphorylation/dimerization signal transfer to liberate the DNA-binding activity of the C-terminal domain, not only serving as a dimerization interface.

J Bacteriol, 2003 Apr, 185(8), 2503 - 11
Membrane topology of PssT, the transmembrane protein component of the type I exopolysaccharide transport system in Rhizobium leguminosarum bv . trifolii strain TA1; Mazur A et al.; The pssT gene was identified as the fourth gene located upstream of the pssNOP gene cluster possibly involved in the biosynthesis, polymerization, and transport of exopolysaccharide (EPS) in Rhizobium leguminosarum bv . trifolii strain TA1 . The hydropathy profile and homology searches indicated that PssT belongs to the polysaccharide-specific transport family of proteins, a component of the type I system of the polysaccharide transport . The predicted membrane topology of the PssT protein was examined with a series of PssT-PhoA fusion proteins and a complementary set of PssT-LacZ fusions . The results generally support a predicted topological model for PssT consisting of 12 transmembrane segments, with amino and carboxyl termini located in the cytoplasm . A mutant lacking the C-terminal part of PssT produced increased amounts of total EPS with an altered distribution of high- and low-molecular-weight forms in comparison to the wild-type RtTA1 strain . The PssT mutant produced an increased number of nitrogen fixing nodules on clover.

Eur J Biochem, 2003 Apr, 270(7), 1365 - 80
Surface polysaccharide involvement in establishing the rhizobium-legume symbiosis; Fraysse N et al.; When the rhizosphere is nitrogen-starved, legumes and rhizobia (soil bacteria) enter into a symbiosis that enables the fixation of atmospheric dinitrogen . This implies a complex chemical dialogue between partners and drastic changes on both plant roots and bacteria . Several recent works pointed out the importance of rhizobial surface polysaccharides in the establishing of the highly specific symbiosis between symbionts . Exopolysaccharides appear to be essential for the early infection process . Lipopolysaccharides exhibit specific roles in the later stages of the nodulation processes such as the penetration of the infection thread into the cortical cells or the setting up of the nitrogen-fixing phenotype . More generally, even if active at different steps of the establishing of the symbiosis, all the polysaccharide classes seem to be involved in complex processes of plant defense inhibition that allow plant root invasion . Their chemistry is important for structural recognition as well as for physico-chemical properties.

Tree Physiol, 2000 Jan, 20(1), 33 - 40
Effects of nitrogen source and defoliation on growth and biological dinitrogen fixation of Gliricidia sepium seedlings; Nygren P et al.; Effects of four N sources and two defoliation treatments on growth and nitrogenase activity of Gliricidia sepium (Jacq.) Walp seedlings were studied in a greenhouse . All nutrients were supplied in irrigation water to the sterile growing medium . The N sources were: (1) 100 mg l(-1) of N supplied as NO(3) (-) (high-NO(3) (-)), (2) 50 mg l(-1) of N supplied as NO(3) (-) and inoculation with Rhizobium spp . medium-NO(3) (-)), (3)100 mg l(-1) of N supplied as NH(4)NO(3), and (4) inoculation with Rhizobium spp without mineral N (N(2)) . At 35 weeks after sowing, mean total biomass was 130.5, 50.5, 22.9 and 17.4 g seedling(-1) in the NH(4)NO(3), N(2), medium-NO(3) (-) and high-NO(3) (-) treatments, respectively . The root/shoot ratio was high in all of the N treatments (1.73-2.77) because the seedlings had big taproots . The medium-NO(3) (-) treatment completely inhibited nodulation, whereas seedlings in the N(2) treatment were profusely nodulated . At 32 weeks after sowing, groups of seedlings in the N(2) and high-NO(3) (-) treatments were subjected to 50 or 100% defoliation . Closed-chamber acetylene reduction assays of intact root systems were conducted to compare nitrogenase activity at 7, 14 and 28 days after defoliation (DAD) . At 7 and 14 DAD, nitrogenase activity of completely and partially defoliated seedlings was about 10 and 60%, respectively, of that of undefoliated controls . At 28 DAD, nitrogenase activity of completely defoliated seedlings was twice the predefoliation value, whereas nitrogenase activity of partially defoliated seedlings was only 87% of the predefoliation value . Recovery of nitrogenase activity was strongly correlated with foliage regrowth in the completely defoliated seedlings, but not in the partially defoliated seedlings . Abundant belowground C and N reserves in the large taproot probably contributed to the rapid recovery from defoliation . Accumulation of belowground biomass may also improve defoliation tolerance of mature trees.

Plant Physiol, 2003 Mar, 131(3), 1124 - 36
Expression of the apyrase-like APY1 genes in roots of Medicago truncatula is induced rapidly and transiently by stress and not by Sinorhizobium meliloti or Nod factors; Navarro-Gochicoa MT et al.; The model legume Medicago truncatula contains at least six apyrase-like genes, five of which (MtAPY1;1, MtAPY1;2, MtAPY1;3, MtAPY1;4, and MtAPY1;5) are members of a legume-specific family, whereas a single gene (MtAPY2) has closer homologs in Arabidopsis . Phylogenetic analysis has revealed that the proteins encoded by these two plant gene families are more similar to yeast (Saccharomyces cerevisiae) GDA1 and to two proteins encoded by newly described mammalian genes (ENP5 and 6) than they are to mammalian CD39- and CD39-like proteins . Northern analyses and analyses of the frequencies of expressed sequence tags (ESTs) in different cDNA libraries suggest that in roots, leaves, and flowers, the more highly expressed genes are MtAPY1;3/MtAPY2, MtAPY1;3/MtAPY1;5 and MtAPY1;2/MtAPY1;3 respectively . In roots, at least four of the MtAPY1 genes are induced transiently within 3 to 6 h by a stress response that seems to be ethylene independent because it occurs after treatment with an ethylene synthesis inhibitor and also in the skl ethylene-insensitive mutant . This response also occurs in roots of the following symbiotic mutants: dmi1, dmi2, dmi3, nsp, hcl, pdl, lin, and skl . No evidence was obtained for a rapid, transient, and specific induction of the MtAPY genes in roots in response to rhizobia or rhizobial lipochitooligosaccharidic Nod factors . Thus, our data suggest that the apyrase-like genes, which in several legumes have been implicated to play a role in the legume-rhizobia symbiosis (with some members being described as early nodulin genes), are not regulated symbiotically by rhizobia in M . truncatula.

Plant Physiol, 2003 Mar, 131(3), 1080 - 90
Proteome analysis . Novel proteins identified at the peribacteroid membrane from Lotus japonicus root nodules; Wienkoop S et al.; The peribacteroid membrane (PBM) forms the structural and functional interface between the legume plant and the rhizobia . The model legume Lotus japonicus was chosen to study the proteins present at the PBM by proteome analysis . PBM was purified from root nodules by an aqueous polymer two-phase system . Extracted proteins were subjected to a global trypsin digest . The peptides were separated by nanoscale liquid chromatography and analyzed by tandem mass spectrometry . Searching the nonredundant protein database and the green plant expressed sequence tag database using the tandem mass spectrometry data identified approximately 94 proteins, a number far exceeding the number of proteins reported for the PBM hitherto . In particular, a number of membrane proteins like transporters for sugars and sulfate; endomembrane-associated proteins such as GTP-binding proteins and vesicle receptors; and proteins involved in signaling, for example, receptor kinases, calmodulin, 14-3-3 proteins, and pathogen response-related proteins, including a so-called HIR protein, were detected . Several ATPases and aquaporins were present, indicating a more complex situation than previously thought . In addition, the unexpected presence of a number of proteins known to be located in other compartments was observed . Two characteristic protein complexes obtained from native gel electrophoresis of total PBM proteins were also analyzed . Together, the results identified specific proteins at the PBM involved in important physiological processes and localized proteins known from nodule-specific expressed sequence tag databases to the PBM.

Plant Physiol, 2003 Mar, 131(3), 1054 - 63
crinkle, a novel symbiotic mutant that affects the infection thread growth and alters the root hair, trichome, and seed development in Lotus japonicus; Tansengco ML et al.; To elucidate the mechanisms involved in Rhizobium-legume symbiosis, we examined a novel symbiotic mutant, crinkle (Ljsym79), from the model legume Lotus japonicus . On nitrogen-starved medium, crinkle mutants inoculated with the symbiont bacterium Mesorhizobium loti MAFF 303099 showed severe nitrogen deficiency symptoms . This mutant was characterized by the production of many bumps and small, white, uninfected nodule-like structures . Few nodules were pale-pink and irregularly shaped with nitrogen-fixing bacteroids and expressing leghemoglobin mRNA . Morphological analysis of infected roots showed that nodulation in crinkle mutants is blocked at the stage of the infection process . Confocal microscopy and histological examination of crinkle nodules revealed that infection threads were arrested upon penetrating the epidermal cells . Starch accumulation in uninfected cells and undeveloped vascular bundles were also noted in crinkle nodules . Results suggest that the Crinkle gene controls the infection process that is crucial during the early stage of nodule organogenesis . Aside from the symbiotic phenotypes, crinkle mutants also developed morphological alterations, such as crinkly or wavy trichomes, short seedpods with aborted embryos, and swollen root hairs . crinkle is therefore required for symbiotic nodule development and for other aspects of plant development.

Plant Physiol, 2003 Mar, 131(3), 1027 - 32
Identification and characterization of nodulation-signaling pathway 2, a gene of Medicago truncatula involved in Nod actor signaling; Oldroyd GE et al.; Bacterially derived Nod factor is critical in the establishment of the legume/rhizobia symbiosis . Understanding the mechanisms of Nod factor perception and signal transduction in the plant will greatly advance our understanding of this complex interaction . Here, we describe the identification of a new locus, nodulation-signaling pathway 2 (NSP2), of Medicago truncatula that is involved in Nod factor signaling . Mutants at this locus are blocked for Nod factor-induced gene expression and show a reduced root hair deformation response . nsp2 plants also show a complete absence of infection and cortical cell division following Sinorhizobium meliloti inoculation . Nod factor-induced calcium spiking, one of the earliest responses tested, is still functional in these mutant plants . We conclude that the gene NSP2 is a component of the Nod factor signal transduction pathway that lies downstream of the calcium-spiking response.

Microbiology, 2003 Feb, 149(Pt 2), 537 - 46
The mid genes of Rhizobium sp strain TAL1145 are required for degradation of mimosine into 3-hydroxy-4-pyridone and are inducible by mimosine; Borthakur D et al.; Mimosine is a toxin present in the tree-legume leucaena (Leucaena leucocephala), including its root nodules and the root exudates . The leucaena-nodulating Rhizobium sp . strain TAL1145 degrades mimosine (Mid(+)) and utilizes it as a source of carbon and nitrogen . Twelve TAL1145 mutants defective in mimosine degradation (Mid(-)) were made through Tn3Hogus, TnphoA or kanamycin-resistance-cassette insertions . A 5.0 kb PstI fragment of TAL1145, subcloned from a cosmid clone containing mid genes for mimosine degradation, complemented most of the Mid(-) mutants . Sequencing this fragment and the adjacent 0.9 kb PstI fragment identified five genes, midA, midB, midC, midD and midR, of which the first three genes encode ABC transporter proteins involved in mimosine uptake, while midD encodes an aminotransferase required for degrading mimosine into 3-hydroxy-4-pyridone, and midR is a regulatory gene encoding a LysR-type transcriptional activator . The location of MidA in the periplasm was shown by making two midA : : phoA fusions, which made active alkaline phosphatase in the periplasm . The various mid : : gus and midA : : phoA fusions were inducible by mimosine, and a midD : : gus fusion mutant showed beta-glucuronidase activity in the leucaena nodules, indicating that midD is expressed in the nodules . Similarly, a midA : : phoA fusion expressed alkaline phosphatase activity in the leucaena nodules, indicating that mimosine induces midA transcription in the bacteroids . mid genes are specific for the Mid(+) strains of leucaena Rhizobium and are absent in strains of other Rhizobium, Sinorhizobium and Bradyrhizobium spp.

Appl Environ Microbiol, 2003 Mar, 69(3), 1573 - 80
Bacterial rRNA genes associated with soil suppressiveness against the plant-parasitic nematode Heterodera schachtii; Yin B et al.; The goal of this study was to identify bacteria involved in soil suppressiveness against the plant-parasitic nematode Heterodera schachtii . Since H . schachtii cysts isolated from the suppressive soil can transfer this beneficial property to nonsuppressive soils, analysis of the cyst-associated microorganisms should lead to the identification of the causal organisms . Our experimental approach was to identify bacterial rRNA genes (rDNA) associated with H . schachtii cysts obtained from soil mixtures with various levels of suppressiveness . We hypothesized that we would be able to identify bacteria involved in the suppressiveness by correlating population shifts with differing levels of suppressiveness . Soil treatments containing different amounts of suppressive and fumigation-induced nonsuppressive soils exhibited various levels of suppressiveness after two nematode generations . The 10%-suppressive-soil treatment contained numbers of eggs per gram of soil similar to those of the 100%-suppressive-soil treatment, indicating that the suppressive factor(s) had been transferred . Bacterial rDNA associated with H . schachtii cysts were identified using a culture-independent method termed oligonucleotide fingerprinting of rRNA genes . Bacteria from five major taxonomic groups (Actinobacteria, Cytophaga-Flexibacter-Bacteroides, alpha-Proteobacteria, beta-Proteobacteria, and gamma-Proteobacteria) were identified . Three bacterial rDNA groups contained clones that were more prevalent in the highly suppressive soil treatments than in the less suppressive treatments, indicating a potential involvement in the H . schachtii suppressiveness . When these three groups were examined with specific PCR analyses performed on H . schachtii cysts that developed in soils treated with three biocidal compounds, only one bacterial rDNA group with moderate to high sequence identity to rDNA from several Rhizobium species and uncultured alpha-proteobacterial clones was consistently associated with the highly suppressive treatments . A quantitative PCR analysis confirmed the association of this Rhizobium-like rDNA group with the H . schachtii suppressiveness.

FEMS Microbiol Lett, 2003 Feb 28, 219(2), 225 - 32
Activation of the nodA promoter by the nodD genes of Rhizobium galegae induced by synthetic flavonoids or Galega orientalis root exudate; Suominen L et al.; Rhizobial nodD genes produce transcriptional regulators that, together with appropriate inducer compounds, activate the other symbiotic nodulation (nod) genes and initiate the nodule formation process . Two nodD homologues, nodD1 and nodD2, are present in the Rhizobium galegae strain HAMBI 1174 . In this work we analysed their ability to induce the nodA promoter with synthetic inducers known to activate nod genes in other rhizobia . According to phylogenetic analysis, the inducer-specific carboxy-terminal part of the R . galegae nodD protein sequence groups together with those of Rhizobium leguminosarum and Sinorhizobium meliloti . However, the respective inducer compounds for their NodD proteins are not highly effective with R . galegae nodD products . The best inducer discovered with R . galegae nodD1 was the root exudate of the host plant of R . galegae, Galega orientalis . HPLC analyses revealed the presence of many divergent flavonoid compounds in the G . orientalis root exudate . The most effective HPLC fractions induced R . galegae nodD1 up to the level obtained by intact G . orientalis root exudate while apigenin and luteolin, which were also present in the root exudate, were only moderate inducers . A UV-Vis diode array spectrum of the most active peak indicated that the main inducer present in the G . orientalis root exudate is an unidentified chalcone-type compound . In the Galega-R . galegae interaction the first recognition between the NodD protein and the flavonoid inducer secreted from the roots of Galega is specific for these organisms, and thus partly responsible of the strict host specificity of this symbiosis.

Genome Biol . 2003;4(2):R15 . Epub 2003 Jan 31.
Transcriptome analysis of Sinorhizobium meliloti during symbiosis; Ampe F et al.; BACKGROUND: Rhizobia induce the formation on specific legumes of new organs, the root nodules, as a result of an elaborated developmental program involving the two partners . In order to contribute to a more global view of the genetics underlying this plant-microbe symbiosis, we have mined the recently determined Sinorhizobium meliloti genome sequence for genes potentially relevant to symbiosis . We describe here the construction and use of dedicated nylon macroarrays to study simultaneously the expression of 200 of these genes in a variety of environmental conditions, pertinent to symbiosis . RESULTS: The expression of 214 S . meliloti genes was monitored under ten environmental conditions, including free-living aerobic and microaerobic conditions, addition of the plant symbiotic elicitor luteolin, and a variety of symbiotic conditions . Five new genes induced by luteolin have been identified as well as nine new genes induced in mature nitrogen-fixing bacteroids . A bacterial and a plant symbiotic mutant affected in nodule development have been found of particular interest to decipher gene expression at the intermediate stage of the symbiotic interaction . S . meliloti gene expression in the cultivated legume Medicago sativa (alfalfa) and the model plant M . truncatula were compared and a small number of differences was found . CONCLUSIONS: In addition to exploring conditions for a genome-wide transcriptome analysis of the model rhizobium S . meliloti, the present work has highlighted the differential expression of several classes of genes during symbiosis . These genes are related to invasion, oxidative stress protection, iron mobilization, and signaling, thus emphasizing possible common mechanisms between symbiosis and pathogenesis.

J Bacteriol, 2003 Mar, 185(6), 1841 - 50
A Rhizobium leguminosarum AcpXL mutant produces lipopolysaccharide lacking 27-hydroxyoctacosanoic acid; Vedam V et al.; The structure of the lipid A from Rhizobium etli and Rhizobium leguminosarum lipopolysaccharides (LPSs) lacks phosphate and contains a galacturonosyl residue at its 4' position, an acylated 2-aminogluconate in place of the proximal glucosamine, and a very long chain omega-1 hydroxy fatty acid, 27-hydroxyoctacosanoic acid (27OHC28:0) . The 27OHC28:0 moiety is common in lipid A's among members of the Rhizobiaceae and also among a number of the facultative intracellular pathogens that form chronic infections, e.g., Brucella abortus, Bartonella henselae, and Legionella pneumophila . In this paper, a mutant of R . leguminosarum was created by placing a kanamycin resistance cassette within acpXL, the gene which encodes the acyl carrier protein for 27OHC28:0 . The result was an LPS containing a tetraacylated lipid A lacking 27OHC28:0 . A small amount of the mutant lipid A may contain an added palmitic acid residue . The mutant is sensitive to changes in osmolarity and an increase in acidity, growth conditions that likely occur in the nodule microenvironment . In spite of the probably hostile microenvironment of the nodule, the acpXL mutant is still able to form nitrogen-fixing root nodules even though the appearance and development of nodules are delayed . Therefore, it is possible that the acpXL mutant has a host-inducible mechanism which enables it to adapt to these physiological changes.

Rev Argent Microbiol, 2002 Oct-Dec, 34(4), 186 - 92
{Characterization of Rhizobium isolates from acid and alkaline soils in semi-arid regions of Pernambuco}; Da Silva ML et al.; The genetic variety of the Rhizobium isolates from acid and alkaline soils in the semiarid zone of Pernambuco state was evaluated through the use of 17 primers of arbitrary sequence . Amplified products were separated by electrophoresis in agarose gel at 1.4% and visualized by ethidium bromide coloration . The results obtained suggest a high genetic variety of the isolates in relation to the standard strain . Data were analyzed by UPGMA (unweighted pair-group method with arithmetic average), based on Jaccard's coefficient and visualized through dendrograms . The strains isolated from the acid soils were included in one group whereas the strains from alkaline soils were located in other three groups . Meanwhile, one of the groups formed by strain Isol-14, isolated from acid soils is more related to the groups of strains isolated from acid soils than to the remaining groups from alkaline soils.

J Exp Bot, 2003 Mar, 54(384), 1085 - 91
Leghaemoglobin oxygenation gradients in alfalfa and yellow sweetclover nodules; Denison RF et al.; Respiration in support of N(2) fixation by rhizobia in legume root nodules depends on an adequate supply of O(2), but excessive O(2) can damage nitrogenase, the key enzyme . The movement of O(2) into and within the nodule is driven by gradients in the concentration of O(2) or in the oxygenation of the O(2)-carrier, leghaemoglobin . Steeper gradients may increase flux to the sites of respiration, but gradients also raise the possibility of inadequate O(2) in some nodule zones and excessive O(2) in others . No detailed study of O(2) gradients in the interior of nodules has been published previously . Spectral changes in leghaemoglobin with oxygenation, previously used to measure the average O(2) status of the nodule interior, were used to map longitudinal gradients in O(2) and in respiratory capacity in the elongated nodules of alfalfa (Medicago sativa L.) and sweetclover (Melilotus officinalis L.) . Variability among nodules under air in the magnitude and direction of internal O(2) gradients was seen in both species . Despite consistently higher respiratory capacity near the meristematic tip, a majority of nodules had higher O(2) towards the tip than towards the base . These results contrast with a previous report, apparently based on limited data, but they are consistent with anatomical and tracer studies showing higher gas permeability near the tip.

Indian J Exp Biol, 2002 Jul, 40(7), 796 - 801
Growth behaviour and bioproduction of indole acetic acid by a Rhizobium sp . isolated from root nodules of a leguminous tree Dalbergia lanceolaria; Ghosh AC et al.; The Rhizobium sp . isolated from healthy and mature root nodules of a leguminous tree, Dalbergia lanceolaria Linn . f., preferred mannitol and KNO3 for growth as carbon and nitrogen sources, respectively . The bacterium produced a high amount (22.3 microg/ml) of indole acetic acid (IAA) from L-tryptophan supplemented basal medium . Growth and IAA production started simultaneously . IAA production was maximum at 20 hr when the bacteria reached the stationary phase of growth . Cultural requirements were optimized for maximum growth and IAA production . The IAA production by the Rhizobium sp . was increased by 270.8% over control when the medium was supplemented with mannitol (1%,w/v), SDS (1 microg/ml), L-asparagine (0.02%,w/v) and biotin (1 microg/ml) in addition to L-tryptophan (2.5 mg/ml) . The possible role of IAA production in the symbiosis is discussed.

Indian J Exp Biol, 2002 Jul, 40(7), 755 - 64
Role of rhizobial biosynthetic pathways of amino acids, nucleotide bases and vitamins in symbiosis; Randhawa GS et al.; Rhizobia require the availability of 20 amino acids for the establishment of effective symbiosis with legumes . Some of these amino acids are synthesized by rhizobium, whereas the remaining are supplied by the host plant . The supply from plant appears to be plant-type specific . Alfalfa provides arginine, cysteine, isoleucine, valine and tryptophan, and cowpea and soybean provide histidine . The production of ornithine and anthranilic acid, the intermediates in the biosynthetic pathways of arginine and tryptophan, respectively, seems to be essential for effective symbiosis of Sinorhizobium meliloti with alfalfa . The expression of ilvC gene of S . meliloti is required for induction of nodules on the roots of alfalfa plants . An undiminished metabolic flow through the rhizobial pathways for the synthesis of purines and pyrimidines and the synthesis of biotin, nicotinic acid, riboflavin and thiamine by rhizobium appear to be requirements for normal symbiosis . To the best of our knowledge, this is the first review article on the role of rhizobial biosynthetic pathways of amino acids, nucleotide bases and vitamins in rhizobium-legume symbiosis . The scientific developments of about 35 years in this field have been reviewed.

J Biol Chem, 2003 May 2, 278(18), 16356 - 64 Epub 2003 Feb 17.
A mannosyl transferase required for lipopolysaccharide inner core assembly in Rhizobium leguminosarum . Purification, substrate specificity, and expression in Salmonella waaC mutants; Kanipes MI et al.; The lipopolysaccharide (LPS) core domain of Gram-negative bacteria plays an important role in outer membrane stability and host interactions . Little is known about the biochemical properties of the glycosyltransferases that assemble the LPS core . We now report the purification and characterization of the Rhizobium leguminosarum mannosyl transferase LpcC, which adds a mannose unit to the inner 3-deoxy-d-manno-octulosonic acid (Kdo) moiety of the LPS precursor, Kdo(2)-lipid IV(A) . LpcC containing an N-terminal His(6) tag was assayed using GDP-mannose as the donor and Kdo(2)-{4'-(32)P}lipid IV(A) as the acceptor and was purified to near homogeneity . Sequencing of the N terminus confirmed that the purified enzyme is the lpcC gene product . Mild acid hydrolysis of the glycolipid generated in vitro by pure LpcC showed that the mannosylation occurs on the inner Kdo residue of Kdo(2)-{4'-(32)P}lipid IV(A) . A lipid acceptor substrate containing two Kdo moieties is required by LpcC, since no activity is seen with lipid IV(A) or Kdo-lipid IV(A) . The purified enzyme can use GDP-mannose or, to a lesser extent, ADP-mannose (both of which have the alpha-anomeric configuration) for the glycosylation of Kdo(2)-{4'-(32)P}lipid IV(A) . Little or no activity is seen with ADP-glucose, UDP-glucose, UDP-GlcNAc, or UDP-galactose . A Salmonella typhimurium waaC mutant, which lacks the enzyme for incorporating the inner l-glycero-d-manno-heptose moiety of LPS, regains LPS with O-antigen when complemented with lpcC . An Escherichia coli heptose-less waaC-waaF deletion mutant expressing the R . leguminosarum lpcC gene likewise generates a hybrid LPS species consisting of Kdo(2)-lipid A plus a single mannose residue . Our results demonstrate that heterologous lpcC expression can be used to modify the structure of the Salmonella and E . coli LPS cores in living cells.

