Rhizobium strains differ considerably in outer membrane permeability and polymyxin B resistance.

Six rhizobium (Rhizobium leguminosarum bv. Trifolii TA1, Sinorhizobium meliloti 1021, Mesorhizobium huakuii IFO 15243(T), Ochrobactrum lupini LUP 21(T), Bradyrhizobium japonicum USDA110 and B. elkanii USDA 76) and two Escherichia coli strains (E. coli ATCC 25922 and E. coli HB 101) were compared in respect to polymyxin B and EDTA resistance, as well as bacterial outer membrane (OM) permeability to a fluorescent hydrophobic agent (N-phenyl-1-naphthylamine - NPN). TEM (Transmission Electron Microscopy) and a microbial test demonstrated that all the rhizobia were much more resistant to polymyxin B in comparison with E. coli strains. EDTA and polymyxin B enhance permeability of B. japonicum and O. lupini OM. Other rhizobia incorporated NPN independently of the presence of membrane-deteriorating agents; however, the level of fluorescence (measured as NPN absorption) was strain dependent.

Nodulation outer proteins: double-edged swords of symbiotic rhizobia.

Rhizobia are nitrogen-fixing bacteria that establish a nodule symbiosis with legumes. Nodule formation depends on signals and surface determinants produced by both symbiotic partners. Among them, rhizobial Nops (nodulation outer proteins) play a crucial symbiotic role in many strain-host combinations. Nops are defined as proteins secreted via a rhizobial T3SS (type III secretion system). Functional T3SSs have been characterized in many rhizobial strains. Nops have been identified using various genetic, biochemical, proteomic, genomic and experimental approaches. Certain Nops represent extracellular components of the T3SS, which are visible in electron micrographs as bacterial surface appendages called T3 (type III) pili. Other Nops are T3 effector proteins that can be translocated into plant cells. Rhizobial T3 effectors manipulate cellular processes in host cells to suppress plant defence responses against rhizobia and to promote symbiosis-related processes. Accordingly, mutant strains deficient in synthesis or secretion of T3 effectors show reduced symbiotic properties on certain host plants. On the other hand, direct or indirect recognition of T3 effectors by plant cells expressing specific R (resistance) proteins can result in effector triggered defence responses that negatively affect rhizobial infection. Hence Nops are double-edged swords that may promote establishment of symbiosis with one legume (symbiotic factors) and impair symbiotic processes when bacteria are inoculated on another legume species (asymbiotic factors). In the present review, we provide an overview of our current understanding of Nops. We summarize their symbiotic effects, their biochemical properties and their possible modes of action. Finally, we discuss future perspectives in the field of T3 effector research.

Efectividad de cepas rizobianas de frijolbajo diferentes regímenes de fósforo / Effectiveness of cowpea rhizobial strains under different phosphorus regimes

En Venezuela, el frijol representa una alternativa a la proteína animal, debido a su alto consumo y valor nutritivo, por ello se ha estimulado la implementación de programas para reactivar la economía de los pequeños y medianos productores, a fin de incrementar su producción y así tener mayor disponibilidad de proteína de alta calidad a bajo costo; de manera que, los estudios encaminados a mejorar su cultivo, son acertados. Se evaluó la efectividad de cepas rizobianas de crecimiento lento (cl) y rápido (cr) en frijol (Vigna unguiculata (L.) Walp.) cultivar TC9-6 en varios regímenes de fósforo (0, 20, 40 y 80 kgP2O5 ha-1), con un diseño experimental de bloques al azar con arreglo factorial. Las plantas se cultivaron en 4 kg de suelo de sabana 45 días y las cepas en caldo de levadura y manitol: 5 (cr: JV91) y 10 (cl: JV94) días. La inoculación (2 ml cada vez) fue aplicada a la siembra y 6 días más tarde. La utilización de fósforo (40-80 kgP2O5 ha-1) incrementó la nodulación (número, peso seco total e individual de nódulos) y favoreció la aparición de nódulos rojos; así mismo, acrecentó el peso de la materia seca, la altura, el número de hojas y la concentración de nitrógeno del vástago. Los valores fueron similares con ambos tipos de cepas (efectividad similar) y para las dos concentraciones (40-80 kgP2O5 ha-1), con las menores estimaciones para 0 y 20 kgP2O5 ha-1. De acuerdo con los resultados las concentraciones de 40 y 80 kgP2O5 ha-1 fueron las más favorables para el crecimiento y la nodulación de frijol.

In Venezuela, cowpea is an alternative to animal protein due to its high consumption and nutritious value, so it has stimulated the implementation of programs to reactivate the small and medium producers economy, in order to increase its production and to have major high quality protein availability at low cost; so that, the studies carry on to improve its cultivation, are well-aimed. The effectiveness of slow (sg) and fast (fg) growing rhizobial strains was evaluated in cowpea (Vigna unguiculata (L.) Walp) cultivar TC9-6 at various phosphorus regimes (0, 20, 40 and 80 kgP2O5 ha-1): randomized block design with factorial arrangement. Plants were cultivated in 4 kg savannah soil: 45 days, and the strains in yeast and mannitol broth: 5 (fg: JV91) and 10 (sg: JV94) days. The inoculation (2 ml each time) was applied at sowing time and 