J Biol Chem, 2003 May 2, 278(18), 16365 - 71 Epub 2003 Feb 17.
Relaxed sugar donor selectivity of a Sinorhizobium meliloti ortholog of the Rhizobium leguminosarum mannosyl transferase LpcC . Role of the lipopolysaccharide core in symbiosis of Rhizobiaceae with plants; Kanipes MI et al.; The lpcC gene of Rhizobium leguminosarum and the lpsB gene of Sinorhizobium meliloti encode protein orthologs that are 58% identical over their entire lengths of about 350 amino acid residues . LpcC and LpsB are required for symbiosis with pea and Medicago plants, respectively . S . meliloti lpsB complements a mutant of R . leguminosarum defective in lpcC, but the converse does not occur . LpcC encodes a highly selective mannosyl transferase that utilizes GDP-mannose to glycosylate the inner 3-deoxy-D-manno-octulosonic acid (Kdo) residue of the lipopolysaccharide precursor Kdo(2)-lipid IV(A) . We now demonstrate that LpsB can also efficiently mannosylate the same acceptor substrate as does LpcC . Unexpectedly, however, the sugar nucleotide selectivity of LpsB is greatly relaxed compared with that of LpcC . Membranes of the wild-type S . meliloti strain 2011 catalyze the glycosylation of Kdo(2)-{4'-(32)P}lipid IV(A) at comparable rates using a diverse set of sugar nucleotides, including GDP-mannose, ADP-mannose, UDP-glucose, and ADP-glucose . This complex pattern of glycosylation is due entirely to LpsB, since membranes of the S . meliloti lpsB mutant 6963 do not glycosylate Kdo(2)-{4'-(32)P}lipid IV(A) in the presence of any of these sugar nucleotides . Expression of lpsB in E . coli using a T7lac promoter-driven construct results in the appearance of similar multiple glycosyl transferase activities seen in S . meliloti 2011 membranes . Constructs expressing lpcC display only mannosyl transferase activity . We conclude that LpsB, despite its high degree of similarity to LpcC, is a much more versatile glycosyltransferase, probably accounting for the inability of lpcC to complement S . meliloti lpsB mutants . Our findings have important implications for the regulation of core glycosylation in S . meliloti and other bacteria containing LpcC orthologs.

Indian J Exp Biol, 2002 Sep, 40(9), 981 - 8
Nitrogen control of bacterial signal production in Rhizobium meliloti-alfalfa symbiosis; Dusha I; Under nitrogen-depleted conditions nitrogen-fixing soil bacteria of the family Rhizobiaceae are able to induce symbiotic nodules on the roots of leguminous plants where bacteroids convert atmospheric nitrogen to ammonia . The presence of exogenous nitrogen source inhibits the development and the functioning of bacterium-plant symbiosis . Earlier experiments demonstrated that nitrate inhibited all stages of symbiotic interaction, affecting primarily the host functions . The investigation of the possible involvement of the microsymbiont in nitrogen regulation showed that two signalling steps were controlled by ammonium . The synthesis of the first bacterial signal, the Nod factor was repressed by ammonium . The nitrogen signal is conveyed to nodulation (nod) genes by the general nitrogen regulatory (ntr) system and by the nodD3-syrM self-amplifying system . The fine control also involves a negative regulatory factor, ntrR . When ntrR is mutated, more efficient nodule formation and nitrogen fixation is observed in symbiosis with alfalfa even in the presence of ammonium . The biosynthesis of the second bacterial signal succinoglycan is also controlled by ammonium . SyrM, a common regulatory factor for nod and exo gene expression, may contribute to the adjustment of the amount of succinoglycan and the ratio of its biologically active form.

Syst Appl Microbiol, 2002 Dec, 25(4), 592 - 602
Soils of the Chinese Hubei province show a very high diversity of Sinorhizobium fredii strains; Camacho M et al.; Biodiversity studies of native soybean-nodulating rhizobia in soils from the Chinese Hubei province (Honghu county; pH 8, alluvial soil) have been carried out . Inoculation of an American (Williams) and an Asiatic (Peking) soybean cultivar with eleven soil samples led to the isolation of 167 rhizobia strains . The ratio (%) of slow-/fast-growing isolates was different depending on the trap plant used . All isolates were able to nodulate both cultivars, although the N2-fixation efficiency (measured as plant-top dry weight) was different among them . A total of thirty-three isolates were selected for further characterisation on the basis of physiological parameters, PCR-RFLP of symbiotic genes and Low Molecular Weight RNA, lipopolysaccharide, protein and plasmid profiles . Low Molecular Weight RNA profiling indicates that all the isolates belong to species Sinorhizobium fredii . The dendrogram obtained with the physiological parameters has been useful to classify the isolates at strain level, although plasmid profiling was the most discriminating technique to detect differences among the analysed soybean-rhizobia isolates, showing there is not two isolates identical each other . Plasmid profile analyses also revealed that some of the investigated strains contain low molecular weight plasmids (7-8-kb) . They are, to our knowledge, the smallest ever found in rhizobia and they could be the starting point for the construction of the first group of vectors based on a native rhizobia replicon.

Theor Appl Genet, 2002 Jun, 104(8), 1312 - 1316 Epub 2002 May 8.
Mapping of the nodulation loci sym9 and sym10 of pea ( Pisum sativum L.); Schneider A et al.; Several mutants defective in the nodulation process during rhizobial or endomycorrhizal endosymbiosis of pea have been identified previously . We have integrated the map positions of two such nodulation mutations, sym9 and sym10, into the molecular map of pea by applying molecular-marker techniques combined with bulked segregant analysis (BSA) . Lines P2 and P54 were found to carry alleles of sym9, line P56 carried an allele of sym10 . F2 populations were derived from crosses of P2, P54 and P56, to JI281 and JI15, two of the parental lines that have been used previously to generate a molecular map of pea . sym9 was located on linkage group IV by AFLP-BSA analysis and subsequently mapped by RFLP in both F2 populations, P2 x JI281 and P54 x JI281 . RFLP-BSA analysis was applied to assign sym10 to linkage group I . The RFLP marker locus, chs2, co-segregates with sym10 in the F2 population of P56 x JI15.

Mol Plant Microbe Interact, 2003 Jan, 16(1), 83 - 91
Salicylic acid inhibits indeterminate-type nodulation but not determinate-type nodulation; van Spronsen PC et al.; LCOs (lipochitin oligosaccharides, Nod factors) produced by the rhizobial symbiote of Vicia sativa subsp . nigra (vetch, an indeterminate-type nodulating plant) are mitogenic when carrying an 18:4 acyl chain but not when carrying an 18:1 acyl chain . This suggests that the 18:4 acyl chain specifically contributes to signaling in indeterminate-type nodulation . In a working hypothesis, we speculated that the 18:4 acyl chain is involved in oxylipin signaling comparable to, for example, signaling by derivatives of the 18:3 fatty acid linolenic acid (the octadecanoid pathway) . Because salicylic acid (SA) is known to interfere with oxylipin signaling, we tested whether nodulation of vetch could be affected by addition of 10(-4) M SA . This concentration completely blocked nodulation of vetch by Rhizobium leguminosarum bv . viciae and inhibited the mitogenic effect of 18:4 LCOs but did not affect LCO-induced root-hair deformation . SA did not act systemically, and only biologically active SA derivatives were capable of inhibiting nodule formation . SA also inhibited R . leguminosarum bv . viciae association with vetch roots . In contrast, addition of SA to Lotus japonicus (a determinate-type nodulating plant responding to 18:1 LCOs) did not inhibit nodulation by Mesorhizobium loti . Other indeterminate-type nodulating plants showed the same inhibiting response toward SA, whereas SA did not inhibit the nodulation of other determinate-type nodulating plants . SA may be a useful tool for studying fundamental differences between signal transduction pathways of indeterminate- and determinate-type nodulating plants.

Mol Plant Microbe Interact, 2003 Jan, 16(1), 65 - 73
BacS: an abundant bacteroid protein in Rhizobium etli whose expression ex planta requires nifA; Jahn OJ et al.; Rhizobium etli CFN42 bacteroids from bean nodules possessed an abundant 16-kDa protein (BacS) that was found in the membrane pellet after cell disruption . This protein was not detected in bacteria cultured in tryptone-yeast extract . In minimal media, it was produced at low oxygen concentration but not in a mutant whose nifA was disrupted . N-terminal sequencing of the protein led to isolation of a bacS DNA fragment . DNA hybridization and nucleotide sequencing revealed three copies of the bacS gene, all residing on the main symbiotic plasmid of strain CFN42 . A stretch of 304 nucleotides, exactly conserved upstream of all three bacS open reading frames, had very close matches with the NifA and sigma 54 consensus binding sequences . The only bacS homology in the genetic sequence databases was to three hypothetical proteins of unknown function, all from rhizobial species . Mutation and genetic complementation indicated that each of the bacS genes gives rise to a BacS polypeptide . Mutants disrupted or deleted in all three genes did not produce the BacS polypeptide but were Nod+ and Fix+ on Phaseolus vulgaris.

J Plant Res, 2002 Dec, 115(6), 439 - 47 Epub 2002 Sep 26.
A white clover nodulin gene, dd23b, encoding a cysteine cluster protein, is expressed in roots during the very early stages of interaction with Rhizobium leguminosarumbiovar trifolii and after treatment with chitolipooligosaccharide Nod factors; Crockard A et al.; An early nodulin cDNA, dd23b, was isolated from white clover root tissue by differential display RT-PCR . Its full-length sequence of 340 nucleotides encodes a predicted 72-amino-acid protein of molecular mass 8.3 kDa, with a polypeptide region containing cysteine pairs spaced in the manner of a cysteine cluster protein . This feature, which is shared by some other late and early nodulins from pea and broad bean, suggests a role in metal ion binding and membrane transport . Temporal and spatial expression patterns were determined during infection and nodulation by the homologous microsymbiont . No expression was found in unchallenged root tissue over a 7-day sampling period . Expression was first detectable in roots by RT-PCR 6 h post-inoculation with Rhizobium leguminosarum biovar trifolii, placing dd23b among the earliest nodulins to be detected to date . In root nodules, expression occurred primarily in the central symbiotic zone, but also in some host cells within the infection zone . Addition of purified wild-type chitolipooligosaccharide Nod factor to axenic white clover roots induced dd23b expression, providing further evidence for the role of this gene in the early plant response to infection by rhizobia.

Int J Immunopathol Pharmacol, 2003 Jan-Apr, 16(1), 55 - 60
Purification and characterisation of two GST's forms from Rhizobium leguminosarum with a high affinity to herbicides; Faraone A et al.; Cytosolic glutathione transferases are a family of multifunctional proteins that catalyse the conjugation of GSH to a large variety of endogenous and exogenous compounds . These enzymes have been widely studied in mammals and, to a lesser extent, in plants . In plants, GSTs can detoxify herbicides; they are also induced by pathogenic infection and are likely to be involved in defence responses . GSTs are found in pathogenic and not pathogenic prokaryotes but the functional role played by these enzymes in the cell still remains to be clarified . Here we report the purification and characterisation of two GST forms from Rhizobium leguminosarum that play a very important role in agriculture by inducing nitrogen-fixing nodules on the roots of legumes . These bacterial GSTs from R . leguminosarum have immunological characteristics that are different among them and they are characterised both by a high affinity to herbicides.

Mol Plant Microbe Interact, 2003 Feb, 16(2), 159 - 68
Genetic analysis of a pH-regulated operon from Rhizobium tropici CIAT899 involved in acid tolerance and nodulation competitiveness; Vinuesa P et al.; Rhizobium tropici CIAT899 is highly acid tolerant and a good competitor for Phaseolus vulgaris nodule occupancy at low pH values . Using Tn5 mutagenesis, we identified an operon required for acid tolerance and nodulation competitiveness . The insertion was mapped to the 5' end of atvA, encoding a product with high sequence identity to the agro-bacterial AcvB virulence protein . Complementation analyses indicated that atvA is an ortholog of acvB, both genes being required for acid tolerance . A Ser/Ala substitution in the LIPASE_SER motif of AtvA resulted in an acid sensitive Fix+ but very poorly competing strain, demonstrating that Ser-313 is essential for AtvA function . atvA is the second gene in an operon that is transcriptionally upregulated by acid shock . The acid-responsive promoter was mapped to a 469-bp intergenic region located upstream of lpiA, the first gene in the operon . lpiA-like genes are found in several alpha, beta, and gamma Proteobacteria that interact with eukaryotic host cells, and they are predicted to encode membrane proteins related to the FmtC/MprF family from low G+C Firmicutes . The latter proteins are involved in resistance to cationic antimicrobial peptides . A nonpolar deletion in lpiA caused a sevenfold decrease in relative nodulation competitiveness.

Appl Environ Microbiol, 2003 Feb, 69(2), 1067 - 74
Symbiotic and genetic diversity of Rhizobium galegae isolates collected from the Galega orientalis gene center in the Caucasus; Andronov EE et al.; This paper explores the relationship between the genetic diversity of rhizobia and the morphological diversity of their plant hosts . Rhizobium galegae strains were isolated from nodules of wild Galega orientalis and Galega officinalis in the Caucasus, the center of origin for G . orientalis . All 101 isolates were characterized by genomic amplified fragment length polymorphism fingerprinting and by PCR-restriction fragment length polymorphism (RFLP) of the rRNA intergenic spacer and of five parts of the symbiotic region adjacent to nod box sequences . By all criteria, the R . galegae bv . officinalis and R . galegae bv . orientalis strains form distinct clusters . The nod box regions are highly conserved among strains belonging to each of the two biovars but differ structurally to various degrees between the biovars . The findings suggest varying evolutionary pressures in different parts of the symbiotic genome of closely related R . galegae biovars . Sixteen R . galegae bv . orientalis strains harbored copies of the same insertion sequence element; all were isolated from a particular site and belonged to a limited range of chromosomal genotypes . In all analyses, the Caucasian R . galegae bv . orientalis strains were more diverse than R . galegae bv . officinalis strains, in accordance with the gene center theory.

Appl Environ Microbiol, 2003 Feb, 69(2), 884 - 93
Rhizobium etli and Rhizobium gallicum nodulate common bean (Phaseolus vulgaris) in a traditionally managed milpa plot in Mexico: population genetics and biogeographic implications; Silva C et al.; The stability of the genetic structure of rhizobial populations nodulating Phaseolus vulgaris cultivated in a traditionally managed milpa plot in Mexico was studied over three consecutive years . The set of molecular markers analyzed (including partial rrs, glnII, nifH, and nodB sequences), along with host range experiments, placed the isolates examined in Rhizobium etli bv . phaseoli and Rhizobium gallicum bv . gallicum . Cluster analysis of multilocus enzyme electrophoresis and plasmid profile data separated the two species and identified numerically dominant clones within each of them . Population genetic analyses showed that there was high genetic differentiation between the two species and that there was low intrapopulation differentiation of the species over the 3 years . The results of linkage disequilibrium analyses are consistent with an epidemic genetic structure for both species, with frequent genetic exchange taking place within conspecific populations but not between the R . etli and R . gallicum populations . A subsample of isolates was selected and used for 16S ribosomal DNA PCR-restriction fragment length polymorphism analysis, nifH copy number determination, and host range experiments . Plasmid profiles and nifH hybridization patterns also revealed the occurrence of lateral plasmid transfer among distinct multilocus genotypes within species but not between species . Both species were recovered from nodules of the same plants, indicating that mechanisms other than host, spatial, or temporal isolation may account for the genetic barrier between the species . The biogeographic implications of finding an R . gallicum bv . gallicum population nodulating common bean in America are discussed.

Planta, 2003 Feb, 216(4), 674 - 85 Epub 2002 Sep 26.
Nod factors activate both heterotrimeric and monomeric G-proteins in Vigna unguiculata (L.) Walp; Kelly MN et al.; Nod factors are lipo-chito-oligosaccharides secreted by rhizobia that initiate many responses in the root hairs of the legume hosts, culminating in deformed hairs . The heterotrimeric G-protein agonists mastoparan, Mas7, melittin, compound 48/80 and cholera toxin provoke root hair deformation, whereas the heterotrimeric G-protein antagonist pertussis toxin inhibits mastoparan and Nod factor NodNGR{S}- (from Rhizobiumsp . NGR234) induced root hair deformation . Another heterotrimeric G-protein antagonist, isotetrandrine, only inhibited root hair deformation provoked by mastoparan and melittin . These results support the notion that G-proteins are implicated in Nod factor signalling . To study the role of G-proteins at a biochemical level, we examined the GTP-binding profiles of root microsomal membrane fractions isolated from the nodulation competent zone of Vigna unguiculata(L.) Walp . GTP competitively bound to the microsomal membrane fractions labelled with {(35)S}GTPgammaS, yielding a two-site displacement curve with displacement constants ( K(i)) of 0.58 micro M and 0.16 mM . Competition with either ATP or GDP revealed a one-site displacement curve with K(i) of 4.4 and 29 micro M, respectively, whereas ADP and UTP were ineffective competitors . The GTP-binding profiles of microsomal membrane fractions isolated from roots pretreated with either NodNGR{S} or the four-sugar, N- N'- N"- N'"-tetracetylchitotetraose (TACT) backbone of Nod factors were significantly altered compared with control microsomal fractions . To identify candidate proteins, membrane proteins were separated by SDS-PAGE and electrotransferred to nitrocellulose . GTP overlay experiments revealed that membrane fractions isolated from roots pretreated with NodNGR{S} or TACT contained two proteins (28 kDa and 25 kDa) with a higher affinity for GTPgammaS than control membrane fractions . Western analysis demonstrated that membranes from the pretreated roots contained more of another protein (~55 kDa) recognised by Galpha(common) antisera . These results provide pharmacological and biochemical evidence supporting the contention that G-proteins are involved in Nod factor signalling and, importantly, implicate monomeric G-proteins in this process.

J Biol Chem, 2003 Apr 11, 278(15), 12946 - 54 Epub 2003 Feb 03.
Sinorhizobium meliloti acpXL mutant lacks the C28 hydroxylated fatty acid moiety of lipid A and does not express a slow migrating form of lipopolysaccharide; Sharypova LA et al.; Lipid A is the hydrophobic anchor of lipopolysaccharide (LPS) in the outer membrane of Gram-negative bacteria . Lipid A of all Rhizobiaceae is acylated with a long fatty acid chain, 27-hydroxyoctacosanoic acid . Biosynthesis of this long acyl substitution requires a special acyl carrier protein, AcpXL, which serves as a donor of C28 (omega-1)-hydroxylated fatty acid for acylation of rhizobial lipid A (Brozek, K.A., Carlson, R.W., and Raetz, C . R . (1996) J . Biol . Chem . 271, 32126-32136) . To determine the biological function of the C28 acylation of lipid A, we constructed an acpXL mutant of Sinorhizobium meliloti strain 1021 . Gas-liquid chromatography and mass spectrometry analysis of the fatty acid composition showed that the acpXL mutation indeed blocked C28 acylation of lipid A . SDS-PAGE analysis of acpXL mutant LPS revealed only a fast migrating band, rough LPS, whereas the parental strain 1021 manifested both rough and smooth LPS . Regardless of this, the LPS of parental and mutant strains had a similar sugar composition and exposed the same antigenic epitopes, implying that different electrophoretic profiles might account for different aggregation properties of LPS molecules with and without a long acyl chain . The acpXL mutant of strain 1021 displayed sensitivity to deoxycholate, delayed nodulation of Medicago sativa, and a reduced competitive ability . However, nodules elicited by this mutant on roots of M . sativa and Medicago truncatula had a normal morphology and fixed nitrogen . Thus, the C28 fatty acid moiety of lipid A is not crucial, but it is beneficial for establishing an effective symbiosis with host plants . acpXL lies upstream from a cluster of five genes, including msbB (lpxXL), which might be also involved in biosynthesis and transfer of the C28 fatty acid to the lipid A precursor.

Indian J Exp Biol, 2000 Sep, 38(9), 856 - 62
Mechanism of plant growth promotion by rhizobacteria; Gupta A et al.; Plant growth results from interaction of roots and shoots with the environment . The environment for roots is the soil or planting medium which provide structural support as well as water and nutrients to the plant . Roots also support the growth and functions of a complex of microorganisms that can have a profound effect on the growth anti survival of plants . These microorganisms constitute rhizosphere microflora and can be categorized as deleterious, beneficial, or neutral with respect to root/plant health . Beneficial interactions between roots and microbes do occur in rhizosphere and can be enhanced . Increased plant growth and crop yield can be obtained upon inoculating seeds or roots with certain specific root-colonizing bacteria- 'plant growth promoting rhizobacteria' . In this review, we discuss the mechanisms by which plant growth promoting rhizobacteria may stimulate plant growth.

Ying Yong Sheng Tai Xue Bao, 2002 Sep, 13(9), 1145 - 9
{Conformation transformation of lead in rhizosphere}; Lin Q et al.; With rhizoboxes, this study dealt with the distribution and conformation transformation of lead in the rhizosphere and non-rhizosphere of wheat and rice grown on red soil . The results showed that the predominant forms of lead were acid dissolved and Carb-Pb (bound to carbonates), Exch-Pb (exchangeable-Pb), and FeMnO-Pb (boud to iron and manganese oxides) . Exch-Pb in the rhizosphere of both rice and wheat was much higher than those in the non-rhizosphere, which means that the activation process in the rhizosphere was very strong, and bio-available Pb greatly increased . Various levels of Pb treatment and Pb-Cd interaction also had certain effects on the distribution of lead . In treatment of low Pb concentration, FeMnO-Pb was higher than Exch-Pb, but the contrary result was observed in treatment of high Pb concentration . Both Exch-Pb and FeMnO-Pb in wheat rhizosphere decreased with an increase of Cd . Exch-Pb in rice rhizosphere was correlative to the Cd content in soil . Compared with the treatment in the absence of Cd, the activation of Exch-Pb in rice rhizosphere was weaker in 5 mg Cd.kg-1 treatment, but stronger in 10 mg Cd.kg-1 treatment.

Ying Yong Sheng Tai Xue Bao, 2002 Sep, 13(9), 1095 - 8
{Obstacles of soybean continuous cropping, III . Mechanism of soybean yield increment by marine actinomyces MB-97}; Hu J et al.; An isolate of marine actinomyces MB-97 identified as Streptomyces microflavus could successfully colonize in the rhizoshpere of soybean, and inhibit Penicillium purpurogenum, a soybean deleterious rhizospheric microorganisms . After applied MB-97, the ratio of bacteria/fungi in the rhizosphere of soybean was increased, and the soil became to be "Bacterial type" from "Fungal type" . The populations of P . purpurogenum were apparently suppressed about 80%, and the harm of toxins in soil was weak . The soybean root rot caused by soilborne fungi such as Fusarium was decreased 50%, and MB-97 could stimulate the growth of soybean seedlings . In field study, the mean yield of soybean raised by 15.2%, implying that Streptomyces microflavus was an effective plant growth-promoting rhizobacteria on soybean.

Can J Microbiol, 2002 Nov, 48(11), 947 - 54
Strategies used by rhizobia to lower plant ethylene levels and increase nodulation; Ma W et al.; Agriculture depends heavily on biologically fixed nitrogen from the symbiotic association between rhizobia and plants . Molecular nitrogen is fixed by differentiated forms of rhizobia in nodules located on plant roots . The phytohormone, ethylene, acts as a negative factor in the nodulation process . Recent discoveries suggest several strategies used by rhizobia to reduce the amount of ethylene synthesized by their legume symbionts, decreasing the negative effect of ethylene on nodulation . At least one strain of rhizobia produces rhizobitoxine, an inhibitor of ethylene synthesis . Active 1-aminocyclopropane-1-carboxylate (ACC) deaminase has been detected in a number of other rhizobial strains . This enzyme catalyzes the cleavage of ACC to alpha-ketobutyrate and ammonia . It has been shown that the inhibitory effect of ethylene on plant root elongation can be reduced by the activity of ACC deaminase.

Wei Sheng Wu Xue Bao, 1999 Aug, 39(4), 287 - 95
{Study on polyphasic taxonomy of rhizobia isolated from Lespedeza species}; Yao Z et al.; The diversity of rhizobia isolated from Lespedeza spp . was determined on the basis of numerical analysis of phenotypic characteristics, sodium dodecyl sulfate-polyacrylarmide gel electrophoresis (SDS-PAGE) of proteins, DNA-DNA homology and restriction fragment length polymorphism (RFLP) analysis of 16S-ribosomal DNA genes . According to numerical analysis of 125 phenotypic characteristics, strains were divided into two groups at a similarity level of 67% . Group I included all the fast-growing strains, group II included all the slow-growing strains . Above the similarity level of 80%, four subgroups could be further divided . Subgroup I was fast-growing rhizobia containing representative strain of Sinorhizobium saheli . Subgroup II, III, IV were slow-growing rhizobia . Subgroup II composed of strains isolated from Lespedeza cuneata in Beijing area and these isolates produced acid in medium containing mannitol . Subgroup III included type strain of Bradyrhizobium japonicum . The DNA G + C contents and DNA-DNA homology of the members of above four subgroups were determined . The subgroup I shared the same DNA homologous group with S . saheli, subgroup III belonged to B . japonicum, subgroup IV belonged to B . elkanii, subgroup II was an unique DNA homologous group which showed low level of DNA relatedness with other slow-growing rhizobia species . RFLP analysis of 16S rDNA genes verified that the subgroup II was a distinctive genealine and showed genetic variation within the strains in it.

Wei Sheng Wu Xue Bao, 1999 Oct, 39(5), 416 - 25
{Cloning, sequencing and expression of Sinorhizobium meliloti strain 042B gene nodD}; Yang X et al.; S . meliloti strain 042B is a rhizobium strain which can form nodules both on alfalfa and on soybean . In this study, nodD gene of 042B was cloned into pBBR1MCS-5 . By functional analysis in the system of R . leguminosarum bv . viciae LPR5045 (pSym-, pMP154), it was found that the NodD has responses both to luteolin and to genistein . This result showed that the 042B nodD gene is probably a specific nodulation determinant which determined its capability of nodulation on both alfalfa and soybean . The nodD fragment was then cloned into the expressional vector pThioHis A, B and C, and three recombinant plasmids pXDA, pXDB and pXDC were constructed . The plasmid pXDC was identified to be in the same open reading frame with the trxA gene of pThioHis C throuth sequencing analysis . Inducing IPTG and analyzing with SDS-PAGE, it was found that the fusion protein expressed from E . coli Top10(pXDC) with the molecular weight of TrXA and NodD together . The Western bolt result demonstrated that the expression result is the target gene nodD product.

Wei Sheng Wu Xue Bao, 1999 Oct, 39(5), 387 - 95
{A study on taxonomy of rhizobia isolated from Kummerwia sp . and Indigofera sp.}; Wei G et al.; The rhizobial strains isolated from Kummerowia and Indigofera and the known reference strains were classified by performing numerical taxonomy . New isolated strains were divided into two new clusters at 83% similarity level . Based on the numerical taxonomy, additional isolates in each cluster were studied by using SDS-PAGE of whole-cell protein . Twenty-four strains isolated from Kummerowia fell into cluster 1 . Twenty strains isolated from Indigofera fell into cluster 2 . The results of G + C mol% and DNA homology analysis showed that the DNA homologies between the central strain SH713 and SHL042 and the 13 type strains were less than 61% . Thus, the rhizobial strains from Kummerowia and Indigofera were two new individual species of Rhizobium.

Wei Sheng Wu Xue Bao, 1999 Apr, 39(2), 95 - 9
{Studies on DNA-DNA hybridization and 16S rDNA sequence of rhizobia isolated from Shapotou desert soil in Ningxia autonomous region of China}; Li Y et al.; Based on the previous studies on numerical taxonomy and multilocus enzymes electrophoresis patterns of the 12 rhizobial strains isolated from Shapotou region, the contents of G + C mol%, DNA-DNA relatedness and 16SrDNA sequence of the representative strain were tested . The DNA G + C content of the members of this group ranged from 56.4 to 62.2 . The values of DNA-DNA hybridization within the group were above 70%, and relatendness between representative strains of this group and known rhizobial species was below 66.6% . The full-length of 16S rDNA sequence of representative strain N220 was compared with the type strains of all known rhizobia species and related bacteria by the PHLIP version 3.572c composed a unrooted phylogenetic tree, the strain N220, R . galegea, two unnamed rhizobial strains(SH19312, SH22623) and three A-grobacterium strains constituted a branch in this tree . The similarity values of 16S rDNA sequence between strain N220 and other strains in this branch were above 95%.

Wei Sheng Wu Xue Bao, 1999 Feb, 39(1), 43 - 8
{Using lux genes marker technique to track Pseudomonas chlororaphis PL9L in cotton rhizosphere}; Bai J et al.; Tn7-luxCDABE marker system was successfully transferred into Pseudomonas chlororaphis (strain PL9) by means of transformation and conjugation and a stable lux-marked strain PL9L was obtained . The colonizing dynamics and distribution of the luminescent bacteria PL9L in the rhizosphere of cotton planted in pots and rhizoboxes were studied by the methods of X-ray film imaging and enumeration of luminescent colonies on agar media, The results of pot culture experiment showed that PL9L successfully colonized in the rhizosphere of cotton . In pot cultures of sterile soil the highest colonizing level(3.1 x 10(2) cfu/g root soil) was reached on 6th day after seeds sown; On 56th day, the population of PL9L tended to stable and decreased to 1.7 x 10(9) cfu/g root soil) but in pot cultures of unsterile soil, the highest colonizing level(1.1 x 10(9) cfu/g root soil) was reached on 8th day . On 46th day, the population of PL9L tended to a stationary state, the numbers of them were 1.4 x 10(2) cfu/g root soil . The results of rhizobox culture experiment showed that PL9L spread from seeds toward the direction of root tip, but not synchronized with the stretch of roots . 6 days after seeds sown, in rhizobox culture of sterile soil, PL9L spread 12.0 cm below seeds, but in non-sterile soil was 11.0 cm . In the region of cotton root tip, PL9L were not detected.

BMC Microbiol . 2003 Jan 28;3(1):1.
Characterization of the nodulation plasmid encoded chemoreceptor gene mcpG from Rhizobium leguminosarum; Yost CK et al.; BACKGROUND: In general, chemotaxis in Rhizobium has not been well characterized . Methyl accepting chemotaxis proteins are sensory proteins important in chemotaxis of numerous bacteria, but their involvement in Rhizobium chemotaxis is unclear and merits further investigation . RESULTS: A putative methyl accepting chemotaxis protein gene (mcpG) of Rhizobium leguminosarum VF39SM was isolated and characterized . The gene was found to reside on the nodulation plasmid, pRleVF39d . The predicted mcpG ORF displayed motifs common to known methyl-accepting chemotaxis proteins, such as two transmembrane domains and high homology to the conserved methylation and signaling domains of well-characterized MCPs . Phenotypic analysis of mcpG mutants using swarm plates did not identify ligands for this putative receptor . Additionally, gene knockouts of mcpG did not affect a mutant strain's ability to compete for nodulation with the wild type . Notably, mcpG was found to be plasmid-encoded in all strains of R . leguminosarum and R . etli examined, though it was found on the nodulation plasmid only in a minority of strains . CONCLUSIONS: Based on sequence homology R . leguminosarum mcpG gene codes for a methyl accepting chemotaxis protein . The gene is plasmid localized in numerous Rhizobium spp . Although localized to the sym plasmid of VF39SM mcpG does not appear to participate in early nodulation events . A ligand for McpG remains to be found . Apparent McpG orthologs appear in a diverse range of proteobacteria . Identification and characterization of mcpG adds to the family of mcp genes already identified in this organism.

Wei Sheng Wu Xue Bao, 2001 Aug, 41(4), 421 - 6
{Studies on transference of hydrogenase genes of Rhizobium arachis}; Wang Z et al.; The hydrogen-uptake genes were transferred into wild Rhizobium arachis Ra strains (Hup-, Nif+, Apr) by triparental mating using pRK2013 as help plasmid . A transconjugant R . arachis Rz34-2(Hup+, Nif+, Apr, Tcr) which expressed high activities of hydrogenase and nitrogenase under free-living and symbiotic state was screened . Peanut inoculation test with recipient R . arachis Ra34, transcojugant Rz34-2 and control strain R . arachis L8-3 (Hup+, Nif+) was carried out respectively . The results showed that, compare to treatment without inoculation, inoculation with R . arachis Ra34 and R . arachis L8-3, the dry weight of leaf inoculated with transconjuant Rz34-2 increased 6.2%, 7.6% and 6.3% respectively; the N-content of seed increased 8.8%, 10.0% and 6.0%; the output increased 18.8%, 10.5% and 10.7% . This suggested that legume plants inoculated with Rhizobium strains (Hup+) were more efficient to accumulate N and to increase its output.

Wei Sheng Wu Xue Bao, 2001 Dec, 41(6), 662 - 8
{Use transposon and parDE through in vivo cloning to promote the genetic stability of plasmid pCPP430}; Zhang L et al.; Plasmid pCPP430 carrying the hrp gene cluster after transformated into 308R (Pantoea agglomerans) can cause the hypersensitive response, and simultaneously induce the plant resistance to disease . It was genetically unstable in P . agglomerans . 0.8 kb parDE region of the broad-host range plasmid RK2 is responsible for plasmid partition . It can mediate plasmid maitenance of many kinds in Rhizobium meliloti, and also can promote the genetic stability of recombinant plasmid in the biocontrol bacteria P . agglomerans . In this paper, we cloned parDE into pCPP430 in vivo through transposition to promote its genetic stability . parDE was amplified by PCR, inserted into pGEM-T vector and cut out and religated to NotI-cut transposon vector pUT/mini-Tn5 Km to get a parDE containing mini-Tn5, pTnp . After conjugation between S17-1/lambda pir (pTnp) and 308R (pCPP430), parDE was cloned in vivo into plasmid pCPP430 to obtain pRTnp . It was demonstrated that the insertion of parDE in pCPP430 increased significantly the plasmid's stability in P . agglomerans.

Wei Sheng Wu Xue Bao, 1998 Jun, 38(3), 225 - 8
{Cloning and analysis of the regulatory gene fragment induced with seed extract in Rhizobium huakuii strain 7653R}; Nong G et al.; The large plasmids of strain 7653R were digested with restriction enzyme EcoRI . Their DNA fragments were cloned into the expression vector pMP220 to construct a lacZ fusion pool, which were transferred into the recipient strain 7653R . Tri-transconjugants were selected onto plates containing X-gal and seed extract . Five blue colonies were assayed of their beta-galactosidase activity after incubation with or without seed extract . A positive induced strain HN18 was obtained . Hybridization of nodDABC probe on the recombinant plasmid pHN18 showed a 1.7 kb positive band . The evidence makes a deduction that the pHN18 contains a promoter of nod operon.

Wei Sheng Wu Xue Bao, 1998 Jun, 38(3), 219 - 24
{Studies on fermentation conditions of cytokinin produced by rhizobia strain 4012a}; Jia X et al.; The conditions of cytokinin fermentation of the rhizobia strain 4012a were detected by the ELISA . The results indicated that the optimal medium for cytokinin production by strain 4012a was composed of glucose 10 g/L, (NH4)2SO4 1.0, K2HPO4.3H2O 0.6, MgSO4.7H2O 0.1, CaCl2.2H2O 0.4, FeCl3.6H2O 0.04, Na2MoO4.2H2O 0.1 mg/L, calcium pantothenate 100 micrograms/L, adenine 200 mg/L . When strain 4012a was grown in 250 ml flask containing 50 ml of the medium on the rotary shaker (150 r/min) at 27 degrees C for 96 h, the yield of CTK 908 micrograms/L culture solution was obtained . It displayed bioactivity kinetin equivalents (KE) 1 mg/L medium with the radish cotyledon expansion test.

Wei Sheng Wu Xue Bao, 1998 Jun, 38(3), 213 - 8
{luxAB genes as marker for detecting Rhizobium fredii HN01 nodulation functions}; Mo C et al.; A suicide plasmid pHNC3 which contains Tn5-luxAB was transferred into Rhizobium fredii HN01 by the help of pRK2013 . Then Tn5-luxAB inserted on the genome of HN01 and gave luminescence activity . The luminescence colonies were picked up and the Eckhardt gel was performed for plasmids profile detection . The location of Tn5-luxAB on the genome was determined using the luxAB as probe . The colonies which were marked by Tn5-luxAB on the chromosome and different plasmids of HN01 were chosen for pot experiment, and a chromosome labelled strain HN01LC02 was detected by soil pot experiment . The detections included the nodulation occupancy and the luminescent nodules distribution on the root system formed by the luxAB-marked rhizobia.

Wei Sheng Wu Xue Bao, 2001 Feb, 41(1), 113 - 6
{Analysis on 16S rDNA sequence of rhizobia isolated from Kummerowla sp.}; Wei G et al.; Based on the previous studies on numerical taxonomy, SDS-PAGE of whole-cell protein and DNA hybridization, the rhizobial strains isolated from Kummerowia sp . in semi-arid area of North-west constituted a new subgroup, the 16S rDNA sequence of representative strain SH714 were tested . The unrooted phylogenetic tree was produced . In this tree, the strain SH714 with Sinorhizobium xinjiangensis, S . fredii, S . meliloti, S . medicae, S . saheli and S . teranga constituted a branch of Sinorhizobium . Within this branch, the similarity valuse of 16S rDNA sequence between strain SH714 and S . xinjiangesis, S . fredii, S . meliloti, S . medicae, S . saheli and S . teranga were 97.4%, 97.5%, 96.8%, 96.7%, 97.2% and 95.6% respectively, the values were more than 95%, this indicated that these known species should belong to the same genus . The values of DNA homology between type strains of these species were less than 70% . Thus, the strain SH714 represented a new rhizobial species, and there were some diversity between SH714 and known rhizobial species in phenotypic feature and composition of protein.

Wei Sheng Wu Xue Bao, 2001 Jun, 41(3), 287 - 92
{Introduction of the chromogenic gene to the plant growth-promoting rhizobacteria of cucumber}; Chen X et al.; Using a bicomponent transposition system with the E . coli lacZY gene cloned between Tn7 termini, a sensitive, selectable marker based on expression of the E . coli lac operon genes encoding beta-galactosidase and lactose permease was transformed into the rifampicin resistant mutant of plant growth-promoting rhizobacteria of cucumber, Pseudomonas aeruginosa CN116 and Pseudomonas corrugata CN31, respectively . Transformants were conferred the ability to utilize lactose as a sole carbon source and the ability to cleave the chromogenis substrate X-Gal to show a specific blue color . Southern blotting analysis showed that lacZY gene was inserted into the genome DNA of target strains . Compared with the wild type strains, the cultural characters, morphological features, growth promoting and disease control effects of transformants were almost unchanged, except the new marked phenotype . This marker system enabled the detection of lac+ transformants at sensitivity of 10 CFU/g soil, which makes the further studies on PGPR more easily.

Wei Sheng Wu Xue Bao, 2000 Jun, 40(3), 296 - 300
{Study on the improvement of plasmid stability in soybean rhizobia}; Liu M et al.; By using luxAB as the report genes, 3.2 kb parCBA/DE gene fragment from pTR102 was inserted into pLAFR3 which contained a 3.7 kb enhancing fragment and deleted its cos site . Recombinant plasmids pHN155 and pHN156 were obtained . Contrasted plasmids pHN157 and pHN158 which contained cos site were also constructed . These four plasmids were transferred into Sinorhizobium fredii HN01 by tri-parental mating, and plasmid pHN155 and pHN158 were introduced into Bradyrhizobium japonicum TA11 by two-parental mating . The stability of the above plasmids was compared under free-living conditions and the results showed that the parCBA/DE could obviously enhance plasmid stability both in S . fredii and B . japonicum, and deletion of cos site showed only less effect.

Wei Sheng Wu Xue Bao, 2000 Aug, 40(4), 339 - 45
{Genetic diversity of rhizobia of Medicago edgeworthii by AFLP and RFLP analysis}; Feng R; Two hundred and ninety one isolates of rhizobia sampled from root nodules of Medicago edgeworthii which came from Yun Nan Province in china were analyzed by amplified restriction fragment polymorphism(AFLP) technique . According to the AFLP banding patterns, the results showed most of these isolates were genetically diversity . Ninety isolates were selected as representing the diversity among the 291 isolates . The 90 isolates were clustered into three groups at the level of similarity of 79% by computer analysis of the data . Additional representative isolates and reference strains were identified by restriction fragment length polymorphism (RFLP) analysis of PCR-Amplified 16S rDNA . Two different 16S rDNA PCR-RFLP types were found, which indicated that the isoltes were phylogentically closely related to Rhizobium mongolense.

Wei Sheng Wu Xue Bao, 2000 Oct, 40(5), 513 - 7
{Isolation of moniliformin-degrading bacterium Ochrobactrum sp . and analysis of its functional properties}; Chen W et al.; A moniliformin(MON)-degrading bacterium strain, named as Y21-2, was isolated from the mycotoxin-contaminated soil from Heilongjiang Province by the enrichment microculture technique . This strain can grow with MON as its sole carbon and energy source . In the minimal medium with 500 micrograms/mL MON, the number of cells increased from 10(7) to 10(10) . Traditional taxonomy, assays of its G + C content and 16S rDNA sequence homology identified Y21-2 as Rhizobiaceae, Ochrobactrum sp . Resting cell suspensions prepared from induced Y21-2 can degrade MON with great speed, which also suggested the existence of enzymes committed to MON degradation in the cell.

Environ Pollut, 2003, 122(3), 447 - 54
Changes of copper speciation in maize rhizosphere soil; Tao S et al.; Chemical forms of copper in the rhizosphere and bulk soil of maize were investigated using rhizobox cultivation and sequential extraction techniques . The copper accumulations were also determined . The results demonstrated that there were continuous changes in copper fractionation within the maize rhizosphere . Initially, the amount of exchangeable copper increased before dropping below the initial level after 40 days or so . Carbonate associated copper followed a similar trend of change, but with a slower pace than the exchangeable copper . The increase in carbonate associated copper only become evident after 30 days, with the net loss occurring after 60 days . There were also initial increases in oxide bound copper as well as decreases in the organic matter associated copper, both followed by a turnover after 40-50 days . The accumulation of copper in the maize plant was found to be biomass dependent . The amount of accumulated copper absorbed in the plant material exceeded the initial quantity of the exchangeable copper in the soil, revealing a transformation from less bioavailable to more bioavailable fractions . During cultivation, decreases in redox potential and increases in pH, dissolved organic carbon (DOC), and microbial activity in the maize rhizosphere were observed . The change in copper speciation may result from root-induced changes in DOC, redox potential, and microbial activity in the rhizosphere.

Microb Ecol, 2003 Feb, 45(2), 137 - 44 Epub 2003 Jan 28.
Arbuscular mycorrhizal symbiosis changes the colonization pattern of Acacia tortilis spp . Raddiana rhizosphere by two strains of rhizobia; Andre S et al.; The aim of the study was to assess the effect of the mycorrhizosphere of A . tortillis spp . raddiana mycorrhized with Glomus intraradices on the root nodulation by Sinorhizobium terangae (ORS 1009) and/or Mesorhizobium plurifarium (ORS 1096) in two different culture substrates (sandy soil and sand) . The endomycorrhizal fungus only stimulated plant growth in the sandy soil . Moreover, arbuscular mycorrhizal infection enhanced the nodulation process in both culture substrates . Beside the stimulatory effects of the mycorrhizosphere on both rhizobia development, fungal symbiosis induces two different dynamics of each bacterial strains in the sand-grown plants . These results suggest specific relationships could occur during the development of the tripartite symbiosis, at physiological and molecular level . From a practical point of view, the role of arbuscular mycorrhizas in improving nodulation and N2 fixation is universally recognized . The fungal symbiosis could modify the development of bacterial inoculants along the root systems . This effect is of particular interest in the controlled inoculation of selected rhizobia.

Carbohydr Res, 2003 Jan 31, 338(3), 237 - 50
Structural determination of the lipo-chitin oligosaccharide nodulation signals produced by Rhizobium giardinii bv . giardinii H152; Soria-Diaz ME et al.; Rhizobium giardinii bv . giardinii is a microsymbiont of plants of the genus Phaseolus and produces extracellular signal molecules that are able to induce deformation of root hairs and nodule organogenesis . We report here the structures of seven lipochitooligosaccharide (LCO) signal molecules secreted by R . giardinii bv . giardinii H152 . Six of them are pentamers of GlcNAc carrying C 16:0, C 18:0, C 20:0 and C 18:1 fatty acyl chains on the non-reducing terminal residue . Four are sulfated at C-6 of the reducing terminal residue and one is acetylated in the same position . Six of them are N-methylated on the non-reducing GlcN residue and all the nodulation factors are carbamoylated on C-6 of the non-reducing terminal residue . The structures were determined using monosaccharide composition and methylation analyses, 1D- and 2D-NMR experiments and a range of mass spectrometric techniques . The position of the carbamoyl substituent on the non-reducing glucosamine residue was determined using a CID-MSMS experiment and an HMBC experiment.

J Biol Chem, 2003 Apr 4, 278(14), 12109 - 19 Epub 2003 Jan 15.
An outer membrane enzyme that generates the 2-amino-2-deoxy-gluconate moiety of Rhizobium leguminosarum lipid A; Que-Gewirth NL et al.; The structures of Rhizobium leguminosarum and Rhizobium etli lipid A are distinct from those found in other Gram-negative bacteria . Whereas the more typical Escherichia coli lipid A is a hexa-acylated disaccharide of glucosamine that is phosphorylated at positions 1 and 4', R . etli and R . leguminosarum lipid A consists of a mixture of structurally related species (designated A-E) that lack phosphate . A conserved distal unit, comprised of a diacylated glucosamine moiety with galacturonic acid residue at position 4' and a secondary 27-hydroxyoctacosanoyl (27-OH-C28) as part of a 2' acyloxyacyl moiety, is present in all five components . The proximal end is heterogeneous, differing in the number and lengths of acyl chains and in the identity of the sugar itself . A proximal glucosamine unit is present in B and C, but an unusual 2-amino-2-deoxy-gluconate moiety is found in D-1 and E . We now demonstrate that membranes of R . leguminosarum and R . etli can convert B to D-1 in a reaction that requires added detergent and is inhibited by EDTA . Membranes of Sinorhizobium meliloti and E . coli lack this activity . Mass spectrometry demonstrates that B is oxidized in vitro to a substance that is 16 atomic mass units larger, consistent with the formation of D-1 . The oxidation of the lipid A proximal unit is also demonstrated by matrix-assisted laser desorption ionization time-of-flight mass spectrometry in the positive and negative modes using the model substrate, 1-dephospho-lipid IV(A) . With this material, an additional intermediate (or by product) is detected that is tentatively identified as a lactone derivative of 1-dephospho-lipid IV(A) . The enzyme, presumed to be an oxidase, is located exclusively in the outer membrane of R . leguminosarum as judged by sucrose gradient analysis . To our knowledge, an oxidase associated with the outer membranes of Gram-negative bacteria has not been reported previously.

Planta, 2003 Jan, 216(3), 437 - 45 Epub 2002 Nov 15.
A host-specific bacteria-to-plant signal molecule (Nod factor) enhances germination and early growth of diverse crop plants; Prithiviraj B et al.; Lipo-chitooligosaccharides (LCOs), or Nod factors, are host-specific bacteria-to-plant signal molecules essential for the establishment of a successful N(2)-fixing legume-rhizobia symbiosis . At submicromolar concentrations Nod factors induce physiological changes in host and non-host plants . Here we show that the Nod factor Nod Bj V(C18:1,MeFuc) of Bradyrhizobium japonicum 532C enhances germination of a variety of economically important plants belonging to diverse botanical families: Zea mays, Oryza sativa (Poaceae), Beta vulgaris (Chenopodaceae), Glycine max, Phaseolus vulgaris (Fabaceae), and Gossypium hirsutum (Malvaceae), under laboratory, greenhouse and field conditions . Similar increases in germination were observed for filtrates of genistein-induced cultures of B . japonicum 532C, while non-induced B . japonicum, induced Bj 168 (a nodC mutant of B . japonicum deficient in Nod factor synthesis) or the pentamer of chitin did not invoke such responses, demonstrating the role of Nod factor in the observed effects . In addition, three out of four synthetic LCOs evaluated also promoted germination of corn, soybean and Arabidopsis thaliana seeds . LCO also enhanced the early growth of corn seedlings under greenhouse conditions . These findings suggest the possible use of LCOs for improved crop production.

Zh Obshch Biol, 2002 Nov-Dec, 63(6), 451 - 72
{Comparative genetics and evolutionary morphology of symbiosis formed by plants with nitrogen-fixing microbes and endomycorrhizal fungi}; Provorov NA et al.; Results of comparative morphological and genetic analyses are described for two major plant-microbe endosymbioses: N2-fixing nodules (with rhizobia or actinomycetes Frankia) and arbuscular mycorrhiza (with Glomales fungi) . Development from the primordia formed de novo in root tissues is common for all known types of N2-fixing nodules . However, their structure varies greatly with respect to: (i) tissue topology (location of vascular bundles is peripheral in legumes but central in non-legumes); (ii) position of nodule primordium (inner or outer cortex in legumes, whereas pericycle in non-legumes); (iii) stability of apical meristem (persistent in the indeterminate nodules, transient in the determinate ones) . In addition, legumes vary in ability to form compartments harboring endosymbiotic rhizobia that can be located intercellularly (infection threads) and intracellularly (symbiosomes) . Using pea (Pisum sativum) symbiotic mutants, the nodule developmental program is dissected into a range of spatially and temporarily differentiated steps composing four sub-programs (development of endosymbiotic compartments; nodule histogenesis; autoregulation of nodulation; bacteroid differentiation) . The developmental mutations are suggested in some cases to reverse the endosymbiotic system into the morphologically simpler forms some of which may correspond to the ancestral stages of nodule evolution . Origination of legume-rhizobial and actinorhizal symbioses is suggested to be based on a set of preadaptations many of which had been evolved in angiosperms during coevolution with arbuscular mycorrhizal fungi (e.g . inter- and intracellular maintenance of symbionts, their control via defence-like reactions and recognition of chitin-like molecules) . Analysis of parallel morphological variation in symbiotic mutants and wild-growing legume species enables us to reconstruct the major stages of evolution for N2-fixing symbioses . This evolution proceeded to a sufficient degree independently from the basic physiological function of nodules (symbiotic N2-fixation) and possibly a recruiting of plant genes that initially fulfilled various "non-symbiotic" functions into the genetic networks monitoring plant-microbe interactions.

Int J Syst Evol Microbiol, 2002 Nov, 52(Pt 6), 2231 - 9
Rhizobium indigoferae sp . nov . and Sinorhizobium kummerowiae sp . nov., respectively isolated from Indigofera spp . and Kummerowia stipulacea; Wei GH et al.; Forty-eight rhizobial isolates from root nodules of Indigofera and Kummerowia, two genera of annual or perennial wild legumes growing in the Loess Plateau in north-western China, were characterized by a polyphasic approach . Two main groups, cluster 1 and cluster 2, were defined based upon the results of numerical taxonomy, SDS-PAGE of whole-cell proteins and DNA relatedness . All the isolates within cluster 1 were isolated from Indigofera and they were identified as Rhizobium strains by 16S rRNA gene analysis . DNA relatedness of 29.5-48.9% was obtained among the cluster 1 isolates and the reference strains for defined Rhizobium species . Cluster 2 consisted of isolates from Kummerowia stipulacea and was identified as belonging to Sinorhizobium by 16S rRNA gene analyses . DNA relatedness varied from 5.2 to 41.7% among the isolates of cluster 2 and reference strains for Sinorhizobium species . Considering the existence of distinctive features among these two groups and related species within the genera Rhizobium and Sinorhizobium, we propose two novel species, Rhizobium indigoferae sp . nov . for cluster 1, with isolate CCBAU 71714(T) (= AS 1.3046(T)) as the type strain, and Sinorhizobium kummerowiae sp . nov . for cluster 2, with isolate CCBAU 71042(T) (= AS 1.3045(T)) as the type strain.

Int J Syst Evol Microbiol, 2002 Nov, 52(Pt 6), 2219 - 30
Characterization of rhizobia that nodulate legume species of the genus Lespedeza and description of Bradyrhizobium yuanmingense sp . nov; Yao ZY et al.; Legume species belonging to the genus Lespedeza are annual or perennial herb or shrub plants that grow in the northern hemisphere . They are known for the formation of root nodules, but little information is available about their microsymbionts . In this study, 58 root-nodule isolates from Lespedeza spp., obtained from China and the USA, were characterized using numerical taxonomic analysis of phenotypic features, SDS-PAGE analysis of whole-cell proteins, DNA-DNA hybridization, 16S rRNA gene sequence analysis and cross-nodulation with selected legume species . From the results generated using these approaches, it was concluded that Lespedeza spp . were promiscuous hosts for rhizobia . Four main clusters of bacteria, which included 35 of the strains isolated from Lespedeza spp., were defined upon numerical taxonomic analysis; these groups corresponded to those determined from analyses of protein electrophoretic and DNA-DNA hybridization data . The four clusters were found to define strains belonging to one of four species, Sinorhizobium saheli, Bradyrhizobium japonicum, Bradyrhizobium elkanii or a novel species of the genus Bradyrhizobium . The strains of B . japonicum and B . elkanii were all from the USA soil samples, and their representative strains could not nodulate soybean . The seven strains found to represent the novel Bradyrhizobium sp . were from China . These were differentiated from recognized species of the genus Bradyrhizobium by all of the taxonomic methods used here; hence, it is proposed that the novel strains isolated from Lespedeza spp . represent a novel species of the genus Bradyrhizobium, Bradyrhizobium yuanmingense . The type strain of the novel species, CCBAU 10071(T) (= CFNEB 101(T)), formed ineffective nodules on Medicago sativa and Melilotus albus but did not nodulate soybean . The other 23 bacterial strains isolated from Lespedeza spp . were found to form single branches or small groups (two to three strains) that were related to Bradyrhizobium, Mesorhizobium, Rhizobium and Sinorhizobium spp . on the basis of numerical taxonomic analysis, indicating the possibility that other rhizobial species are also associated with Lespedeza spp.

Int J Syst Evol Microbiol, 2002 Nov, 52(Pt 6), 1953 - 60
Farnesyl diphosphate synthase gene of three phototrophic bacteria and its use as a phylogenetic marker; Cantera JJ et al.; Farnesyl diphosphate (FPP) synthase is essential not only for phototrophic bacteria in carotenoid biosynthesis, but also for non-phototrophic bacteria in the biosynthesis of physiologically important compounds . The gene encoding FPP synthase was assessed as a molecular marker to investigate the intermingled relationship between the phototropic and non-phototropic bacteria in the alpha-Proteobacteria based on 16S rRNA analysis . The FPP synthase amino acid sequences from three phototropic bacteria, Rhodobacter sphaeroides ATCC 11167(T), Rhodobacter capsulatus ATCC 11166(T) and Rhodovulum sulfidophilum W4(T), were determined and used in conjunction with sequences of other representative members of the alpha-, gamma- and epsilon-Proteobacteria and the low-G+C Gram-positive bacteria for phylogenetic analyses by the neighbour-joining and maximum-likelihood methods . The overall topology of the FPP synthase gene tree is consistent with that of the 16S rRNA tree, producing a distinct cluster of the three phototropic bacteria . A minor discordance between the two trees was observed in the cluster of the non-phototrophic Bradyrhizobiumjaponicum USDA 110 and Mesorhizobium loti MAFF 303099; the FPP synthase genes of these two rhizobial species are highly homologous as compared with their respective 16S rRNA . The results suggest that the FPP synthase and 16S rRNA genes have the same evolutionary pattern, evolving vertically from each common ancestral gene; the FPP synthase gene, therefore, could possibly be used for further study on the molecular systematics of photosynthetic bacteria.

Water Res, 2003 Jan, 37(2), 441 - 9
Metabolism of the soil and groundwater contaminants, ethylene dibromide and trichloroethylene, by the tropical leguminous tree, Leuceana leucocephala; Doty SL et al.; Ethylene dibromide (EDB; dibromoethane) and trichloroethylene (TCE) are hazardous environmental pollutants . The use of plants to treat polluted sites and groundwater, termed phytoremediation, requires plants that can both effectively remove the pollutant as well as grow in the climatic region of the site . In this paper, we report that the tropical leguminous tree, Leuceana leucocephala var . K636, is able to take up and metabolize EDB and TCE . The plants were grown in sterile hydroponic solution without its symbiont, Rhizobium . EDB and TCE were both metabolized by the plant, as indicated by the formation of bromide ion from EDB and trichloroethanol from TCE . Each plant organ was independently capable of debromination of EDB . L . leucocephala is being used to treat perched groundwater as part of a remedial alternative to address an accidental EDB spill in Hawaii . Bromide levels of plant tissues from the trees grown in the phytoremediation treatment cells at the Hawaii Site were elevated, indicating uptake and degradation of brominated compounds in the trees . This report is the first evidence of a tropical tree effectively metabolizing these common organic pollutants.

J Gen Appl Microbiol, 1999 Oct, 45(5), 213 - 220
Characterization of high temperature-tolerant rhizobia isolated from Prosopis juliflora grown in alkaline soil; Kulkarni S et al.; A method was developed for the fast screening and selection of high-temperature tolerant rhizobial strains from root nodules of Prosopis juliflora growing in alkaline soils . The high-temperature tolerant rhizobia were selected from 2,500 Rhizobium isolates with similar growth patterns on yeast mannitol agar plates after 72 h incubation at 30 and 45 degrees C, followed by a second screening at 47.5 degrees C . Seventeen high-temperature tolerant rhizobial strains having distinguishable protein band patterns were finally selected for further screening by subjecting them to temperature stress up to 60 degrees C in yeast mannitol broth for 6 h . The high-temperature tolerant strains were NBRI12, NBRI329, NBRI330, NBRI332, and NBRI133 . Using this procedure, a large number of rhizobia from root nodules of P . juliflora were screened for high-temperature tolerance . The assimilation of several carbon sources, tolerance to high pH and salt stress, and ability to nodulate P . juliflora growing in a glasshouse and nursery of the strains were studied . All five isolates had higher plant dry weight in the range of 29.9 to 88.6% in comparison with uninoculated nursery-grown plants . It was demonstrated that it is possible to screen in nature for superior rhizobia exemplified by the isolation of temperature-tolerant strains, which established effective symbiosis with nursery-grown P . juliflora . These findings indicate a correlation between strain performance under in vitro stress in pure culture and strain behavior under symbiotic conditions . Pure culture evaluation may be a useful tool in search for Rhizobium strains better suited for soil environments where high temperature, pH, and salt stress constitutes a limitation for symbiotic biological nitrogen fixation.

Acta Crystallogr D Biol Crystallogr, 2003 Jan, 59(Pt 1), 166 - 7 Epub 2002 Dec 19.
Crystallization and preliminary X-ray crystallographic analysis of malonyl-CoA decarboxylase from Rhizobium leguminosarum bv . trifolii; Jung JS et al.; Malonyl-CoA decarboxylase (MCD), which catalyzes the conversion of malonyl-CoA to acetyl-CoA, is an evolutionarily distinct and highly conserved enzyme . MCD does not share sequence homology with other known decarboxylases, while the enzymes from different species exhibit at least >30% sequence identity to each other . In order to provide a canonical structure of the enzyme for detailed study of its structure-function relationship, the MCD of Rhizobium leguminosarum bv . trifolii was overexpressed and crystallized . The crystals belong to the orthorhombic space group P2(1)2(1)2, with unit-cell parameters a = 133.45, b = 127.10, c = 66.37 A . The asymmetric unit is likely to contain two molecules of MCD (molecular weight of 51 418 Da), with a crystal Volume per protein weight (V(M)) of 2.69 A(3) Da(-1) and a solvent content of about 54.3% by Volume . A native data set to 3.0 A resolution was obtained using a rotating-anode X-ray generator.

Proc Natl Acad Sci U S A, 2002 Dec 24, 99(26), 16678 - 83 Epub 2002 Dec 16.
A polyphosphate kinase (PPK2) widely conserved in bacteria; Zhang H et al.; Synthesis of inorganic polyphosphate (poly P) from the terminal phosphate of ATP is catalyzed reversibly by poly P kinase (PPK, now designated PPK1) initially isolated from Escherichia coli . PPK1 is highly conserved in many bacteria, including some of the major pathogens such as Pseudomonas aeruginosa . In a null mutant of P . aeruginosa lacking ppk1, we have discovered a previously uncharacterized PPK activity (designated PPK2) distinguished from PPK1 by the following: synthesis of poly P from GTP or ATP, a preference for Mn2+ over Mg2+, and a stimulation by poly P . The reverse reaction, a poly P-driven nucleoside diphosphate kinase synthesis of GTP from GDP, is 75-fold greater than the forward reaction, poly P synthesis from GTP . The gene encoding PPK2 (ppk2) was identified from the amino acid sequence of the protein purified near 1,000-fold, to homogeneity . The 5'-end is 177 bp upstream of the annotated genome sequence of a "conserved hypothetical protein"; ppk2 (1,074 bp) encodes a protein of 357 aa with a molecular mass of 40.8 kDa . Sequences homologous to PPK2 are present in two other proteins in P . aeruginosa, in two Archaea, and in 32 other bacteria (almost all with PPK1 as well); these include rhizobia, cyanobacteria, Streptomyces, and several pathogenic species . Distinctive features of the poly P-driven nucleoside diphosphate kinase activity and structural aspects of PPK2 are among the subjects of an accompanying report.

Mol Plant Microbe Interact, 2002 Dec, 15(12), 1245 - 52
Analysis of differences between Sinorhizobium meliloti 1021 and 2011 strains using the host calcium spiking response; Wais RJ et al.; In the Rhizobium-legume symbiosis, compatible partners recognize each other through an exchange of signals . Plant inducers act together with bacterial transcriptional activators, the NodD proteins, to regulate the expression of bacterial biosynthetic nodulation (nod) genes . These genes direct the synthesis of a lipochito-oligosaccharide signal called Nod factor (NF) . NFs elicit an early host response, root hair calcium spiking, that is initiated in root hair cells within 15 min of NF or live Rhizobium inoculation . We used calcium spiking as an assay to compare two closely related strains of Sinorhizobium meliloti, Rm1021 and Rm2011, derived from the same field isolate . We found that the two strains show a kinetic difference in the calcium spiking assay: Rm1021 elicits calcium spiking in host root hairs as rapidly as purified NF, whereas Rm2011 shows a significant delay . This difference can be overcome by raising expression levels of either the NodD transcriptional activators or GroEL, a molecular chaperone that affects expression of the biosynthetic nod genes . We further demonstrate that the delay in triggering calcium spiking exhibited by Rm2011 is correlated with a reduced amount of nod gene expression compared with Rm1021 . Therefore, calcium spiking is a useful tool in detecting subtle differences in bacterial gene expression that affect the early stages of the Rhizobium-legume symbiosis.

Mol Plant Microbe Interact, 2002 Dec, 15(12), 1228 - 35
Mutational and transcriptional analysis of the type III secretion system of Bradyrhizobium japonicum; Krause A et al.; Sequencing the symbiotic region of Bradyrhizobium japonicum revealed a gene cluster (tts) encoding a type III secretion system (TTSS) that is similar to those found in Mesorhizobium loti MAFF303099 and Rhizobium strain NGR234 . In addition to genes that are likely to encode structural core components of the TTSS, the cluster contains several open reading frames that are found exclusively in rhizobia or that are specific to B . japonicum . Depending on the host, mutations within this cluster affected nodulation capacity to different extents . One of the genes likely encodes a transcriptional activator (TtsI) of the two-component regulatory family . Upstream of ttsI, a nod box promoter was identified . Expression of ttsI could be induced by genistein . This induction depended on the transcriptional activator protein NodW as well as the nodD1nodD2nolA gene region . TtsI was found to be involved in transcriptional regulation of the tts gene cluster . Sequence comparison revealed a conserved tts box element within putative promoter regions of several genes . Here, we propose a model of the regulatory cascade leading to the induction of the tts gene cluster.

Microbiology, 2002 Dec, 148(Pt 12), 4059 - 71
RirA, an iron-responsive regulator in the symbiotic bacterium Rhizobium leguminosarum; Todd JD et al.; Mutations in a Rhizobium leguminosarum gene, rirA (rhizobial iron regulator), caused high-level, constitutive expression of at least eight operons whose transcription is normally Fe-responsive and whose products are involved in the synthesis or uptake of siderophores, or in the uptake of haem or of other iron sources . Close homologues of RirA exist in other rhizobia and in the pathogen Brucella; many other bacteria have deduced proteins with more limited sequence similarity . None of these homologues had been implicated in Fe-mediated gene regulation . Transcription of rirA itself is about twofold higher in cells grown in Fe-replete than in Fe-deficient growth media . Mutations in rirA reduced growth rates in Fe-replete and -depleted medium, but did not appear to affect symbiotic N(2) fixation.

FEMS Microbiol Lett, 2002 Dec 17, 217(2), 255 - 61
Oleomonas sagaranensis gen . nov., sp . nov., represents a novel genus in the alpha-Proteobacteria; Kanamori T et al.; A Gram-negative bacterium was previously isolated from an oil field in Shizuoka, Japan, and designated strain HD-1 . Here we have performed detailed characterization of the strain, and have found that it represents a novel genus . The 16S rRNA sequence of strain HD-1 displayed highest similarity to various uncultured species (86.7-99.7%), along with 86.2-88.2% similarity to sequences from Azospirillum, Methylobacterium, Rhizobium, and Hyphomicrobium, all members of the alpha-Proteobacteria . Phylogenetic analysis revealed that HD-1 represented a deep-branched lineage among the alpha-Proteobacteria . DNA-DNA hybridization analysis with Azospirillum lipoferum and Hyphomicrobium vulgare revealed low levels of similarity among the strains . We further examined the biochemical properties of the strain under aerobic conditions . Among carbon sources, ethanol, n-propanol, n-butanol, and n-tetradecanol were the most preferred, while acetate, propionate, and pyruvate also supported high levels of growth . The strain could also grow on aromatic compounds such as toluene, benzene and phenol, and aliphatic hydrocarbons such as n-octane and n-tetradecane . In contrast, glycerol and various sugars, including glucose, fructose, maltose, and lactose, failed to support growth of HD-1 . Under an anaerobic gas phase with butanol as the carbon source, little increase in cell weight was observed with the addition of several possible electron acceptors . As strain HD-1 represents a novel genus in the alpha-Proteobacteria, we designated the strain as Oleomonas sagaranensis gen . nov., sp . nov., strain HD-1 .

Biochem Biophys Res Commun, 2002 Dec 20, 299(5), 780 - 6
RNase E is involved in 5'-end 23S rRNA processing in alpha-Proteobacteria; Klein F et al.; In Rhodobacter capsulatus and Rhizobium leguminosarum, an internal transcribed spacer consisting of helices 9 and 10 is removed during 23S rRNA processing, which leads to the occurrence of a 5.8S-like rRNA . The particular rRNA maturation steps are not known, with exception of the initial RNase III cleavage in helix 9 . We found that GC-rich stem-loop structures of helix 9, which are released by RNase III, are immediately degraded . The degradation of helix 10 is slower and its kinetics differs in both species . Nevertheless, the helix 10 processing mechanism is conserved and includes cleavages by RNase E.

J Gen Appl Microbiol, 2002 Aug, 48(4), 181 - 91
Symbiotic root nodule bacteria isolated from yam bean (Pachyrhizus erosus); Fuentes JB et al.; A total of 25 isolates from root nodules of yam bean (Pachyrhizus erosus L . Urban), a tuber-producing leguminous plant, were characterized . All isolates formed effective nodules mainly on lateral roots while edible tubers were developed on the taproot . The root nodules formed were identified as the typical determinate type . By an analysis of the partial sequences of the 16S rRNA gene (approximately 300 bp) of 10 strains which were selected randomly, the isolated root nodule bacteria of yam bean were classified into two different genera, Rhizobium and Bradyrhizobium . Two strains, YB2 (Bradyrhizobium group) and YB4 (Rhizobium group) were selected and used for further analyses . The generation time of each strain was shown to be 22.5 h for strain YB2 and 0.8 h for strain YB4, respectively . Differences between strains YB2 and YB4 were also reflected in the bacteroid state in the symbiosome . Symbiosome in nodule cells for the strain YB4 contained one bacteroid cell in a peribacteroid membrane, whereas a symbiosome for strain YB2 contained several bacteroid cells.

Microb Ecol, 2003 Jan, 45(1), 72 - 87 Epub 2002 Dec 10.
Inability to find consistent bacterial biocontrol agents of Pythium aphanidermatum in cucumber using screens based on ecophysiological traits; Folman LB et al.; A collection of 821 rhizobacteria from cucumber, originating from different root locations and stages of plant development, was screened for potential biocontrol agents of Pythium aphanidermatum (Edson) Fitzp . The screening procedure exploited carbon source utilization profiles and growth rates of bacteria as indicators of a partial niche overlap with the pathogen . The bacteria were tested for growth on nine carbon sources (glucose, fucose, sucrose, maltose, asparagine, alanine, galacturonic acid, succinic acid, and linoleic acid), most of which are reported to be used by the zoospores of P . aphanidermatum in the infection process . The isolates were classified as fast- or slow-growing, depending on their growth rate in 1/10 strength TSB . By nonhierarchical cluster analysis, 20 clusters were generated of bacteria with similar profiles of carbon source utilization . Redundancy analysis showed that the type of root sample explained 47% of the variance found in the relative abundance of bacteria from the clusters . Bacteria from clusters using none or few of the carbon sources, e.g., maltose and linoleic acid, with many slow-growing isolates, showed a preference for plants in the vegetative or generative stage, or for old root regions (root base) . Bacteria from clusters with fast-growing isolates, using many carbon sources, were relatively abundant in the seedling stage . A selection of 127 bacteria from the different clusters was tested for disease suppressive capabilities in bioassays on young cucumber plants in nutrient solution, inoculated with zoospores of P . aphanidermatum . Nine of these bacteria produced biosurfactants, and 27 showed antibiosis against mycelial growth in plate assays . For 31 isolates, significant positive effects on plant biomass were shown, as analyzed with a general linear regression model . For most isolates, these effects occurred only in one of two replicate assays and no reductions in the degree of root and crown rot were found . Of the isolates that used many of the tested carbon sources, only four had positive effects on plant biomass . The majority of the isolates that positively affected plant biomass used few to moderate numbers of carbon sources and did not produce antibiotics or biosurfactants . In conclusion, competition for the tested carbon sources with the zoospores did not play a decisive role in disease suppression, and no clear relation was found between ecophysiological traits and disease suppression . Only isolate 3.1T8, isolated from root tips in the generative stage of plant growth, significantly increased plant biomass and suppressed root and crown rot symptoms in five out of six bioassays . The isolate produced an antifungal substance in plate assays and showed biosurfactant production in several (cucumber-derived) media.

Glycobiology, 2002 Nov, 12(11), 741 - 8
Symbiotic conditions induce structural modifications of Sinorhizobium sp . NGR234 surface polysaccharides; Fraysse N et al.; When the rhizosphere is starved of nitrogen, the soil bacteria Rhizobium are able to infect legume roots and invade root nodules, where they can fix atmospheric nitrogen . Nod boxes, the nod gene promoters located on the rhizobial symbiotic plasmid, are activated by means of flavonoids present in the legume root exudates, leading to the synthesis of lipochitooligomers: the Nod factors . Several recent works pointed out the importance of rhizobial surface polysaccharides in establishing the highly specific symbiosis between rhizobia and legumes . Lipopolysaccharides (LPSs) exhibit specific active roles in the later stages of the nodulation processes, such as the penetration of the infection thread into the cortical cells or the setting up of the nitrogen-fixing phenotype . The study reported here concerns the structural modifications affecting surface (lipo)polysaccharides when Sinorhizobium sp . NGR234 strains are grown with nod gene induction under nitrogen starvation . In the absence of induction, NGR234 only produces fast-migrating LPSs . When cultured in the presence of flavonoids, the same strain produces large quantities of a high-molecular-weight rhamnose-rich lipopolysaccharide (RLPS) . Because the synthesis of this compound seems to be coded by the symbiotic plasmid under direct or indirect gene induction by flavonoids, this RLPS is thought to be biologically relevant.

Appl Environ Microbiol, 2002 Dec, 68(12), 5877 - 81
Identification of an iron-regulated, hemin-binding outer membrane protein in Sinorhizobium meliloti; Battistoni F et al.; Rhizobia are soil bacteria that are able to establish symbiotic associations with leguminous hosts . In iron-limited environments these bacteria can use iron present in heme or heme compounds (hemoglobin, leghemoglobin) . Here we report the presence in Sinorhizobium meliloti of an iron-regulated outer membrane protein that is able to bind hemin but not hemoglobin . Protein assignment was done by matrix-assisted laser desorption ionization-time of flight mass spectrometry . Tryptic peptides correlated with the mass measurements obtained accounted for 54% of the translated sequence of a putative heme receptor gene present in the chromosome of S . meliloti 1021 . The results which we obtained suggest that this protein (designated ShmR for Sinorhizobium heme receptor) is involved in high-affinity heme-mediated iron transport.

Antonie Van Leeuwenhoek, 2002 Aug, 81(1-4), 607 - 16
The influence of the symbiotic plasmid pRL1JI on the distribution of GM rhizobia in soil and crop rhizospheres, and implications for gene flow; Clark IM et al.; The distribution of two genetically modified Rhizobium leguminosarum strains was investigated in the field . One, RSM2004, released in 1987, carries a TnS marker on its conjugative symbiotic plasmid (pSym) . The second, CT0370, released at the same site in 1994, has a gusA gene integrated into its chromosome but no pSym . Plate counts indicated that the CT0370 population became established at a higher level than RSM2004 . However, when peas, alfalfa and barley were grown, RSM2004 was found to outnumber CT0370 on all roots and by 100-fold on pea . Although the transfer of pSym from RSM2004 to CT0370 could be detected on plates and in microcosm studies with high inoculum densities, no transfer was detected in the field . Subsequent transfer of pSym from RSM2004 to CT0370 demonstrated that it conferred an advantage in the rhizosphere . In addition to increasing host fitness, plasmids may transfer, or mobilise other genetic elements, to other bacteria . This is more likely in sites such as the rhizosphere, where cells are active and numbers are high . The distribution of pSym and other genetic elements associated with rhizobia, in bacterial sub-populations from the soil and roots of the different plants, was investigated using PCR . The genetic elements studied were: ISRm3, an insertion element from Sinorhizobium meliloti; pSB 102, a broad host range mer plasmid; the Rhizobium nodC gene (carried on pSym) and plasmid replication origins repCI and repCII . As expected, ISRm3 was detected in rhizoflora cultured from alfalfa but not the other plants . The mer gene was ubiquitous but the transfer region of pSB 102 was not detected . The nodC and both repC primers amplified products from all the plants, giving further evidence for the occurrence of plasmids originating from Rhizobium in the rhizoflora of non-host plants . Despite the abundance of elements associated with transferable plasmids in rhizobia, none was detected in either inoculant strain.

Antonie Van Leeuwenhoek, 2002 Aug, 81(1-4), 397 - 407
Quorum-sensing in Rhizobium; Wisniewski-Dye F et al.; Quorum-sensing signals are found in many species of legume-nodulating rhizobia . In a well-characterized strain of R . leguminosarum biovar viciae, a variety of autoinducers are synthesised, and all have been identified as N-acyl-homoserine lactones . One of these N-acyl-homoserine lactones, is N-(3-hydroxy-7-cis-tetradecenoyl)-L-homoserine lactone, previously known as small bacteriocin, which inhibits the growth of several R . leguminosarum strains . The cinRI locus is responsible for the production of small bacteriocin . CinR induces cinl in response to the AHL made by Cinl, thus forming a positive autoregulatory induction loop . A complex cascade of quorum-sensing loops was characterized, in which the cinMR locus appears to be the master control for three other AHL-dependent quorum-sensing control systems . These systems include the rail/raiR, trallyriR and rhiI/rhiR . Other rhizobial strains appear to share some of these quorum sensing loci, but not all loci are found in all strains . Small bacteriocin along with the other N-acyl-homoserine lactones produced by these three AHL-based control systems regulate (i) growth inhibition of sensitive strains, (ii) transfer of the symbiotic plasmid pRL1JI, and (iii) expression of the rhizosphere-expressed (rhi) genes that influence nodulation . Some of the genes regulated by these systems have been identified . While the functions of some, such as the trb operon regulated by triR are clear, several of the regulated genes have no homologues of known function . It is anticipated that several other genes regulated by these systems have yet to be identified . Therefore, despite the regulation of one of the most complex quorum-sensing cascade being understood, several of the functions regulated by the quorum-sensing genes remain to be elucidated.

Antonie Van Leeuwenhoek, 2002 Aug, 81(1-4), 385 - 95
Signal transduction in plant-beneficial rhizobacteria with biocontrol properties; Haas D et al.; Biological control of root pathogens--mostly fungi--can be achieved by the introduction of selected bacterial inoculants acting as 'biopesticides' . Successful inoculants have been identified among Gram-negative and Gram-positive bacteria, often belonging to Pseudomonas spp . and Bacillus spp., respectively . Biocontrol activity of a model rhizobacterium, P . fluorescens CHAO, depends to a considerable extent on the synthesis of extracellular antimicrobial secondary metabolites and exoenzymes, thought to antagonize the pathogenicity of a variety of phytopathogenic fungi . The regulation of exoproduct formation in P . fluorescens (as well as in other bacteria) depends essentially on the GacS/GacA two-component system, which activates a largely unknown signal transduction pathway . However, recent evidence indicates that GacS/GacA control has a major impact on target gene expression at a post-transcriptional level, involving an mRNA target sequence (typically near the ribosome binding site), two RNA binding proteins (designated RsmA and RsmE), and a regulatory RNA (RsmZ) capable of binding RsmA . The expression and activity of the regulatory system is stimulated by at least one low-molecular-weight signal . The timing and specificity of this switch from primary to secondary metabolism are essential for effective biocontrol.

Mol Cells, 2002 Oct 31, 14(2), 261 - 6
Symbiotic effects of deltamatB Rhizobium leguminosarum bv . trifolii mutant on clovers; An JH et al.; The role of malonate in symbiotic nitrogen metabolism has long been controversial, although it is known to occur in legume roots, especially in the nodules . Here we report that malonate metabolism plays a key role in the differentiation of bacteroids Rhizobium leguminosarum bv . trifolii in clover nodules . An operon, mat, that consists of three consecutive genes (matABC) has been discovered . Mat encodes enzymes that catalyze the uptake and conversion of malonate to acetyl-CoA through malonyl-CoA . A mutant bacteria, which replaced matB that encodes malonyl-CoA synthetase with a kanamycin resistant gene, was generated and infected with white clover . Clover growth was considerably reduced, even though nodules were formed . However, the nodules were filled with vacuoles, but not with bacteroids . This indicates that malonate metabolism is an important requirement for the formation of mature nodules that are filled with bacteroids.

Nature, 2002 Nov 28, 420(6914), 422 - 6 Epub 2002 Nov 06.
Shoot control of root development and nodulation is mediated by a receptor-like kinase; Krusell L et al.; In legumes, root nodule organogenesis is activated in response to morphogenic lipochitin oligosaccharides that are synthesized by bacteria, commonly known as rhizobia . Successful symbiotic interaction results in the formation of highly specialized organs called root nodules, which provide a unique environment for symbiotic nitrogen fixation . In wild-type plants the number of nodules is regulated by a signalling mechanism integrating environmental and developmental cues to arrest most rhizobial infections within the susceptible zone of the root . Furthermore, a feedback mechanism controls the temporal and spatial susceptibility to infection of the root system . This mechanism is referred to as autoregulation of nodulation, as earlier nodulation events inhibit nodulation of younger root tissues . Lotus japonicus plants homozygous for a mutation in the hypernodulation aberrant root (har1) locus escape this regulation and form an excessive number of nodules . Here we report the molecular cloning and expression analysis of the HAR1 gene and the pea orthologue, Pisum sativum, SYM29 . HAR1 encodes a putative serine/threonine receptor kinase, which is required for shoot-controlled regulation of root growth, nodule number, and for nitrate sensitivity of symbiotic development.

FEMS Microbiol Lett, 2002 Nov 5, 216(2), 139 - 44
Partial 16S rRNA gene sequence diversity and numerical taxonomy of slow growing pigeonpea (Cajanus cajan L Millsp) nodulating rhizobia; Ramsubhag A et al.; An investigation was carried out to determine the diversity of 30 isolates of slow growing pigeonpea nodulating rhizobia based on variations in partial sequences of the 16S rRNA gene and numerical analysis of 80 phenotypic traits . Phylogenetic analysis using molecular sequences of 23 isolates showed that ARPE1 separated from the other isolates at an average distance of >14% divergence level . The other isolates were all within 5% divergence from each other but separated into four main groups, with group 1 containing 16 of the 23 isolates . Comparisons to sequences of reference strains revealed that the group 1 isolates were phylogenetically closely related to the slow growing soybean nodulating rhizobia belonging to Bradyrhizobium elkanii, although only three of these isolates were able to nodulate soybean . Numerical analysis of phenotypic data of 19 isolates showed that 14 isolates clustered together in one branch of the phenogram, which included the group 1, group 2 and group 4 isolates from the phylogenetic analysis . The group 3 isolates were highly variable in the phenogram with similarity levels lower than 50% among these isolates.

J Bacteriol, 2002 Dec, 184(23), 6681 - 9
A Sinorhizobium meliloti lipopolysaccharide mutant altered in cell surface sulfation; Keating DH et al.; The Rhizobium-legume symbiosis involves the formation of a novel plant organ, the nodule, in which intracellular bacteria reduce molecular dinitrogen in exchange for plant photosynthates . Nodule development requires a bacterial signal referred to as Nod factor, which in Sinorhizobium meliloti is a beta-(1,4)-linked tetramer of N-acetylglucosamine containing N-acyl and O-acetyl modifications at the nonreducing end and a critical 6-O-sulfate at the reducing end . This sulfate modification requires the action of three gene products: nodH, which catalyzes the sulfonyl transfer, and nodPQ, which produce the activated form of sulfate, 3'-phosphoadenosine-5'-phosphosulfate . It was previously reported that S . meliloti cell surface polysaccharides are also covalently modified by sulfate in a reaction dependent on NodPQ . We have further characterized this unique form of bacterial carbohydrate modification . Our studies have determined that one of the nodPQ mutant strains used in the initial study of sulfation of cell surface harbored a second unlinked mutation . We cloned the gene affected by this mutation (referred to as lps-212) and found it to be an allele of lpsL, a gene previously predicted to encode a UDP-glucuronic acid epimerase . We demonstrated that lpsL encoded a UDP-glucuronic acid epimerase activity that was reduced in the lps-212 mutant . The lps-212 mutation resulted in an altered lipopolysaccharide structure that was reduced in sulfate modification in vitro and in vivo . Finally, we determined that the lps-212 mutation resulted in a reduced ability to elicit the formation of plant nodules and by altered infection thread structures that aborted prematurely.

Meded Rijksuniv Gent Fak Landbouwkd Toegep Biol Wet, 2001, 66(2b), 655 - 62
Influence of plant species on the biological control activity of the antagonistic rhizobacterium Rhizobium etli strain G12 toward the root-knot nematode, Meloidogyne incognita; Mahdy M et al.; The influence of plant species on the antagonistic activity of the rhizosphere bacterium Rhizobium etli G12 towards the root-knot nematode Meloidogyne incognita was studied . The crops tested were tomato, cucumber, cotton, soybean and pepper . The plants were evaluated for the following parameters: root gall-index, total number of galls and egg masses of M . incognita, as well as shoot and root fresh weight and root length . Results indicated a clear influence of plant species on the ability of R . etli G12 to reduce nematode infection . Based on the root gall index, nematode control by R . etli G12 was higher on vegetables (tomato, cucumber, pepper) than on field crops (soybean, cotton) . Reduction in galling ranged from 17% for cotton to 50% for tomato . R . etli G12 also reduced the actual number of galls produced . The reduction in the number of galls produced between crops was not affected significantly as was seen when a galling index was used to measure activity . The reduction in the number of galls was similar in level for all the crops studied and ranged from 34% for cucumber to 47% for tomato . There was a significant reduction in the number of egg masses produced by the females ranging from 37% for soybean to 70% for pepper . This indicated a direct effect on female development in the root after penetration or delayed penetration on certain crops . The bacteria caused significant increases in shoot fresh weight from 11% for soybean to 31% for pepper and in root fresh weight from 3% for soybean to 39% for tomato and in root length from 11% for cucumber to 24% for pepper . R . etli G12 gave significant control of M . incognita on a broad range of host plants, but the level of control varied . The suitability of each plant species, therefore, must be examined before R . etli G12 can be recommend for control of this nematode.

Meded Rijksuniv Gent Fak Landbouwkd Toegep Biol Wet, 2001, 66(2a), 195 - 203
Potential of induced resistance to control Oidium lycopersici on tomato and tobacco; Achuo AE et al.; Powdery mildew is widespread on greenhouse-cultivated tomato especially in Europe . Rhizobacteria, particularly Pseudomonas strains are well known for inducing resistance to many diseases on field crops . Plant disease resistance can also be induced by treatment with benzothiadiazole (BTH) which specifically induces the salicylic acid-dependent defence pathway . We investigated the possibility of inducing resistance to Oidium lycopersici on tomato and tobacco by treatment with BTH or with resistance-inducing rhizobacteria . Tomato (Lycopersicon esculentum) cv . Moneymaker and NahG tomato (a transgenic tomato which cannot accumulate salicylic acid (SA)) were planted in soil treated with P . aeruginosa KMPCH, a SA-producing mutant of P . aeruginosa 7NSK2, at 10(8) CFU/g . Four weeks-old plants were inoculated by exposure to an atmosphere heavily infested with O . lycopersici . P . aeruginosa 7NSK2 and KMPCH; and P . fluorescens WCS 417 were similarly tested on tobacco cv . Xanthi and Samsun NN . The results showed that the rhizobacteria had no effect on the levels of infection of the plants by O . lycopersici . There were also no differences between NahG and wild type plants . We conclude that the induced resistance pathways activated by rhizobacteria do not lead to enhanced resistance of tomato and tobacco to O . lycopersici . BTH was tested on tomato by soil application at 0.1 ppm or foliar spray with 100 microM solution 4 or 7 days before inoculation (dbi) and tobacco was treated by foliar spray with 1, 10 or 100 microM BTH, 4 dbi . While a dose-dependent suppression of O . lycopersici by BTH was registered on tobacco, tomato infection by the powdery mildew was not affected by BTH . This difference indicates that BTH probably affects the tobacco-Oidium and the tomato-Oidium systems differently . We are currently investigating whether or not this difference can be associated with the production of different PR proteins.

Carbohydr Res, 2002 Oct 11, 337(19), 1785 - 9
13C NMR spectroscopic analysis on the chiral discrimination of N-acetylphenylalanine, catechin and propranolol induced by cyclic-(1-->2)-beta-D-glucans (cyclosophoraoses); Lee S et al.; Cyclosophoraoses (cyclic-(1-->2)-beta-D-glucans) produced by Rhizobium meliloti were used as a novel chiral NMR solvating agent . 13C NMR spectroscopic analysis as an enantiodiscriminating tool was carried out where NMR signal splittings were observed on the interactions of cyclosophoraoses with the enantiomers of N-acetylphenylalanine, catechin and propranolol . The 13C chemical shifts of cyclosophoraoses induced by the enantiomeric interactions predominantly occurred at the C-1 and C-2 carbons associated with the -glycosidic linkage.

Phytochemistry, 2002 Nov, 61(6), 637 - 44
Formation of pyridine nucleotides under symbiotic and non-symbiotic conditions between soybean nodules and free-living rhizobia; Tezuka T et al.; Enzymatic regulation of pyricline nucleotide formation, under symbiotic and non-symbiotic conditions, was analyzed using soybeans (Glycine max L . cv . 'Akisengoku') and rhizobia (Bradyrhizobia japonicum strain A1017), respectively . It was found that levels of pyridine nucleotides in bacteroids in root nodules were different from those in free-living cells of rhizobia . This difference was associated with differences in activities of enzymes involved in the pathway from L-tryptophan to NAD and NADP . That is, these activities were lower in bacteroids than in free-living bacteria and lower in the nodule cytosol than in root extracts . The optimum pH for NAD synthetase in bacteroids, was 9.0 . Additionally, the optimum pH for ATP-nicotinamide mononucleotide (NMN) adenyltransferase, final step enzyme in NAD formation, was estimated to be 7.6 . In the bacteroid fraction, the K(m) of NAD synthetase (22 microM) was approximately 1/22 of that of ATP-NMN adenyltransferase (482 microM) . Vmax values were estimated to be almost in the same order for both NAD synthetase and ATP-NMN adenyltransferase . This is the first report on the formation of pyridine nucleotides originating from L-tryptophan in bacteroids in soybean nodules and free-living bacteria.

Mol Plant Microbe Interact, 2002 Nov, 15(11), 1147 - 56
Induction of systemic resistance to Botrytis cinerea in tomato by Pseudomonas aeruginosa 7NSK2: role of salicylic acid, pyochelin, and pyocyanin; Audenaert K et al.; The rhizobacterium Pseudomonas aeruginosa 7NSK2 produces secondary metabolites such as pyochelin (Pch), its precursor salicylic acid (SA), and the phenazine compound pyocyanin . Both 7NSK2 and mutant KMPCH (Pch-negative, SA-positive) induced resistance to Botrytis cinerea in wild-type but not in transgenic NahG tomato . SA-negative mutants of both strains lost the capacity to induce resistance . On tomato roots, KMPCH produced SA and induced phenylalanine ammonia lyase activity, while this was not the case for 7NSK2 . In 7NSK2, SA is probably very efficiently converted to Pch . However, Pch alone appeared not to be sufficient to induce resistance . In mammalian cells, Fe-Pch and pyocyanin can act synergistically to generate highly reactive hydroxyl radicals that cause cell damage . Reactive oxygen species are known to play an important role in plant defense . To study the role of pyocyanin in induced resistance, a pyocyanin-negative mutant of 7NSK2, PHZ1, was generated . PHZ1 is mutated in the phzM gene encoding an O-methyltransferase . PHZ1 was unable to induce resistance to B . cinerea, whereas complementation for pyocyanin production or co-inoculation with mutant 7NSK2-562 (Pch-negative, SA-negative, pyocyanin-positive) restored induced resistance . These results suggest that pyocyanin and Pch, rather than SA, are the determinants for induced resistance in wild-type P . aeruginosa 7NSK2.

Mol Microbiol, 2002 Nov, 46(4), 1023 - 32
A site-specific recombinase (RinQ) is required to exert incompatibility towards the symbiotic plasmid of Rhizobium etli; Quintero V et al.; The replication/partition region of the symbiotic plasmid p42d of Rhizobium etli CE3 is characterized by the presence of the repABC operon . A recombinant plasmid containing this region is able to replicate in a R . etli derivative cured from p42d, with the same stability and copy number shown by the parental plasmid . However, when this construct is introduced into the wild-type strain, instead of exerting incompatibility against the p42d, it forms a stable cointegrate with it . In this paper, we show that a site-specific resolvase, and its action sites are essential factors to displace the symbiotic p42d . We propose a model for this novel incompatibility mechanism.

Syst Appl Microbiol, 2002 Oct, 25(3), 440 - 9
Characterization of pigmented methylotrophic bacteria which nodulate Lotononis bainesii; Jaftha JB et al.; Root nodule isolates from a shrubby legume, Lotononis bainesii, were characterized by 16S rRNA gene sequencing and morphologically by substrate utilization patterns . The symbiotic genome of these isolates was analysed by partial sequencing of the nifH gene . Based on the results of numerical taxonomy, the isolates formed a closely related cluster, showing no correspondence to any of the known rhizobial clusters . Analysis of nearly full-length 16S rDNA sequences demonstrated that these isolates were related to Methylobacterium nodulans (SY et al., 2001) . In the absence of nifH sequence data for the genus Methylobacterium, the nifH phylogeny showed these isolates to be related to Azospirillum brasilense . The facultative methylotrophic nature of these isolates was also demonstrated by their ability to grow in the presence of methanol as a sole carbon source.

Syst Appl Microbiol, 2002 Oct, 25(3), 423 - 33
Analysis of genomic diversity among photosynthetic stem-nodulating rhizobial strains from northeast Argentina; Montecchia MS et al.; The genomic diversity among photosynthetic rhizobia from northeast Argentina was assessed . Forty six isolates obtained from naturally occurring stem and root nodules of Aeschynomene rudis plants were analyzed by three molecular typing methods with different levels of taxonomic resolution: repetitive sequence-based PCR (rep-PCR) genomic fingerprinting with BOX and REP primers, amplified 16S rDNA restriction analysis (ARDRA), and 16S-23S rDNA intergenic spacer-restriction fragment length polymorphism (IGS-RFLP) analysis . The in vivo absorption spectra of membranes of strains were similar in the near infrared region with peaks at 870 and 800 nm revealing the presence of light harvesting complex I, bacteriochlorophyll-binding polypeptides (LHI-Bchl complex) . After extraction with acetone-methanol the spectra differed in the visible part displaying peaks belonging to canthaxanthin or spirilloxanthin as the main carotenoid complement . The genotypic characterization by rep-PCR revealed a high level of genomic diversity among the isolates and almost all the photosynthetic ones have identical ARDRA patterns and fell into one cluster different from Bradyrhizobium japonicum and Bradyrhizobium elkanii . In the combined analysis of ARDRA and rep-PCR fingerprints, 7 clusters were found including most of the isolates . Five of those contained only photosynthetic isolates; all canthaxanthin-containing strains grouped in one cluster, most of the other photosynthetic isolates were grouped in a second large cluster, while the remaining three clusters contained a few strains . The other two clusters comprising reference strains of B . japonicum and B . elkanii, respectively . The IGS-RFLP analysis produced similar clustering for almost all the strains . The 16S rRNA gene sequence of one representative isolate was determined and the DNA sequence analysis confirmed the position of photosynthetic rhizobia in a distinct phylogenetic group within the Bradyrhizobium rDNA cluster.

Vet Microbiol, 2002 Dec 20, 90(1-4), 349 - 63
Seeking a niche: putative contributions of the hfq and bacA gene products to the successful adaptation of the brucellae to their intracellular home; Roop RM 2nd et al.; Long-term residence of the brucellae in the phagosomal compartment of host macrophages is essential to their ability to produce disease in both natural and experimental hosts . Correspondingly, the Brucella spp . appear to be well adapted to resist the multiple environmental stresses they encounter in their intracellular home . This brief review will focus on the contributions of the hfq and bacA gene products to this adaptation . Studies with Brucella hfq mutants suggest that stationary phase physiology is critical for successful long-term residence in host macrophages . Analysis of Brucella bacA mutants, on the other hand, reveal very striking parallels between the strategies employed by the rhizobia to establish and maintain protracted intracellular residence in their plant host and those used by the brucellae during their long-term survival in the phagosomal compartment of host macrophages .

Plant J, 2002 Nov, 32(3), 343 - 52
The Nod factor-elicited annexin MtAnn1 is preferentially localised at the nuclear periphery in symbiotically activated root tissues of Medicago truncatula; De Carvalho-Niebel F et al.; The Medicago truncatula MtAnn1 gene, encoding a putative annexin, is transcriptionally activated in root tissues in response to rhizobial Nod factors . To gain further insight into MtAnn1 function during the early stages of nodulation, we have examined in detail both spatio-temporal gene expression patterns and MtAnn1 activity and localisation in root tissues . Analysis of transgenic Medicago plants expressing a pMtAnn1-GUS fusion has revealed a novel pattern of transcription in both outer and inner cell layers of the root following either Nod factor-treatment or rhizobial inoculation . The highest gene expression levels were observed in the endodermis and outer cortex . These transgenic plants also revealed that MtAnn1 expression is associated with lateral root development and cell differentiation in the root apex independent of nodulation . By purifying recombinant MtAnn1 we were able to demonstrate that this plant annexin indeed possesses the calcium-dependent binding to acidic phospholipids typical of the annexin family . Antisera against recombinant MtAnn1 were then used to show that tissue-specific localisation of the MtAnn1 protein in Medicago roots matches the pMtAnn1-GUS expression pattern . Finally, both immunolabelling and in vivo studies using MtAnn1-GFP reporter fusions have revealed that MtAnn1 is cytosolic and in particular localises to the nuclear periphery in cortical cells activated during the early stages of nodulation . In the light of our findings, we discuss the possible role of this annexin in root tissues responding to symbiotic rhizobial signals.

Appl Environ Microbiol, 2002 Nov, 68(11), 5217 - 22
A new species of Devosia that forms a unique nitrogen-fixing root-nodule symbiosis with the aquatic legume Neptunia natans (L.f.) druce; Rivas R et al.; Rhizobia are the common bacterial symbionts that form nitrogen-fixing root nodules in legumes . However, recently other bacteria have been shown to nodulate and fix nitrogen symbiotically with these plants . Neptunia natans is an aquatic legume indigenous to tropical and subtropical regions and in African soils is nodulated by Allorhizobium undicola . This legume develops an unusual root-nodule symbiosis on floating stems in aquatic environments through a unique infection process . Here, we analyzed the low-molecular-weight RNA and 16S ribosomal DNA (rDNA) sequence of the same fast-growing isolates from India that were previously used to define the developmental morphology of the unique infection process in this symbiosis with N . natans and found that they are phylogenetically located in the genus Devosia, not Allorhizobium or RHIZOBIUM: The 16S rDNA sequences of these two Neptunia-nodulating Devosia strains differ from the only species currently described in that genus, Devosia riboflavina . From the same isolated colonies, we also located their nodD and nifH genes involved in nodulation and nitrogen fixation on a plasmid of approximately 170 kb . Sequence analysis showed that their nodD and nifH genes are most closely related to nodD and nifH of Rhizobium tropici, suggesting that this newly described Neptunia-nodulating Devosia species may have acquired these symbiotic genes by horizontal transfer.

Protein Expr Purif, 2002 Nov, 26(2), 321 - 8
Expression and purification of Rhizobium leguminosarum NodD; Feng J et al.; A Rhizobium expression system was constructed via introducing strong transcriptional elements to an IncP broad host range plasmid pKT230 . Using this system, the nodD gene of Rhizobium leguminosarum biovar viciae was overexpressed in its own in vivo environment . Western blot showed that the NodD yield of the newly constructed expression vector pKNDT was much higher than that of a reported vector pIJ1518 . Based on the result from quantitative gel retardation assay, the specific DNA-binding activity of NodD was estimated to increase by about 80-fold . Purification of NodD was performed by the sequential steps: polyethyleneimine treatment, ammonium sulfate fractional precipitation, and cellulose phosphate P11 chromatography . About 26 mg NodD with a purity about 90% was obtained from 14 g wet weight cell pellet within 2 days.

Crit Rev Biotechnol, 2002, 22(3), 281 - 314
Promiscuity of hosting nitrogen fixation in rice: an overview from the legume perspective; Dey M et al.; The subject area of this review provides extraordinary challenges and opportunities . The challenges relate to the fact that the integration of various fields such as microbiology, biochemistry, plant physiology, eukaryotic as well as bacterial genetics, and applied plant sciences are required to assess the disposition of rice, an alien host, for establishing such a unique phenomenon as biological nitrogen fixation . The opportunities signify that, if successful, the breakthrough will have a significant impact on the global economy and will help improve the environment . This review highlights the literature related to the area of legume-rhizobia interactions, particularly those aspects whose understanding is of particular interest in the perspective of rice . This review also discusses the progress achieved so far in this area of rice research and the possibility of built-in nitrogen fixation in rice in the future . However, it is to be borne in mind that such research does not ensure any success at this point . It provides a unique opportunity to broaden our knowledge and understanding about many aspects of plant growth regulation in general.

Trends Plant Sci, 2002 Oct, 7(10), 440 - 4
Suppression of plant defence in rhizobia-legume symbiosis; Mithofer A; The symbiosis between rhizobia and legumes is characterized by the formation of dinitrogen-fixing root nodules . Although rhizobia colonize roots in a way that is reminiscent of pathogenic microorganisms, no host plant defence reactions are triggered during successful symbioses . Nevertheless, the plants obviously control the invading bacteria; failure in effective nodule formation or infections with rhizobia defective in surface polysaccharides often result in pathogenic responses . This article focuses on whether and how defence responses in effective symbiosis might be suppressed . Recent results suggest a central role for rhizobial polysaccharides acting as antagonists in the negative regulation of defence induction.

Microbiol Res, 2002, 157(3), 157 - 60
In vitro induction of lipo-chitooligosaccharide production in Bradyrhizobium japonicum cultures by root extracts from non-leguminous plants; Lian B et al.; Bradyrhizobium japonicum can form a N2-fixing symbiosis with compatible leguminous plants . It can also act as a plant-growth promoting rhizobacterium (PGPR) for non-legume plants, possibly through production of lipo-chitooligosaccharides (LCOs), which should have the ability to induce disease resistance responses in plants . The objective of this work was to determine whether non-leguminous crop plants can induce LCO formation by B . japonicum cultures . Cultures treated with root extracts of soybean, corn, cotton or winter wheat were assayed for presence and level of LCO . Root extracts of soybean, corn and winter wheat all induced LCO production, with extracts of corn inducing the greatest amounts . Root washings of corn also induced LCO production, but less than the root extract . These results indicated that the stimulation of non-legume plant growth by B . japonicum could be through the production of LCOs, induced by materials excreted by the roots of non-legume plants.

Biotechniques, 2002 Oct, 33(4), 782, 784, 786 - 8
In vivo cloning strategy for Rhizobium plasmids; Hernandez-Lucas I et al.; We have developed a simple system to clone indigenous Rhizobium plasmids into E . coli . The strategy consists of three matings: the first is to insert Tn5 in the plasmid to be cloned, the second incorporates the integrative vector into the inserted Tn5 in the native Rhizobium plasmid, and the last mating transfers the target plasmid directly into E . coli . This mating-based system was successfully used to clone plasmids of Rhizobium species with sizes ranging from 150 to 270 kb . In addition, a 500-kb fragment of a 600-kb megaplasmid was also cloned . This strategy could be used for cloning indigenous replicons of other gram-negative bacteria into a different host.

Proc Natl Acad Sci U S A, 2002 Nov 12, 99(23), 15206 - 10 Epub 2002 Oct 23.
A Lotus basic leucine zipper protein with a RING-finger motif negatively regulates the developmental program of nodulation; Nishimura R et al.; The developmental program of nodulation is regulated systemically in leguminous host species . A mutant astray (Ljsym77) in Lotus japonicus has lost some sort of its ability to regulate this symtem, and shows enhanced and early nodulation . In the absence of rhizobia, this mutant exhibits characteristics associated with defects in light and gravity responses . These nonsymbiotic phenotypes of astray are very similar to those observed in photomorphogenic Arabidopsis mutant hy5 . Based on this evidence, we predicted that astray might contain a mutation in the HY5 homologue of L . japonicus . The homologue, named LjBzf, encodes a basic leucine zipper protein in the C-terminal half that shows the highest level of identity with HY5 of all Arabidopsis proteins . It also encodes legume-characteristic combination of motifs, including a RING-finger motif and an acidic region in the N-terminal half . The astray phenotypes were cosegregated with LjBzf, and the failure to splice the intron was detected . Nonsymbiotic and symbiotic phenotypes of astray were complemented by introduction of CaMV35SLjBzf . It is noteworthy that although Arabidopsis hy5 showed an enhancement of lateral root initiation, Lotus astray showed an enhancement of nodule initiation but not of lateral root initiation . Legume-characteristic combination of motifs of ASTRAY may play specific roles in the regulation of nodule development.

J Biotechnol, 2002 Nov 13, 99(3), 279 - 93
A novel bioremediation system for heavy metals using the symbiosis between leguminous plant and genetically engineered rhizobia; Sriprang R et al.; A novel plant-bacterial remediation system for heavy metals (HM) was developed by expression of tetrameric human metallothionein (MTL4) in Mesorhizobium huakuii subsp . rengei B3, a strain which infects and forms nodules on a green manure, Astragalus sinicus . The MTL4 gene was fused to the nifH and nolB promoters, which generated nodule- specific expression of the MTL4 gene . The expression analysis of the MTL4 gene was demonstrated in free-living cells in the presence of Cd(2+) and Cu(2+), under the low oxygen condition . The MTL4 under the nifH and nolB promoters was expressed and increased the accumulation of Cd(2+), but not Cu(2+) in free-living cells . The expression of the integrated nifH-MTL4 gene in the chromosome of strain B3 was also expressed stably and accumulated Cd(2+) in the bacterial cells . The MTL4 transcripts were detected by in situ hybridization in bacteroids of mature nodules of A . sinicus containing nifH-MTL4 and nolB-MTL4 fusion gene . Moreover the MTL4 protein was detected by immunostaining . By infection of the recombinant B3, A . sinicus established symbiosis with the recombinant B3 that was grown in Cd(2+) and Cu(2+)-polluted soils . The symbionts increased Cd(2+) accumulation in nodules 1.7-2.0-fold, whereas, no significantly increase in Cu(2+) accumulation was noted.

Gene, 2002 Sep 4, 297(1-2), 159 - 68
Isolation and characterization of the glnD gene of Gluconacetobacter diazotrophicus, encoding a putative uridylyltransferase/uridylyl-removing enzyme; Perlova O et al.; The glnD gene of Gluconacetobacter diazotrophicus was isolated by complementation of the Azotobacter vinelandii glnD (nfrX) mutant strain MV17 using a pLAFR3 cosmid library . The 5 kb chromosomal DNA region encoding the glnD gene on cosmid pAD401 was identified by introduction of deletions as well as subcloning of restriction fragments followed by subsequent DNA sequencing . Three open reading frames were identified with the deduced amino acid sequence of ORF1 showing significant homologies to known GlnD proteins of other proteobacteria such as Sinorhizobium meliloti, Rhizobium tropici, Escherichia coli and Azotobacter vinelandii.A mutagenesis of the chromosomal glnD gene was carried out by insertion of an interposon carrying the kanamycin resistance gene of Tn5 . Mutants carrying the cassette inserted into a central region of glnD could not be isolated, while an interposon mutation at the 3' end of glnD was successful . The resulting strain showed a prolonged generation time in complex growth medium and was unable to utilize ammonium as sole nitrogen source . This phenotype appears to be pleiotropic, since the addition of single amino acids to the minimal medium was not sufficient to allow growth . Furthermore, the glnD mutant was able to express nitrogenase under diazotrophic as well as repressing growth conditions.

Plasmid, 2002 Sep, 48(2), 104 - 16
Rhizobium etli CFN42 contains at least three plasmids of the repABC family: a structural and evolutionary analysis; Cevallos MA et al.; In this paper, we report the identification of replication/partition regions of plasmid p42a and p42b of Rhizobium etli CFN42 . Sequence analysis reveals that both replication/partition regions belong to the repABC family . Phylogenetic analysis of all the complete repABC replication/partition regions reported to date, shows that repABC plasmids coexisting in the same strain arose most likely by lateral transfer instead of by duplication followed by divergence . A model explaining how new incompatibility groups originate, is proposed.

Cell Stress Chaperones, 2002 Apr, 7(2), 130 - 6
Rhizobium leguminosarum chaperonin 60.3, but not chaperonin 60.1, induces cytokine production by human monocytes: activity is dependent on interaction with cell surface CD14; Lewthwaite J et al.; As part of a program of work to understand the interaction of bacterial chaperonins with human leukocytes, we have examined 2 of the 3 chaperonin 60 (Cpn 60) gene products of the nonpathogenic plant symbiotic bacterium, Rhizobium leguminosarum, for their capacity to induce the production of pro- and antiinflammatory cytokines by human cells . Recombinant R . leguminosarum Cpn 60.1 and 60.3 proteins were added to human monocytes at a range of concentrations, and cytokine production was measured by sandwich enzyme-linked immunosorbent assay . In spite of the fact that the 2 R . leguminosarum Cpn 60 proteins share 74.5% amino acid sequence identity, it was found that Cpn 60.3 induced the production of interleukin (IL)-1beta, tumor necrosis factor alpha, IL-6, IL-8, IL-10, and IL-12, but not IL-4, interferon gamma, or GM-CSF (granulocyte-macrophage colony-stimulating factor), whereas the Cpn 60.1 protein failed to demonstrate any cytokine-inducing activity . The use of neutralizing monoclonal antibodies showed that the cytokine-inducing activity of Cpn 60.3 was dependent on its interaction with CD14 . This demonstrates that CD14 mediates not only lipopolysaccharide but also R . leguminosarum Cpn 60.3 cell signaling in human monocytes.

J Bacteriol, 2002 Nov, 184(21), 5979 - 86
Real-time imaging of fluorescent flagellar filaments of Rhizobium lupini H13-3: flagellar rotation and pH-induced polymorphic transitions; Scharf B; The soil bacterium Rhizobium lupini H13-3 has complex right-handed flagellar filaments with unusual ridged, grooved surfaces . Clockwise (CW) rotation propels the cells forward, and course changes (tumbling) result from changes in filament speed instead of the more common change in direction of rotation . In view of these novelties, fluorescence labeling was used to analyze the behavior of single flagellar filaments during swimming and tumbling, leading to a model for directional changes in R . lupini . Also, flagellar filaments were investigated for helical conformational changes, which have not been previously shown for complex filaments . During full-speed CW rotation, the flagellar filaments form a propulsive bundle that pushes the cell on a straight path . Tumbling is caused by asynchronous deceleration and stops of individual filaments, resulting in dissociation of the propulsive bundle . R . lupini tumbles were not accompanied by helical conformational changes as are tumbles in other organisms including enteric bacteria . However, when pH was experimentally changed, four different polymorphic forms were observed . At a physiological pH of 7, normal flagellar helices were characterized by a pitch angle of 30 degrees, a pitch of 1.36 micro m, and a helical diameter of 0.50 micro m . As pH increased from 9 to 11, the helices transformed from normal to semicoiled to straight . As pH decreased from 5 to 3, the helices transformed from normal to curly to straight . Transient conformational changes were also noted at high viscosity, suggesting that the R . lupini flagellar filament may adapt to high loads in viscous environments (soil) by assuming hydrodynamically favorable conformations.

Acta Biochim Pol, 2002, 49(2), 537 - 46
Nitrate reduction and nitrogen fixation in symbiotic association Rhizobium-legumes; Lucinski R et al.; The inhibitory effect of nitrate on nitrogenase activity in root nodules of legume plants has been known for a long time . The major factor inducing changes in nitrogenase activity is the concentration of free oxygen inside nodules . Oxygen availability in the infected zone of nodule is limited, among others, by the gas diffusion resistance in nodule cortex . The presence of nitrate may cause changes in the resistance to O2 diffusion . The aim of this paper is to review literature data concerning the effect of nitrate on the symbiotic association between rhizobia and legume plants, with special emphasis on nitrogenase activity . Recent advances indicate that symbiotic associations of Rhizobium strains characterized by a high nitrate reductase activity are less susceptible to inhibition by nitrate . A thesis may be put forward that dissimilatory nitrate reduction, catalyzed by bacteroid nitrate reductase, significantly facilitates the symbiotic function of bacteroids.

J Biochem Mol Biol, 2002 Sep 30, 35(5), 443 - 51
Malonate metabolism: biochemistry, molecular biology, physiology, and industrial application; Kim YS; Malonate is a three-carbon dicarboxylic acid . It is well known as a competitive inhibitor of succinate dehydrogenase . It occurs naturally in biological systems, such as legumes and developing rat brains, which indicates that it may play an important role in symbiotic nitrogen metabolism and brain development . Recently, enzymes that are related to malonate metabolism were discovered and characterized . The genes that encode the enzymes were isolated, and the regulation of their expression was also studied . The mutant bacteria, in which the malonate-metabolizing gene was deleted, lost its primary function, symbiosis, between Rhizobium leguminosarium bv trifolii and clover . This suggests that malonate metabolism is essential in symbiotic nitrogen metabolism, at least in clover nodules . In addition to these, the genes matB and matC have been successfully used for generation of the industrial strain of Streptomyces for the production of antibiotics.

Lett Appl Microbiol, 2002, 35(4), 347 - 52
Genetic diversity of rhizobia isolated from Caragana intermedia in Maowusu sandland, north of China; Gao LF et al.; AIMS: Thirty-three rhizobial strains isolated from nodules of Caragana intermedia in Maowusu sandland were examined for their genetic diversity and putative phylogenetic position . METHODS AND RESULTS: Isolates from Caragana intermedia were classified into 12 genotypes by 16S rDNA polymerase chain reaction-restriction fragment length polymorphism (RFLP), which showed no distinct relationships with those of the reference strains . The genotypes of rhizobia were not related to geographical location . Thr 16S rDNA sequence of representative strain GH2001 from dominant genotype 2 shared high homologuey with some Rhizobium species: Rh . giardinii (96.4%), Rh . huautlense (95.3%), Rh . galegae (95.7%), Rh . yanglingense (95.2%), Rh . mongolense (95.6%), Rh . radiobacter (99%) and Rh . rubi (98.3%) . CONCLUSIONS: A high degree of genetic diversity existed among rhizobia nodulating Caragana intermedia in Maowusu sandland . Most of the new isolates might belong to Rhizobium . SIGNIFICANCE AND IMPACT OF THE STUDY: The results suggest that the rich diversity of rhizobia might have contributed to the adaptation of the arid region . These strains could be valuable at the economic and ecosystem level.

Plant Cell, 1990 Feb, 2(2), 139 - 151
Cooperative Action of Rhizobium meliloti Nodulation and Infection Mutants during the Process of Forming Mixed Infected Alfalfa Nodules; Kapp D et al.; Alfalfa plants co-inoculated with Rhizobium meliloti nodulation (Nod-) and infection mutants deficient in exopolysaccharide production (Inf-EPS-) formed mixed infected nodules that were capable of fixing atmospheric nitrogen . The formation of infected nodules was dependent on close contact between the inoculation partners . When the partners were separated by a filter, empty Fix- nodules were formed, suggesting that infection thread formation in alfalfa is dependent on signals from the nodulation and infection genes . In mixed infected nodules, both nodulation and infection mutants colonized the plant cells and differentiated into bacteroids . The formation of bacteroids was not dependent on cell-to-cell contact between the mutants . Immunogold/silver staining revealed that the ratio of the two mutants varied considerably in colonized plant cells following mixed inoculation . The introduction of an additional nif/fix mutation into one of the inoculation partners did not abolish nitrogen fixation in mixed infected nodules . The expression of nif D::lacZ fusions additionally demonstrated that mutations in the nodulation and infection genes did not prevent the nif genes from being expressed in the mutant bacteroids.

Plant Cell, 1990 Dec, 2(12), 1157 - 1170
Rhizobium meliloti Genes Encoding Catabolism of Trigonelline Are Induced under Symbiotic Conditions; Boivin C et al.; Rhizobium meliloti trc genes controlling the catabolism of trigonelline, a plant secondary metabolite often abundant in legumes, are closely linked to nif-nod genes on the symbiotic megaplasmid pSym {Boivin, C., Malpica, C., Rosenberg, C., Denarie, J., Goldman, A., Fleury, V., Maille, M., Message, B., and Tepfer, D . (1989) . In Molecular Signals in the Microbe-Plant Symbiotic and Pathogenic Systems . (Berlin: Springer-Verlag), pp . 401-407} . To investigate the role of trigonelline catabolism in the Rhizobium-legume interaction, we studied the regulation of trc gene expression in free-living and in endosymbiotic bacteria using Escherichia coli lacZ as a reporter gene . Experiments performed with free-living bacteria indicated that trc genes were organized in at least four transcription units and that the substrate trigonelline was a specific inducer for three of them . Noninducing trigonelline-related compounds such as betaines appeared to antagonize the inducing effect of trigonelline . None of the general or symbiotic regulatory genes ntrA, dctB/D, or nodD seemed to be involved in trigonelline catabolism . trc fusions exhibiting a low basal and a high induced {beta}-galactosidase activity when present on pSym were used to monitor trc gene expression in alfalfa tissue under symbiotic conditions . Results showed that trc genes are induced during all the symbiotic steps, i.e., in the rhizosphere, infection threads, and bacteroids of alfalfa, suggesting that trigonelline is a nutrient source throughout the Rhizobium-legume association.

FEMS Microbiol Lett, 2002 Sep 10, 214(2), 165 - 70
Construction of a Sinorhizobium meliloti strain carrying a stable and non-transmissible chromosomal single copy of the green fluorescent protein GFP-P64L/S65T; Pistorio M et al.; A single copy of the green fluorescent protein (GFP)-encoding gene gfp-P64L/S65T under the control of the constitutive nptII promoter was introduced in a neutral region of the Sinorhizobium meliloti chromosome, between the genes recA and alaS . Within the same chromosomal region downstream of gfp-P64L/S65T a tetracycline (Tc) resistant cassette was also inserted . Both markers were very stable during at least 40 bacterial generations without any selective pressure . Similarly, the gfp-Tc cassette was stable and functional in all rhizobia that were recovered from alfalfa nodules . The GFP-associated fluorescence derived from the (single copy) chromosomal gfp-P64L/S65T allowed detection of rhizobia during the colonisation of the root, infection thread formation, and nodule development . The gfp-Tc rhizobia showed indistinguishable phenotypes for nodulation, competitiveness, and nitrogen-fixation from the parental strain . The labelling system described here can be used for the stable fluorescent tagging of S . meliloti strains allowing their detection in biologically complex soil environments.

Prikl Biokhim Mikrobiol, 2002 Jul-Aug, 38(4), 427 - 32
{Formation and function of the bean-rhizobium symbiosis in soy plants upon introduction of strains of Azotobacter and Bacillus species}; Mel'nikova NN et al.; The effects of bacteria belonging to the genera Azotobacter and Bacillus in a mixed culture with Bradyrhizobium japonicum strains on formation and function of the legume-rhizobium symbiosis of soybean plants were studied . The data showed that the bacterial compositions B . japonicum 634b + B . subtilis 5, B . japonicum 634b + A . chroococcum 20, and B . japonicum 10k + A . vinelandii 56 with a cell ratio of 1:0.1 increased the number and weight of root nodules as well as the height and weight of the aboveground plant parts in almost all the cases by 22-105% compared with the control variants . These binary microbial cultures may be used for development of combined bacterial preparations for soybean.

Appl Environ Microbiol, 2002 Oct, 68(10), 4915 - 24
Diversity and evolution of hydrogenase systems in rhizobia; Baginsky C et al.; Uptake hydrogenases allow rhizobia to recycle the hydrogen generated in the nitrogen fixation process within the legume nodule . Hydrogenase (hup) systems in Bradyrhizobium japonicum and Rhizobium leguminosarum bv . viciae show highly conserved sequence and gene organization, but important differences exist in regulation and in the presence of specific genes . We have undertaken the characterization of hup gene clusters from Bradyrhizobium sp . (Lupinus), Bradyrhizobium sp . (Vigna), and Rhizobium tropici and Azorhizobium caulinodans strains with the aim of defining the extent of diversity in hup gene composition and regulation in endosymbiotic bacteria . Genomic DNA hybridizations using hupS, hupE, hupUV, hypB, and hoxA probes showed a diversity of intraspecific hup profiles within Bradyrhizobium sp . (Lupinus) and Bradyrhizobium sp . (Vigna) strains and homogeneous intraspecific patterns within R . tropici and A . caulinodans strains . The analysis also revealed differences regarding the possession of hydrogenase regulatory genes . Phylogenetic analyses using partial sequences of hupS and hupL clustered R . leguminosarum and R . tropici hup sequences together with those from B . japonicum and Bradyrhizobium sp . (Lupinus) strains, suggesting a common origin . In contrast, Bradyrhizobium sp . (Vigna) hup sequences diverged from the rest of rhizobial sequences, which might indicate that those organisms have evolved independently and possibly have acquired the sequences by horizontal transfer from an unidentified source.

Plant Cell, 1993 Jun, 5(6), 615 - 620
Rhizobium Lipooligosaccharides Rescue a Carrot Somatic Embryo Mutant; De Jong AJ et al.; At a nonpermissive temperature, somatic embryos of the temperature-sensitive (ts) carrot cell mutant ts11 only proceed beyond the globular embryo stage in the presence of medium conditioned by wild-type embryos . The causative component in the conditioned medium has previously been identified as a 32-kD acidic endochitinase . In search of a function for this enzyme in plant embryogenesis, several compounds that contain oligomers of N-acetylglucosamine were tested for their ability to promote ts11 embryo formation . Of these compounds, only the Rhizobium lipooligosaccharides or nodulation (Nod) factors were found to be effective in rescuing the formation of ts11 embryos . These results suggest that N-acetylglucosamine-containing lipooligosaccharides from bacterial origin can mimic the effect of the carrot endochitinase . This endochitinase may therefore be involved in the generation of plant analogs of the Rhizobium Nod factors.

Mikrobiologiia, 2002 Jul-Aug, 71(4), 521 - 5
{Isolation and phenotypic characteristics of growth-stimulating rhizobacteria (PGPR), with high root-colonizing and phytopathogenic fungi inhibiting abilities}; Kravchenko LV et al.; Some bacterial strains isolated from the plant rhizosphere showed high root-colonizing ability and antiphytopathogenic activity against 6 fungal species . The antifungal activity was species-specific, which could be accounted for by the fact that the isolates differed in the ability to produce lytic enzymes (chitinases, proteases, and lipases) and to secrete cyanide . The possibility of using there rhizobacteria to control phytopathogens is discussed.

Mol Plant Microbe Interact, 2002 Sep, 15(9), 922 - 31
Glycine-rich proteins encoded by a nodule-specific gene family are implicated in different stages of symbiotic nodule development in Medicago spp; Kevei Z et al.; Four genes encoding small proteins with significantly high glycine content have been identified from root nodules of Medicago sativa . All of these proteins as well as their Medicago truncatula homologues carried an amino terminal signal peptide and a glycine-rich carboxy terminal domain . All except nodGRP3 lacked the characteristic repeat structure described for cell wall and stress response-related glycine-rich proteins (GRP) . Expression of these GRP genes was undetectable in flower, leaf, stem, and hypocotyl cells, whereas expression was highly induced during root nodule development, suggesting that GRP genes act as nodulins . Moreover, none of these nodule-expressed GRP genes were activated by hormones or stress treatments, which are inducers of many other GRPs . In Rhizobium-free spontaneous nodules and in nodules induced by a noninfective mutant strain of Sinorhizobium meliloti, all these genes were repressed, while they were induced in Fix- nodules, unaffected in bacterial infection, but halted in bacteroid differentiation . These results demonstrated that bacterial infection but not bacteroid differentiation is required for the induction of the nodule-specific GRP genes . Differences in kinetics and localization of gene activation as well as in the primary structure of proteins suggest nonredundant roles for these GRPs in nodule organogenesis.

Mol Plant Microbe Interact, 2002 Sep, 15(9), 859 - 65
Rapid colorimetric quantification of lipo-chitooligosaccharides from Mesorhizobium loti and Sinorhizobium meliloti; Goedhart J et al.; Nod factors are lipids with a chitinlike headgroup produced by gram-negative Rhizobium bacteria . These lipo-chitooligosaccharides (LCOs) are essential signaling molecules for accomplishing symbiosis between the bacteria and roots of legume plants . Despite their important role in the Rhizobium-legume interaction, no fast and sensitive Nod factor quantification methods exist . Here, we report two different quantification methods . The first is based on the enzymatic hydrolysis of Nod factors to release N-acetylglucosamine (GlcNAc), which can subsequently be quantified . It is shown that the degrading enzyme, glusulase, releases exactly two GlcNAc units per pentameric nodulation factor from Mesorhizobium loti factor, allowing quantification of LCOs from Mesorhizobium loti . The second method is based on a specific type of Nod factors that are sulfated on the reducing GlcNAc, allowing quantification analogous to the quantification of sulfolipids . Here, a two-phase extraction method is used in the presence of methylene blue, which specifically forms an ion pair with sulfated lipids . The blue ion pair partitions into the organic phase, after which the methylene blue signal can be quantified . To enable Nod factor quantification with this method, the organic phase was modified and the partitioning was evaluated using fluorescent and radiolabeled sulfated Nod factors . It is shown that sulfated LCOs can be quantified with this method, using sodium dodecyl sulfate for calibration . Both methods allow Nod factor quantification in parallel enabling a fast and easy detection of nanomole quantities of Nod factors . Accurate Nod factor quantification will be crucial for characterization and cross-comparison of the affinity for Nod factors of newly identified Nod factor binding proteins or putative Nod factor receptors.

J Appl Microbiol, 2002, 93(4), 531 - 40
Phenotypic and molecular characterization of chickpea rhizobia isolated from different areas of Morocco; Maatallah J et al.; AIMS: To determine the biodiversity of rhizobial strains nodulating Cicer arietinum L . in representative soils from various areas of Morocco . METHODS AND RESULTS: Symbiotic traits, utilization of 49 carbohydrate sources, resistance to antibiotics and heavy metals, tolerance to salinity, to extreme temperatures and pH were studied as phenotypic markers . In addition, restriction fragment length polymorphism (RFLP) of PCR-amplified 16S rDNAs were compared with those of reference strains . Numerical analysis of the phenotypic characteristics showed that the 48 strains studied fell into three distinct groups . RFLP analysis of 16S rRNA genes revealed an additional heterogeneity and four ribotypes were identified . CONCLUSIONS: Chickpea rhizobia isolated from Moroccan soils are both phenotypically and genetically diverse . Most of these rhizobia belong to the Mesorhizobium genus . However, some strains originating from a particular soil appeared to have 16S rRNA genes similar to Sinorhizobium as well as very distinct auxanographic characteristics compared with Mesorhizo- bium isolates . SIGNIFICANCE AND IMPACT OF THE STUDY: A well characterized collection of chickpea-nodulating rhizobia in representative soils of Morocco has been generated, which can be used to develop efficient inoculants for this crop . This is the first report evidencing that chickpeas may be nodulated by bacteria from the Sinorhizobium genus.

Res Microbiol, 2002 Jul-Aug, 153(6), 369 - 72
Evaluation of symbiotic effectiveness of various Rhizobium cicer strains; Icgen B et al.; Five local and seven standard strains of Rhizobium cicer were compared in terms of their efficiency in increasing the nitrogen content of the chickpea . Shoot dry weight, nodule number, nodule dry weight, protein and total nitrogen contents were taken as the parameters of plant productivity . Different combinations of the strains that were found to be promising (385, 620, Y-29 and 3379) were next employed . The maximum increase in total nitrogen content was only 3.5-fold in single infection whereas an increase as great as 35-fold was recorded for multiple infections . The double infection with Y-29 and 385 as well as the triple infection with Y-29, 620 and 3379 gave rise to the maximum values . Competitiveness of the strains in mixed infections was determined through their recovery from root surfaces and nodules and their subsequent identification . The effects of soil pH and of varying the concentration of some minerals on the outcome of symbiosis were also reported.

Curr Microbiol, 2002 Nov, 45(5), 378 - 82
Molecular cloning and characterization of nodD genes from Rhizobium sp . SIN-1, a nitrogen-fixing symbiont of Sesbania and other tropical legumes; Rana D et al.; Rhizobium sp . SIN-1, a nitrogen-fixing symbiont of Sesbania aculeata and other tropical legumes, carries two copies of nodD, both on a sym plasmid . We have isolated these two nodD genes by screening a genomic library of Rhizobium sp . SIN-1 with a nodD probe from Sinorhizobium meliloti . Nucleotide sequence and the deduced amino acid sequence analysis indicated that the nodD genes of Rhizobium sp . SIN-1 are most closely related to those of R . tropici and Azorhziobium caulinodans . Rhizobium sp . SIN-1 nodD1 complemented a S . meliloti nodD1 D2 D3 negative mutant for nodulation on alfalfa, but failed to complement a nodD1 mutant of S . fredii USDA191 for soybean nodulation . A hybrid nodD gene, containing the N-terminus of S . fredii USDA191 nodD1 and the C-terminus of Rhizobium sp . SIN-1 nodD1, complemented the nodD1 negative mutant of USDA191 for nodulation on soybean.

Curr Microbiol, 2002 Nov, 45(5), 368 - 77
Effect of drought on the growth and survival of the stress-tolerant bacterium Rhizobium sp . NBRI2505 sesbania and its drought-sensitive transposon Tn 5 mutant; Rehman A et al.; Studies were conducted to elucidate the nature of drought tolerance in the bacterium Rhizobium sp . NBRI2505 sesbania and its transposon Tn 5 induced mutant to assess the role of salt, pH, and temperature stresses in contributing to drought tolerance, and to correlate drought tolerance and symbiotic effectiveness . Rhizobium sp . NBRI2505 sesbania tolerated yeast extract mannitol broth (YEB) containing 28% salt (NaCl; wt/vol) for up to 18 h of incubation at 30 degrees C, survived a 2-h incubation in YEB at 65 degrees C, and when subjected to drought stress, tolerated YEB containing 45% polyethylene glycol 6000 (PEG; wt/vol) for up to 5 days of incubation at 30 degrees C . One drought-sensitive mutant Rhizobium sp . NBRI2505 sesbania T112 (T112) containing a single Tn 5 insertion was selected after screening about 10,000 clones . T112 was specifically defective in its tolerance for drought: when subjected to drought stress, it tolerated YEB containing 45% PEG for up to 2 days of incubation at 30 degrees C . T122 mutant was also more sensitive to the heat and desiccation stresses, compared with Rhizobium sp . NBRI2505 sesbania in the presence of 45% PEG . Our results demonstrated a positive effect of calcium on the survival of Rhizobium sp . sesbania under acidic stress conditions . The observed enhanced survival at pH 3 of Rhizobium sp . NBRI2505 sesbania and T112 in the presence of 5% CaCO(3) suggests the requirement of calcium for growth and survival, which may have an ecological significance in acidic soils . Mutant strain T112 produced ineffective symbiosis with the plant host in the presence of 2.5 and 5% PEG, indicating that drought tolerance is required for effective symbiosis.

Plant Physiol, 1994 Jul, 105(3), 787 - 797
Root Hair Deformation Activity of Nodulation Factors and Their Fate on Vicia sativa; Heidstra R et al.; We used a semiquantitative root hair deformation assay for Vicia sativa (vetch) to study the activity of Rhizobium leguminosarum bv viciae nodulation (Nod) factors . Five to 10 min of Nod factor-root interaction appears to be sufficient to induce root hair deformation . The first deformation is visible within 1 h, and after 3 h about 80% of the root hairs in a small susceptible zone of the root are deformed . This zone encompasses root hairs that have almost reached their maximal size . The Nod factor accumulates preferentially to epidermal cells of the young part of the root, but is not restricted to the susceptible zone . In the interaction with roots, the glucosamine backbone of Nod factors is shortened, presumably by chitinases . NodRlv-IV(C18:4,Ac) is more stable than NodRlv-V(C18:4,Ac) . No correlation was found between Nod factor degradation and susceptibility . Degradation occurs both in the susceptible zone and in the mature zone . Moreover, degradation is not affected by NH4NO3 and is similar in vetch and in the nonhost alfalfa (Medicago sativa).

Plant Physiol, 1994 Jan, 104(1), 91 - 97
Production and Characterization of Monoclonal Antibodies against Aspartate Aminotransferase-P1 from Lupin Root Nodules; Jones WT et al.; Six hybridoma clones were obtained that secreted monoclonal antibodies against the aspartate aminotransferase-P1 (AAT-P1) isoenzyme from root nodules of Lupinus angustifolius {L.} cv Uniharvest . This enzyme is found constitutively in the plant cytosol fraction . The monoclonal antibodies produced were all of the immunoglobulin G1 class, recognized two distinct epitopes on the protein, and represented the major paratopes found in the immunoglobulin fraction of sera taken from mice and rabbits immunized with the pure AAT-P1 protein . One of these epitopes was unique to lupin nodule AAT-P1 . The other epitope was shown to be present on enzyme from lupin bean, white clover and tobacco leaves, lupin roots and nodules, and potato tubers . Both epitopes were recognized by the appropriate monoclonal antibodies in both their native and denatured forms . None of the monoclonal antibodies produced reacted with Rhizobium lupini NZP2257, Escherichia coli extracts, or with the inducible aspartate aminotransferase-P2 (AAT-P2) isoform also found in root nodules . A sandwich enzyme-linked immunosorbent assay utilizing two monoclonal antibodies recognizing the two distinct epitopes was developed and was capable of quantitating AAT-P1 in plant extracts . The limit of detection of AAT-P1 was less than 15 pg/mL and AAT-P1 protein could be quantified in the range 80 to 1000 pg/mL . Using this assay, AAT-P1 protein was shown to remain relatively constant during nodule development . Use of an AAT-P2-specific monoclonal antibody that inhibits the enzyme activity of this isoform enabled the direct determination of AAT-P1 enzyme activity in nodule extracts . Using these assays, specific activities of the individual isoforms were calculated; that of the AAT-P1 isoform was shown to be 7.5-fold higher than that of the AAT-P2 isoform.

Plant Physiol, 1994 Jan, 104(1), 85 - 90
Essentiality of Boron for Symbiotic Dinitrogen Fixation in Pea (Pisum sativum) Rhizobium Nodules; Bolanos L et al.; The effect of boron deficiency on symbiotic nitrogen fixation in pea (Pisum sativum) was examined . The absence of boron in the culture medium resulted in a decrease of the number of nodules and an alteration of nodule development leading to an inhibition of nitrogenase activity . Examination of boron-deficient nodules showed dramatic changes in cell walls and in both peribacteroid and infection thread membranes, suggesting a role for boron in the stability of these structures . These results indicate that boron is a requirement for normal nodule development and functionality.

Plant Physiol, 1993 Nov, 103(3), 925 - 932
Five Nodulation Mutants of White Sweetclover (Melilotus alba Desr.) Exhibit Distinct Phenotypes Blocked at Root Hair Curling, Infection Thread Development, and Nodule Organogenesis; Utrup LJ et al.; In an effort to obtain a developmental sequence of mutations in the Rhizobium-legume interaction within a single legume species, we have characterized the early events of nodule development in 10 nodulation mutants of sweetclover, Melilotus alba Desr . cv U389, representing five genetic loci . Both seed and root exudates from all of the sweetclover mutants induced expression of the nod genes of Rhizobium meliloti . Mutants in three loci were blocked in the early stages of root hair curling . Of these, a mutant in the sym-3 locus exhibited root hair deformations in response to inoculation with R . meliloti but produced no nodules or emerging nodule primordia, suggesting a blockage in the signal transduction events leading to nodule organogenesis . In contrast, mutants in both the sym-1 and sym-5 loci formed ineffective nodules in response to inoculation but differed slightly in the type of root hair response observed . None of these three early mutants formed infection threads . Infection threads were observed in mutant sym-2 as well as in ineffective nodules . Mutant sym-4 also formed infection threads but lacked nodules . The phenotypes observed for mutants from these five loci suggest that a secondary receptor or signal produced by the plant is required for nodule development.

Plant Physiol, 1993 Mar, 101(3), 819 - 824
Alfalfa (Medicago sativa L.) Root Exudates Contain Isoflavonoids in the Presence of Rhizobium meliloti; Dakora FD et al.; Root exudates of alfalfa (Medicago sativa L.) inoculated with symbiotic Rhizobium meliloti bacteria contained three isoflavonoids that were not found in exudates of uninoculated plants . Data from proton nuclear magnetic resonance, mass spectrometry, and ultraviolet-visible absorbance analyses indicated that root exudates of inoculated plants contained aglycone and glycoside forms of the phytoalexin medicarpin and a formononetin-7-O-(6"-O-malonylglycoside), a conjugated form of the medicarpin precursor formononetin . The medicarpin molecules did not induce nod gene transcription in R . meliloti, but the formononetin-7-O-(6"-O-malonylglycoside) induced nod genes regulated by both NodD1 and NodD2 proteins in R . meliloti . Hydrolysis of either the malonyl or the glycosyl linkage from the formononetin conjugate eliminated nod gene-inducing activity . The nod gene-inducing activity of crude root exudates was increased 200 and 65% upon inoculation with R . meliloti or R . leguminosarum bv phaseoli, respectively . When root exudate from uninoculated alfalfa was incubated with R . meliloti, high performance liquid chromatography analyses showed no evidence that bacterial metabolism produced medicarpin . These results indicate that alfalfa responds to symbiotic R . meliloti by exuding a phytoalexin normally elicited by pathogens and that the microsymbiont can use a precursor of the phytoalexin as a signal for inducing symbiotic nod genes.

Plant Physiol, 1995 Dec, 109(4), 1167 - 1177
Sugar-Binding Activity of Pea Lectin Expressed in White Clover Hairy Roots; Diaz CL et al.; Introduction of the pea (Pisum sativum L.) lectin (PSL) gene into white clover (Trifolium repens L.) hairy roots facilitates nodulation by the nitrogen-fixing bacterium Rhizobium leguminosarum biovar viciae, which normally nodulates pea and not white clover (C.L . Diaz, L.S . Melchers, P.J.J . Hooykaas, B.J.J . Lugtenberg, and J.W . Kijne {1989} Nature 338: 579-581) . Here, we show that PSL is functionally expressed in transgenic white clover hairy roots transformed with the PSL gene . PSL could be isolated from these roots by affinity chromatography . Immunoanalysis of PSL showed the presence of polypeptides corresponding to the PSL precursor and its {beta} subunits . In addition, we developed a highly sensitive localization technique based on specific binding of a glycan moiety of rat IgE to PSL . Similar to the situation in pea roots, PSL appeared to be localized on the external cell surface of elongated epidermal cells and on the tips of emerging and growing root hairs of transgenic white clover hairy roots . PSL was not observed on normal white clover roots and on hairy roots without the PSL gene . These results show that (a) in transgenic white clover hairy roots, PSL is correctly processed and targeted to root cells susceptible to rhizobial infection, and (b) like in pea roots, PSL is surface bound with at least one of its two sugar-binding sites available for (rhizobial) ligands.

Plant Physiol, 1995 Aug, 108(4), 1607 - 1614
Lipo-chitooligosaccharide Nodulation Signals from Rhizobium meliloti Induce Their Rapid Degradation by the Host Plant Alfalfa; Staehelin C et al.; Extracellular enzymes from alfalfa (Medicago sativa L.) involved in the degradation of nodulation (Nod) factors could be distinguished by their different cleavage specificities and were separated by lectin affinity chromatography . A particular glycoprotein was able to release an acylated lipo-disaccharide from all tested Nod factors having an oligosaccharide chain length of four or five residues . Structural modifications of the basic lipo-chitooligosaccharide did not affect the cleavage site and had only weak influence on the cleavage efficiency of Nod factors tested . The acylated lipo-trisaccharide was resistant to degradation . When alfalfa roots were preincubated with Nod factors at nanomolar concentrations, the activity of the dimer-forming enzyme was stimulated up to 6-fold within a few hours . The inducing activity of Nod factors decreased in the order NodRm-IV(C16:2,Ac,S) > NodRm-IV(C16:2,S) and NodRm-V(C16:2,Ac,S) > NodRm-V(C16:2,S) > NodRm-IV(C16:0,S) > NodRm-IV(C16:2) . Pretreatment with NodRm-III(C16:2) as well as unmodified chitooligosaccharides did not stimulate the dimer-forming enzyme . Roots preincubated with Rhizobium meliloti showed similar stimulation of the dimer-forming activity . Mutant strains unable to produce Nod factors did not enhance the hydrolytic activity . These results indicate a rapid feedback inactivation of Nod signals after their perception by the host plant alfalfa.

Plant Physiol, 1995 Aug, 108(4), 1547 - 1552
The Rhizobial hemA Gene Is Required for Symbiosis in Species with Deficient {delta}-Aminolevulinic Acid Uptake Activity; McGinnis SD et al.; Most rhizobial hemA mutants induce root nodules on their respective legume hosts that lack nitrogen fixation activity and leghemoglobin expression . However, a Bradyrhizobium japonicum hemA mutant elicits effective nodules on soybean, and we proposed previously that synthesis and uptake of the heme precursor {delta}-aminolevulinic acid (ALA) by the plant and bacterial symbiont, respectively, allow mutant rescue (I . Sangwan, M.R . O'Brian {1991} Science 251: 1220-1222) . In the present work, the B . japonicum hemA mutant MLG1 elicited normal nodules on three hosts, including cowpea, a plant that is not effectively nodulated by a hemA mutant of Rhizobium sp . These data indicate that B . japonicum rather than soybean possesses the unique trait that allows normal nodule development by a hemA mutant . Cowpea expressed glutamate-dependent ALA formation activity in nodules induced by B . japonicum strains I110 or MLG1 and by Rhizobium sp . ANU240 . Exogenous ALA was taken up by B . japonicum bacteroids isolated from soybean or cowpea nodules, and the kinetics of uptake were biphasic . By comparison, Rhizobium sp . ANU240 had very low ALA uptake activity . In addition, ALA uptake was observed in cultured cells of B . japonicum but not in cultured cells of three other rhizobial species tested . We suggest that the differential success of legume-rhizobial hemA symbioses is due to an ALA uptake activity in B . japonicum that is deficient in other rhizobia, thereby further validating the ALA rescue hypothesis.

Plant Physiol, 1995 Aug, 108(4), 1519 - 1525
Rhizobial Nodulation Factors Stimulate Mycorrhizal Colonization of Nodulating and Nonnodulating Soybeans; Xie ZP et al.; Legumes form tripartite symbiotic associations with noduleinducing rhizobia and vesicular-arbuscular mycorrhizal fungi . Co-inoculation of soybean (Glycine max {L.} Merr.) roots with Bradyrhizobium japonicum 61-A-101 considerably enhanced colonization by the mycorrhizal fungus Glomus mosseae . A similar stimulatory effect on mycorrhizal colonization was also observed in nonnodulating soybean mutants when inoculated with Bradyrhizobium japonicum and in wild-type soybean plants when inoculated with ineffective rhizobial strains, indicating that a functional rhizobial symbiosis is not necessary for enhanced mycorrhiza formation . Inoculation with the mutant Rhizobium sp . NGR{delta}nodABC, unable to produce nodulation (Nod) factors, did not show any effect on mycorrhiza . Highly purified Nod factors also increased the degree of mycorrhizal colonization . Nod factors from Rhizobium sp . NGR234 differed in their potential to promote fungal colonization . The acetylated factor NodNGR-V (MeFuc, Ac), added at concentrations as low as 10-9 M, was active, whereas the sulfated factor, NodNGR-V (MeFuc, S), was inactive . Several soybean flavonoids known to accumulate in response to the acetylated Nod factor showed a similar promoting effect on mycorrhiza . These results suggest that plant flavonoids mediate the Nod factor-induced stimulation of mycorrhizal colonization in soybean roots.

Plant Physiol, 1995 Jun, 108(2), 533 - 542
Alfalfa Root Flavonoid Production Is Nitrogen Regulated; Coronado C et al.; Flavonoids produced by legume roots are signal molecules acting both as chemoattractants and nod gene inducers for the symbiotic Rhizobium partner . Combined nitrogen inhibits the establishment of the symbiosis . To know whether nitrogen nutrition could act at the level of signal production, we have studied the expression of flavonoid biosynthetic genes as well as the production of flavonoids in the roots of plants grown under nitrogen-limiting or nonlimiting conditions . We show here that growth of the plant under nitrogen-limiting conditions results in the enhancement of expression of the flavonoid biosynthesis genes chalcone synthase and isoflavone reductase and in an increase of root flavonoid and isoflavonoid production as well as in the Rhizobium meliloti nod gene-inducing activity of the root extract . These results indicate that in alfalfa (Medicago sativa L.) roots, the production of flavonoids can be influenced by the nitrogen nutrition of the plant.

Plant Physiol, 1995 Mar, 107(3), 783 - 790
Role of the Differentiation of Root Epidermal Cells in Nod Factor (from Rhizobium meliloti)-Induced Root-Hair Depolarization of Medicago sativa; Kurkdjian AC; The stage of differentiation of epidermal cells and the development of root hairs was found to be important for the induction of depolarization in root hairs of Medicago sativa by Nod factor {NodRm-IV(S)} isolated from the bacterium Rhizobium meliloti . The electrical membrane response was concentration dependent, having its major effect (amplitude of the depolarization and number of root hairs that responded) at 10-8 and 10-7 M Nod factor . This response was correlated with a morphological effect of Nod factor in the root-hair-deformation bioassay at similar concentrations . The effect of Nod factor on depolarization and root-hair deformation showed specificity with respect to the structure, since unsulfated Nod molecules were inactive, as was the synthetic N,N',N",N"'- tetraacetylchitotetraose . The Nod factor that is O-acetylated at the nonreducing sugar was as efficient in root-hair deformation and membrane depolarization as the sulfated Nod factor.

Plant Physiol, 1995 Jan, 107(1), 53 - 62
TE7, An Inefficient Symbiotic Mutant of Medicago truncatula Gaertn . cv Jemalong; Benaben V et al.; A mutagenesis program using ethylmethane sulfonate on Medicago truncatula Gaertn cv Jemalong, an annual, autogamous and diploid lucerne, permitted the isolation of a mutant (TE7) unable to establish an effective nitrogen-fixing symbiosis, {Nod+Fix-}, with Rhizobium meliloti wild-type strains . The mutant phenotype is characterized by an altered infection process that leads to the formation of two kinds of inefficient nodules on the same root system . A certain proportion of the nodules are small, round, and uninfected, with infection threads limited to the outer root cortical cells . Others develop to a normal elongated shape and are infected; bacterial release occurs but the bacteria do not differentiate into bacteroids . The ratio of invaded to uninvaded nodules depends on the bacterial strain used . Throughout the infection process, certain events correlated with the plant defense response against pathogens can be observed: (a) the presence of polyphenolic compounds associated with the walls of infected cells and also with some parts of infection threads in the root cortex; (b) appositions on infection thread walls during the early stage of infection and also within the central tissue of infected nodules; and (c) autophagy of the plant cells that contain released bacteria . Genetic data suggest that the phenotype of TE7 is under monogenic and recessive control; this gene has been designated Mtsym1.

Infect Immun, 2002 Oct, 70(10), 5540 - 6
Effect of omp10 or omp19 deletion on Brucella abortus outer membrane properties and virulence in mice; Tibor A et al.; The distinctive properties of Brucella outer membrane have been considered to be critical for Brucella sp . virulence . Among the outer membrane molecules possibly related to these properties, Omp10 and Omp19 are immunoreactive outer membrane lipoproteins . Moreover, these proteins of Brucella could constitute a new family of outer membrane proteins specifically encountered in the family RHIZOBIACEAE: We evaluated the impact of omp10 or omp19 deletion on Brucella abortus outer membrane properties and virulence in mice . The omp10 mutant was dramatically attenuated for survival in mice and was defective for growth in minimal medium but was not impaired in intracellular growth in vitro, nor does it display clear modification of the outer membrane properties . Significantly fewer brucellae were recovered from the spleens of mice infected with the omp19 mutant than from those of mice infected with the parent strain at 4 and 8 weeks postinfection . The omp19 mutant exhibited an increase in sensitivity to the polycation polymyxin B and to sodium deoxycholate . These results indicate that inactivation of the omp19 gene alters the outer membrane properties of B . abortus.

Plant Physiol, 1996 Apr, 110(4), 1249 - 1256
Effects of Boron on Rhizobium-Legume Cell-Surface Interactions and Nodule Development; Bolanos L et al.; Boron (B) is an essential micronutrient for the development of nitrogen-fixing root nodules in pea (Pisum sativum) . By using monoclonal antibodies that recognize specific glycoconjugate components implicated in legume root-nodule development, we investigated the effects of low B on the formation of infection threads and the colonization of pea nodules by Rhizobium leguminosarum bv viciae . In B-deficient nodules the proportion of infected host cells was much lower than in nodules from plants supplied with normal quantities of B . Moreover, the host cells often developed enlarged and abnormally shaped infection threads that frequently burst, releasing bacteria into damaged host cells . There was also an over-production of plant matrix material in which the rhizobial cells were embedded during their progression through the infection thread . Furthermore, in a series of in vitro binding studies, we demonstrated that the presence of B can change the affinity with which the bacterial cell surface interacts with the peribacteroid membrane glycocalyx relative to its interaction with intercellular plant matrix glycoprotein . From these observations we suggest that B plays an important role in mediating cell-surface interactions that lead to endocytosis of rhizobia by host cells and hence to the correct establishment of the symbiosis between pea and Rhizobium.

Plant Physiol, 1996 Feb, 110(2), 501 - 510
The Auxin Transport Inhibitor N-(1-Naphthyl)phthalamic Acid Elicits Pseudonodules on Nonnodulating Mutants of White Sweetclover; Wu C et al.; The collection of symbiotic (sym) mutants of white sweetclover (Melilotus alba Desr.) provides a developmental sequence of mutants blocked early in infection or nodule organogenesis . Mutant phenotypes include non-nodulating mutants that exhibit root-hair deformations in response to Rhizobium meliloti, mutants that form ineffective nodules lacking infection threads, and mutants that form infection threads and ineffective nodules . Mutant alleles from both the sym-1 and the sym-3 loci exhibited a non-nodulating phenotype in response to R . meliloti, although one allele in the sym-1 locus formed ineffective nodules at a low frequency . Spot-inoculation experiments on a non-nodulating allele in the sym-3 locus indicated that this mutant lacked cortical cell divisions following inoculation with R . meliloti . The auxin transport inhibitor N-(1-naphthyl)phthalamic acid elicited development of pseudonodules at a high frequency on all of the sweetclover sym mutants, including the non-nodulating mutants, in which the early nodulin ENOD2 was expressed . This suggests that N-(1-naphthyl)phthalamic acid activates cortical cell divisions by circumventing a secondary signal transduction event that is lacking in the non-nodulating sweetclover mutants . The sym-3 locus and possibly the sym-1 locus appear to be essential to early host plant responses essential to nodule organogenesis.

Plant Physiol, 1997 Oct, 115(2), 351 - 359
Sym2 of Pea Is Involved in a Nodulation Factor-Perception Mechanism That Controls the Infection Process in the Epidermis; Geurts R et al.; In pea (Pisum sativum) up to 50 nodulation mutants are known, several of which are affected in the early steps of the symbiotic interaction with Rhizobium sp . bacteria . Here we describe the role of the sym2 gene in nodulation (Nod) factor perception . Our experiments show that the sym2A allele from the wild pea variety Afghanistan confers an arrest in infection-thread growth if the Rhizobium leguminosarum bv viciae strain does not produce Nod factors with a NodX-mediated acetylation at their reducing end . Since the induction of the early nodulin gene ENOD12 in the epidermis and the formation of a nodule primordium in the inner cortex were not affected, we conclude that more than one Nod factor-perception mechanism is active . Furthermore, we show that sym2A-mediated control of infection-thread growth was affected by the bacterial nodulation gene nodO.

Plant Physiol, 1997 Apr, 113(4), 1233 - 1242
P Metabolism in the Bean-Rhizobium tropici Symbiosis; Al-Niemi TS et al.; Nodulated legumes require more P than legumes growing on mineral nitrogen, but little is known about the basis for the higher P requirement . Experiments were conducted to determine how Rhizobium tropici responds to P limitation and to understand how P is partitioned between the symbionts under conditions of adequate or limiting P . Free-living R . tropici responds to P stress by increasing P transport capacity and inducing both an acid and an alkaline phosphatase . This P-stress response occurs when the medium P concentration decreases below 1 {mu}M . Both P-stress-inducible phosphatases are found in bacteroids taken from plants growing with adequate P, suggesting that P levels in the symbiosome space is low enough to induce the expression of these enzymes . Bacteroid alkaline phosphatase-specific activity was highest during vegetative growth of the bean plant, but decreased approximately 75% during the host reproductive stages . In hydroponic experiments 32P-tracer studies showed that in vivo rates of P accumulation were significantly higher in bacteroids from P-limited plants compared with those from plants that had been supplied with adequate P . In contrast, label accumulation in leaves was greatest in plants grown with adequate P.

Mol Genet Genomics, 2002 Aug, 267(6), 820 - 8 Epub 2002 Jul 03.
The Rhizobium etli gene iscN is highly expressed in bacteroids and required for nitrogen fixation; Dombrecht B et al.; Sequence analysis of the rpoN (2)- fixA intergenic region in the genome of Rhizobium etli CNPAF512 has uncovered three genes involved in nitrogen fixation, namely nifU, nifS and nifW . These genes are preceded by an ORF that is highly conserved among nitrogen-fixing bacteria . It encodes a putative gene product of 105 amino acids, belonging to the HesB-like protein family . A phylogenetic analysis of members of the HesB-like protein family showed that the R . etli HesB-like protein clusters with polypeptides encoded by ORFs situated upstream of the nifUS nitrogen fixation regions in the genomes of other diazotrophs . The R . etli ORF that encodes the HesB-like protein was designated iscN . iscN is co-transcribed with nifU and nifS, and is preferentially expressed under free-living microaerobic conditions and in bacteroids . Expression is regulated by the alternative sigma factor RpoN and the enchancer-binding protein NifA . A R . etli iscN mutant displays a reduction in nitrogen fixation capacity of 90% compared to the wild-type strain . This Nif(-) phenotype could be complemented by the introduction of intact copies of R . etli iscN.

J Bacteriol, 2002 Oct, 184(19), 5436 - 48
A monocarboxylate permease of Rhizobium leguminosarum is the first member of a new subfamily of transporters; Hosie AH et al.; Amino acid transport by Rhizobium leguminosarum is dominated by two ABC transporters, the general amino acid permease (Aap) and the branched-chain amino acid permease (Bra) . However, mutation of these transporters does not prevent this organism from utilizing alanine for growth . An R . leguminosarum permease (MctP) has been identified which is required for optimal growth on alanine as a sole carbon and nitrogen source . Characterization of MctP confirmed that it transports alanine (K(m) = 0.56 mM) and other monocarboxylates such as lactate and pyruvate (K(m) = 4.4 and 3.8 micro M, respectively) . Uptake inhibition studies indicate that propionate, butyrate, alpha-hydroxybutyrate, and acetate are also transported by MctP, with the apparent affinity for solutes demonstrating a preference for C3-monocarboxylates . MctP has significant sequence similarity to members of the sodium/solute symporter family . However, sequence comparisons suggest that it is the first characterized permease of a new subfamily of transporters . While transport via MctP was inhibited by CCCP, it was not apparently affected by the concentration of sodium . In contrast, glutamate uptake in R . leguminosarum by the Escherichia coli GltS system did require sodium, which suggests that MctP may be proton coupled . Uncharacterized members of this new subfamily have been identified in a broad taxonomic range of species, including proteobacteria of the beta-subdivision, gram-positive bacteria, and archaea . A two-component sensor-regulator (MctSR), encoded by genes adjacent to mctP, is required for activation of mctP expression.

Sheng Wu Hua Xue Yu Sheng Wu Wu Li Xue Bao (Shanghai), 1997, 29(5), 481 - 488
Sequence Analysis of the exo1 Gene Involved in the Exopolysaccharide Synthesis of Rhizobium huakuii; Huang JB et al.; The 2.6 kb fragment which can complement the Exo(-)Ndv(-)Fix(-) mutants of R . huakuii to Exo(+)Nod(+)Fix(+) was sequenced . The result revealed the presence of an open reading frame exo1, which encodes 340 amino acids . Analysis of the hydrophobicity plot of the putative protein indicated that it was a cytoplasmic protein . Exo1 displayed strong homology to the ExoU protein of R . meliloti, which was a glucosyltransferase . A transcriptional fusion to lacZ using the promoterless vector pMP221 showed that there were two regions with promoter activity in exo1 . Pexo1a was identified upstream of exo1, and Pexo1b in the middle of exo1 . Pexo1a probably contains the promoter of exo1 gene.

Arch Microbiol, 2002 Oct, 178(4), 301 - 5 Epub 2002 Jun 29.
Mesorhizobium amorphae, a rhizobial species that nodulates Amorpha fruticosa, is native to American soils; Wang ET et al.; Amorpha fruticosa was inoculated with rhizosphere soil from Iowa, USA, and 140 rhizobia isolated from root nodules were compared with Mesorhizobium amorphae originating from Chinese soils . PCR-RFLP patterns of the 16S rRNA gene from the isolates and from M . amorphaewere the same . All isolates had a symbiotic plasmid of the same size with a single nifHgene . DNA:DNA hybridization values, DNA G+C content, and induced Nod factor patterns also were similar . We concluded that the four genotypes distinguished among 53 representative American isolates were M . amorphae . Since A . fruticosa is native to the Americas and is highly specific in its nodulation requirement, M . amorphae probably was transmitted to China.

Appl Environ Microbiol, 2002 Sep, 68(9), 4201 - 8
Effect of a Sinorhizobium meliloti strain with a modified putA gene on the rhizosphere microbial community of alfalfa; van Dillewijn P et al.; The success of a rhizobial inoculant in the soil depends to a large extent on its capacity to compete against indigenous strains . M403, a Sinorhizobium meliloti strain with enhanced competitiveness for nodule occupancy, was recently constructed by introducing a plasmid containing an extra copy of a modified putA (proline dehydrogenase) gene . This strain and M401, a control strain carrying the same plasmid without the modified gene, were used as soil inoculants for alfalfa in a contained field release experiment at Leon, Spain . In this study, we determined the effects of these two strains on the indigenous microbial community . 16S rRNA genes were obtained from the rhizosphere of alfalfa inoculated with strain M403 or strain M401 or from noninoculated plants by amplification of DNA from soil with bacterial group-specific primers . These genes were analyzed and compared by restriction fragment length polymorphism and temperature gradient gel electrophoresis . The results allowed us to differentiate between alterations in the microbial community apparently caused by inoculation and by the rhizosphere effect and seasonal fluctuations induced by the alfalfa plants and by the environment . Only moderate inoculation-dependent effects could be detected, while the alfalfa plants appeared to have a much stronger influence on the microbial community.

Ann Bot (Lond), 2002 Aug, 90(2), 175 - 83
Nodule ultrastructure and initial growth of Anadenanthera peregrina (L.) Speg . var . falcata (Benth.) altschul plants infected with rhizobia; Grossi E et al.; The anatomy and ultrastructure of root nodules of Anadenanthera peregrina var . falcata (Leguminosae-Mimosoideae) were analysed, as was plant growth . To ensure that nodules developed, seedlings were inoculated with a mixture of six strains of rhizobia . Nodules were produced that differed in appearance-and probably also effectiveness-but their structure was similar and they showed characteristics typical of indeterminate nodules, such as persistent meristematic tissue and a gradient of cells at different stages of development . Many starch grains were present in inner cortex cells and interstitial cells of infected tissue . Infected cells were densely packed with bacteroids, which contained many poly-beta-hydroxybutyrate granules . The high incidence of these granules, together with high levels of starch accumulation in interstitial cells, suggested low N2-fixation efficiency of the rhizobia isolates used for inoculation . In the symbiosomes of early-senescent infected cells, reticulum-like structures, small vesicles and a fibrillar material were observed; these may be related to bacteroid degradation . In the cytoplasm of late-senescent infected cells, many vesicles and membrane-like structures were observed, probably associated with membrane degradation of bacteroids and peribacteroids . The total biomass of plants inoculated with rhizobia was low and their xylopodia and shoots had low levels of N compared with noninoculated plants fertilized with ammonium nitrate . However, inoculated plants did not show N-deficiency symptoms and grew better than non-inoculated plants without N fertilization . These growth results, together with ultrastructural observations of nodules, suggest that nitrogen fixation of rhizobia isolates associated with Anadenanthera peregrina var . falcata roots is poor.

Biochem Soc Trans, 2002 Aug, 30(4), 771 - 4
Structural studies of the Fur protein from Rhizobium leguminosarum; Kolade OO et al.; The X-ray crystal structure of the apo-form of the Fur protein from Rhizobium leguminosarum has been solved at 2.7 A resolution . Small-angle X-ray scattering was used to give information on the solution conformation of the protein . The Fur homodimer folds into two domains . The N-terminal domain is formed from the packing of two helix-turn-helix motifs while the C-terminal domain appears primarily to stabilize the dimeric state of the protein.

J Bacteriol, 2002 Sep, 184(18), 5067 - 76
A LuxR homolog controls production of symbiotically active extracellular polysaccharide II by Sinorhizobium meliloti; Pellock BJ et al.; Production of complex extracellular polysaccharides (EPSs) by the nitrogen-fixing soil bacterium Sinorhizobium meliloti is required for efficient invasion of root nodules on the host plant alfalfa . Any one of three S . meliloti polysaccharides, succinoglycan, EPS II, or K antigen, can mediate infection thread initiation and extension (root nodule invasion) on alfalfa . Of these three polysaccharides, the only symbiotically active polysaccharide produced by S . meliloti wild-type strain Rm1021 is succinoglycan . The expR101 mutation is required to turn on production of symbiotically active forms of EPS II in strain Rm1021 . In this study, we have determined the nature of the expR101 mutation in S . meliloti . The expR101 mutation, a spontaneous dominant mutation, results from precise, reading frame-restoring excision of an insertion sequence from the coding region of expR, a gene whose predicted protein product is highly homologous to the Rhizobium leguminosarum bv . viciae RhiR protein and a number of other homologs of Vibrio fischeri LuxR that function as receptors for N-acylhomoserine lactones (AHLs) in quorum-sensing regulation of gene expression . S . meliloti ExpR activates transcription of genes involved in EPS II production in a density-dependent fashion, and it does so at much lower cell densities than many quorum-sensing systems . High-pressure liquid chromatographic fractionation of S . meliloti culture filtrate extracts revealed at least three peaks with AHL activity, one of which activated ExpR-dependent expression of the expE operon.

J Biol Chem, 2002 Nov 1, 277(44), 41811 - 6 Epub 2002 Aug 21.
Rhizobium sin-1 lipopolysaccharide (LPS) prevents enteric LPS-induced cytokine production; Vandenplas ML et al.; Endotoxin (lipopolysaccharide (LPS)), a component of Gram-negative bacteria, is among the most potent proinflammatory substances known . The lipid-A region of this molecule initiates the production of multiple host-derived inflammatory mediators, including cytokines (e.g . tumor necrosis factor-alpha (TNFalpha)) . It has been a continuous effort to identify methods of interfering with the interaction between enteric LPS and inflammatory cells using natural and synthetic LPS analogs . Some of these LPS analogs (e.g . Rhodobacter spheroides LPS/lipid-A derivatives) are antagonists in human cells but act as potent agonists with cells of other species . Data reported here indicate that structurally novel LPS from symbiotic, nitrogen-fixing bacteria found in association with the root nodules of legumes do not stimulate human monocytes to produce TNFalpha . Furthermore, LPS from one of these symbiotic bacterial species, Rhizobium sp . Sin-1, significantly inhibits the synthesis of TNFalpha by human cells incubated with Escherichia coli LPS . Rhizobium Sin-1 LPS exerts these effects by competing with E . coli LPS for binding to LPS-binding protein and by directly competing with E . coli LPS for binding to human monocytes . Rhizobial lipid-A differs significantly from previously characterized lipid-A analogs in phosphate content, fatty acid acylation patterns, and carbohydrate backbone . These structural differences define the rhizobial lipid-A compounds as a potentially novel class of LPS antagonists that might well serve as therapeutic agents for the treatment of Gram-negative sepsis.

J Biol Chem, 2002 Nov 1, 277(44), 41802 - 10 Epub 2002 Aug 21.
Characterization of a novel lipid-A from Rhizobium species Sin-1 . A unique lipid-A structure that is devoid of phosphate and has a glycosyl backbone consisting of glucosamine and 2-aminogluconic acid; Jeyaretnam B et al.; The structure of the lipid-A from Rhizobium species Sin-1, a nitrogen-fixing Gram-negative bacterial symbiont of Sesbania, was determined by composition, nuclear magnetic resonance spectroscopic, and mass spectrometric analyses . The lipid-A preparation consisted of a mixture of structures due to differences in fatty acylation and in the glycosyl backbone . There were two different disaccharide backbones . One disaccharide consisted of a distal glucosaminosyl residue beta-linked to position 6 of a proximal 2-aminoglucono-1,5-lactonosyl residue, and in the second disaccharide, the proximal residue was 2-amino-2,3-dideoxy-d-erythro-hex-2-enono-1,5-lactone . For both disaccharides, the distal glucosamine was acylated at C-2' primarily with beta-hydroxypalmitate (beta-OHC16:0) which, in turn, was O-acylated with 27-hydroxyoctacosanoic acid . For some of the lipid-A molecules, the distal glucosaminosyl residue was also acylated at C-3' with beta-hydroxymyristate (beta-OHC14:0), whereas other molecules were devoid of this acyl substituent . Both the 2-aminoglucono-1,5-lactonosyl and 2-amino-2,3-dideoxy-d-erythro-hex-2-enono-1,5-lactonosyl residues were acylated at C-2, primarily with beta-OHC16:0 . Minor amounts of lipid-A molecules contained beta-OHC14:0 at C-3 and/or beta-hydroxystearate (beta-OHC18:0) or beta-hydroxyoctadecenoate (beta-OHC18:1) as the C-2 and C-2' N-acyl substituents.

Mikrobiol Z, 2002 May-Jun, 64(3), 44 - 51
{Species and strain sensitivity of diasotrophs to heavy metals}; Antipchuk AF et al.; The species and strain sensitivity of free and symbiotic nitrogen-fixing agents to heavy metals: cadmium, copper, lead and zinc in the gradient of concentration from 0.5 to 20.0 MPC/l has been studied under the laboratory conditions . Azotobacter (35 strains) and rhizobia (63 strains) have been tested . It has been established that zinc and copper are more toxical for free nitrogen-fixing agents than lead and cadmium . Among symbiotrophic nitrogen-fixing agents the nodule bacteria for alfalfa were most sensitive to cadmium; nodule bacteria of pea and lupine--to copper; those of galega and soya--to zinc . Lead proved to be the least toxic for rhizobia . Negative effect of the heavy-metal mix on diasotrophs was higher than that of each separate metal . The cultures resistant to the effect of heavy metals have been found which may be used when producing preparation for biological reclamation of polluted soils.

Mikrobiol Z, 2002 Mar-Apr, 64(2), 11 - 20
{The study of bacterial glycopolymers using laser spectroscopy}; Varbanets LD et al.; A possibility has been demonstrated to use laser spectroscopy of bacterial glycopolymers by means of measurement of their water solutions fluorescence . Comparative investigations of native lipopolysaccharide (LPS) Ralstonia solanacearum and its structure components permits a supposition to be made that the LPS total spectrum is a result of superposition of the spectrum of O-specific polysaccharide and core oligosaccharide as well as core oligosaccharide and lipid A . The LPS spectrum maximum shift is determined by core oligosaccharide and lipid A luminescence contribution . A decrease as well as complete loss of serological activity as a result of 30 and 60 min UV irradiation of LPS has been established . It has been shown that LPS Rhizobium leguminosarum bv . viciae quenches luminescence of host-plant (pea) lectin depending on the extent of their affinity . Luminescence spectrum of glucan Sinorhizobium meliloti CXM1-188 and two its LPS-mutants differ between themselves both in luminescence intensity and in presence and expression degree of the site 2 with maximum 2.8 eV.

J Biochem Mol Biol Biophys, 2002 Aug, 6(4), 283 - 8
Transcription of matR gene in Rhizobium leguminosarum bv . trifolii; Lee HY et al.; A transcriptional regulator, MatR, encoded on Rhizobium leguminosarum bv . trifolii matR gene was reported to repress mat operon gene expression by binding the upstream operator site . Also, MatR activated its own gene expression by binding the same site that caused the transcriptional repression of the mat operon . This report suggests that MatR plays a dual role in the transcription of matR and matABC with malonate as a positive effector.

Infect Control Hosp Epidemiol, 2002 Aug, 23(8), 462 - 5
Patients in the intensive care unit are exposed to amoeba-associated pathogens; La Scola B et al.; OBJECTIVE: To study the role of amoeba-associated alpha Proteobacteria as infecting agents in intensive care units (ICUs) . DESIGN: Amoeba-associated alpha Proteobacteria were isolated from water samples taken from ICU taps and general hospital reservoir tanks using an amoebal co-culture procedure . Isolates were identified by 16S rDNA gene sequence comparison, and one isolate of each species was used as an antigen in a microimmunofluorescence assay to test the sera of the patients in the ICUs and compare them with those of control subjects . SETTING: The four university hospitals in Marseilles, France . PATIENTS: The sera of 85 patients in the ICUs with nosocomial pneumonia were tested . RESULTS: We isolated 64 bacterial strains that were identified as Afipia species or close relatives within the Rhizobiaceae subgroup of alpha Proteobacteria . These bacteria were assigned to 8 different species . Eleven patients were found to have an elevated antibody titer to either Afipia genospecies 1, or 3 still unnamed bacteria . No specific antibodies were detected in 100 control subjects (P < .01) . CONCLUSION: These preliminary results support the hypothesis that ICU patients are exposed to amoeba-associated alpha Proteobacteria.

Infect Immun, 2002 Sep, 70(9), 5036 - 44
Identification and characterization of a Brucella abortus ATP-binding cassette transporter homolog to Rhizobium meliloti ExsA and its role in virulence and protection in mice; Rosinha GM et al.; Brucella abortus is a facultative intracellular bacterial pathogen that causes abortion in domestic animals and undulant fever in humans . The mechanism of virulence of Brucella spp . is not fully understood yet . Furthermore, genes that allow Brucella to reach the intracellular niche and to interact with host cells need to be identified . Using the genomic survey sequence (GSS) approach, we identified the gene encoding an ATP-binding cassette (ABC) transporter of B . abortus strain S2308 . The deduced amino acid sequence encoded by this gene exhibited 69 and 67% identity with the sequences of the ABC transporters encoded by the exsA genes of Rhizobium meliloti and Mesorhizobium loti, respectively . Additionally, B . abortus ExsA, like R . meliloti and M . loti ExsA, possesses ATP-binding motifs and the ABC signature domain features of a typical ABC transporter . Furthermore, ortholog group analysis placed B . abortus ExsA in ortholog group 6 of ABC transporters more likely to be involved in bacterial pathogenesis . In R . meliloti, ExsA is an exopolysaccharide transporter essential for alfalfa root nodule invasion and establishment of infection . To test the role of ExsA in Brucella pathogenesis, an exsA deletion mutant was constructed . Replacement of the wild-type exsA by recombination was demonstrated by Southern blot analysis of Brucella genomic DNA . Decreased survival in mice of the Brucella DeltaexsA mutant compared to the survival of parental strain S2308 demonstrated that ExsA is critical for full bacterial virulence . Additionally, the B . abortus exsA deletion mutant was used as a live vaccine . Challenge experiments revealed that the exsA mutant strain induced superior protective immunity in BALB/c mice compared to the protective immunity induced by strain S19 or RB51.

Mol Plant Microbe Interact, 2002 Aug, 15(8), 834 - 9
High-affinity nod factor binding site from Phaseolus vulgaris cell suspension cultures; Gressent F et al.; The lipo-chitooligosaccharidic Nod factors produced by rhizobia are key molecules in the establishment of symbiosis with legumes and probably are recognized by the host plant via specific receptors . Here, we report on the presence of a binding site in cell cultures of Phaseolus vulgaris displaying a high affinity for Nod factors from Rhizobium tropici (NodRt-V) (Me, S, C18:1), a symbiont of this legume . The binding site shares common properties with NFBS2, a Nod-factor binding site previously characterised in Medicago varia, in terms of affinity, preferential plasma-membrane location, and sensitivity to proteases and lysine reactive reagents . However, the bean site poorly recognizes the Nod factors produced by Sinorhizobium meliloti, the symbiont of Medicago . The study of selectivity toward the Nod factors reveals that the length and degree of unsaturation of the acyl chain and the length of the oligosaccharidic moiety are important determinants of high affinity binding to the bean site; whereas, the N-methyl and O-sulfuryl groups play a minor role . Thus, the common characteristics of P . vulgaris and M . varia Nod-factor binding sites suggest that they probably correspond to structurally related proteins, but their different selectivity suggests that they may be involved in a differential perception system for Nod factors in legumes.

Int Microbiol, 2002 Jun, 5(2), 81 - 6
Survival of several Rhizobium/Bradyrhizobium strains on different inoculant formulations and inoculated seeds; Temprano FJ et al.; The effect of a variety factors on the survival of several rhizobia strains on inoculants and inoculated seeds has been evaluated . Since the rhizobia strains showed different cell-density-evolution patterns on peat-based inoculants and on inoculated seeds, several inoculant formulations with highly effective Rhizobium/Bradyrhizobium strains (for Lupinus, Hedysarum, Phaseolus and Glycine max.) were monitored under the following storage conditions: (a) the inoculants were kept refrigerated (at 4 degrees C), or (b) at room temperature (25 degrees C) . The effect of water content (30-50%, w/w) in the inoculants as well as that of several seed-coating adhesives were also investigated . Alternative carriers including perlite and vermiculite were tested . For all of the strains, survival on sterile peat-based inoculants was higher than on the corresponding unsterile peat formulation; for the latter, refrigerated storage conditions are recommended to ensure high bacterial densities . The water content of the inoculants had a differential effect on strain survival depending on the sterility of the peat, such that a high water content was more detrimental when unsterilized peat was employed . The best adherent for rhizobia survival was a gum arabic/water solution . Perlite was as effective as peat in maintaining a high population of rhizobia, at least for 6 months of storage.

J Environ Qual, 2002 Jul-Aug, 31(4), 1339 - 48
Growth of alfalfa in sludge-amended soils and inoculated with rhizobia produced in sludge; Rebah FB et al.; The efficiency of rhizobial inoculants produced in wastewater sludge used as a growth medium and as a carrier was compared with that of inoculants produced in yeast mannitol broth (YMB) medium and by using peat as a carrier . Alfalfa (Medicago sativa L.) plants were inoculated with solid and liquid Sinorhizobium meliloti inoculants and grown in pots containing two soil types (Kamouraska clay soil and Saint-Andre sandy soil) . The effect of various levels of sludge amendment (60 and 120 kg N/ha) and nitrogen fertilizer (60 kg N/ ha) was also studied . The sludge-based inoculants showed the same symbiotic efficiency (nodulation and plant yield) as YMB-based inoculants . The inoculation increased the nodulation indexes from 4-6 to 8-12, and the rhizobial number from 10(3) (uninoculated soils) to 10(6)-10(7) cells/g in inoculated soils . However, the shoot dry weights and the nitrogen contents were not increased significantly by the inoculation . Applying sludge as an amendment enhanced the rhizobial number in soils from 10(3) to 10(4) cells/g and improved significantly the plant growth (shoot dry weights and nitrogen contents) . This improvement increased with sludge rate and with the cut (three cuts) . Compared with sludge, N fertilizer gave lower plant yields . The nodulation was not affected by sludge and N-fertilizer application . The texture and physico-chemical properties of soil were found to affect the yield and nitrogen content of the plants . In this study, macroelements and heavy metals were at acceptable levels and were not considered to be negative factors.

Plant Mol Biol, 2002 Sep, 50(2), 197 - 211
Transcripts for genes encoding soluble acid invertase and sucrose synthase accumulate in root tip and cortical cells containing mycorrhizal arbuscules; Blee KA et al.; Arbuscule formation by the arbuscular mycorrhizal fungus Glomus intraradices (Schenck & Smith) was limited to cortical cells immediately adjacent to the endodermis . Because these cortical cells are the first to intercept photosynthate exiting the vascular cylinder, transcript levels for sucrose metabolizing-enzymes were compared between mycorrhizal and non-mycorrhizal roots . The probes corresponded to genes encoding a soluble acid invertase with potential vacuolar targeting, which we generated from Phaseolus vulgaris roots, a Rhizobium-responsive sucrose synthase of soybean and a cell wall acid invertase of carrot . Transcripts in non-mycorrhizal roots were developmentally regulated and abundant in the root tips for all three probes but in differentiated roots of P . vulgaris they were predominantly located in phloem tissues for sucrose synthase or the endodermis and phloem for soluble acid invertase . In mycorrhizal roots increased accumulations of transcripts for sucrose synthase and vacuolar invertase were both observed in the same cortical cells bearing arbuscules that fluoresce . There was no effect on the expression of the cell wall invertase gene in fluorescent carrot cells containing arbuscules . Thus, it appears that presence of the fungal hyphae in the fluorescent arbusculated cell stimulates discrete alterations in expression of sucrose metabolizing enzymes to increase the sink potential of the cell.

Sheng Wu Hua Xue Yu Sheng Wu Wu Li Xue Bao (Shanghai), 1998, 30(1), 47 - 52
Analysis of the Exo(-) Mutant of Rhizobium huakuii and Their Exopolysaccharide Component; Su WY et al.; We have subcloned two plasmids, pWS-6BP1 and pWS-6BP2, from the 8.5 kb DNA fragment of plasmid pJB22-B6 . The plasmid of pWS-6BP1, with a 5.8 kb DNA fragment, can complement the exopolysaccharide-deficient mutant NA06 and NA12 of Rhizobium huakuii strain 107, but the plasmid pWS-6BP2, with a 2.6 kb DNA fragment can only complement NA12 . The effective nodules formed by the wild type strain 107 (Rh107) contain lots of bacteriods; the ineffective nodules formed by the mutant NA06 and NA12 contain few bacteroids . Although the nodule formed by Exo(+) transconjugants are ineffective, the bacteria can be successfully released into the cells and form lots of bacteroids . The Rh107 produces acidic exopolysaccharides (EPS) of high molecular weight (HMW) in large amount with only small amount of low molecular weight (LMW) forms . EPS secreted by mutants NA06 and NA12 are just the other way round with LMW in higher amount than HMW ones . They also produce an EPS with molecular weight in between . The EPS secreted by the Exo(+) transconjugants is more like the EPS of Rh107 than that of mutants, although their component and structure are still different from that of Rh107.

Sheng Wu Hua Xue Yu Sheng Wu Wu Li Xue Bao (Shanghai), 1998, 30(4), 319 - 324
Cloning and Sequencing of Rhizobium huakuii exoA Gene Encoding a Glucosyl-transferase; Gong F et al.; From exoR'-11 which could complement two Exo(-) mutants of R . huakuii 107: NA03 and NA10, a 2.0kb BglI fragment was subcloned in pRK415 . The resulted plasmid pJB-H701 could restore the Exo(-) phenotype of NA03 and NA10 . The complete nucleotide sequence of the fragment was determined, which contains the structural genes of a glucosyl-transferase, as well as the 5'- and 3'- flanking regions . An open reading frame of 984 base pairs was identified as R . huakuii exoA gene . The MW of ExoA, as deduced from the nucleotide sequence, was estimated as 35 kD . Comparison of the nucleotide sequences revealed a high similarity between exoA genes of R . huakuii and R . meliloti . The deduced amino acid sequence of R . huakuii ExoA also showed a high similarity with that of R . meliloti ExoA . Furthermore, the exoA-lacZ transcription fusion gene was constructed and the expression of exoA-lacZ was analyzed.

Int J Syst Evol Microbiol, 2002 Jul, 52(Pt 4), 1267 - 76
Rhizobium sullae sp . nov . (formerly 'Rhizobium hedysari'), the root-nodule microsymbiont of Hedysarum coronarium L; Squartini A et al.; This work is the completion of a series of reports describing the nitrogen-fixing bacterial symbionts of sulla (Hedysarum coronarium L., Leguminosae) and providing the grounds for their proposal as a new taxon . The introduction summarizes a large amount of previous evidence gathered on the physiology, genetics and ecology of such organisms, which have in the past been referred to provisionally as 'Rhizobium hedysari' . Upon adding 16S RNA sequencing, amplified rDNA restriction analysis of the rrn operon, DNA-DNA hybridization homology and analysis of low-molecular-mass RNA species, it is concluded that the group of strains that specifically nodulate sulla consists of a coherent set of isolates that differ from previously described rhizobia to an extent that warrants the constitution of the species boundary . The name Rhizobium sullae sp . nov . is proposed, with isolate 1S123T (=USDA 4950T = DSM 14623T) as the type strain.

J Exp Bot, 2002 Aug, 53(375), 1735 - 45
Effect of exogenous flavonoids on nodulation of pea (Pisum sativum L.); Novak K et al.; Selected flavonoids that are known as inducers and a suppressor of nodulation (nod) genes of the symbiotic bacterium Rhizobium leguminosarum bv . viciae were tested for their effect on symbiosis formation with garden pea as the host . A solid substrate was omitted from the hydroponic growing system in order to prevent losses of flavonoids due to adsorption and degradation . The presumed interaction of the tested flavonoids with nod genes has been verified for the genetic background of strain 128C30 . A stimulatory effect of a nod gene inducer naringenin on symbiotic nodule number formed per plant 14 d after inoculation was detected at concentrations of 0.1 and 1 micro g ml(-1) nutrient solution . At 10 micro g ml(-1), the highest concentration tested, naringenin was already inhibitory . By contrast, nodulation was negatively affected by a nod gene suppressor, quercetin, at concentrations above 1 micro g ml(-1), as well as by another tested nod gene inducer, hesperetin . The deleterious effect of hesperetin might be due to its toxicity or to the toxicity of its degradation product(s) as indicated by the inhibition of root growth . Both the stimulatory effect of naringenin and the inhibitory effect of quercetin on nodule number were more pronounced at earlier stages of nodule development as revealed with specific staining of initial nodules . The lessening of the flavonoid impact during nodule development was ascribed to the plant autoregulatory mechanisms . Feedback regulation of nodule metabolism might also be responsible for the fact that the naringenin-conditioned increase in nodule number was not accompanied by any increase in nitrogenase activity . By contrast, the inhibitory action of quercetin and hesperetin on nodule number was associated with decreases in total nitrogenase activity . Naringenin also stimulated root hair curling (RHC) as one of the earliest nodulation responses at concentrations of 1 and 10 microg ml(-1), however, the same effect was exerted by the nod gene suppressor, quercetin, suggesting that feedback regulatory mechanisms control RHC in the range of nodulation-inhibiting high flavonoid concentrations . The comparison of the effect of the tested flavonoids in planta with nod gene activity response showed a two orders of magnitude shift to higher concentrations . This shift is explained by the absorption and degradation of flavonoids by both the symbionts during 3 d intervals between hydroponic solution changes . The losses were 99, 96.4, and 90% of the initial concentration of 10 micro g ml(-1) for naringenin, hesperetin, and quercetin, respectively.

Appl Environ Microbiol, 2002 Aug, 68(8), 4025 - 34
The diversity of Phaseolus-nodulating rhizobial populations is altered by liming of acid soils planted with Phaseolus vulgaris L . in Brazil; Andrade DS et al.; PCR-mediated restriction fragment length polymorphism (RFLP) analysis of the 16S-23S rRNA internally transcribed spacer (ITS) region and the 16S rRNA gene indicated that the rhizobial populations isolated from common bean (Phaseolus vulgaris L.) nodules in the unlimed soil from a series of five lime rates applied 6 years previously to plots of an acidic oxisol had less diversity than those from plots with higher rates of liming . Isolates affiliated with Rhizobium tropici IIB and Rhizobium leguminosarum bv . phaseoli were predominant independent of lime application . An index of richness based on the number of ITS groups increased from 2.2 to 5.7 along the soil liming gradient, and the richness index based on "species" types determined by RFLP analysis of the 16S rRNA gene varied from 0.5 to 1.4 . The Shannon index of diversity, based on the number of ITS groups, increased from 1.8 in unlimed soil to 2.8 in limed soil, and, based on RFLP analysis of the 16S rRNA gene, ranged from 0.9 to 1.4 . In the limed soil, the subpopulation of R . tropici IIB pattern types contained the largest number of ITS groups . In contrast, there were more R . leguminosarum bv . phaseoli types in the unlimed soil with the lowest pH than in soils with the highest pH . The number of ITS ("strain") groups within R . leguminosarum bv . phaseoli did not change with increased abundance of rhizobia in the soil, while with R . tropici IIB, the number of strain groups increased significantly . Some cultural and biochemical characteristics of Phaseolus-nodulating isolates were significantly related to changes in soil properties caused by liming, largely due to changes in the predominance of the rhizobial species groups.

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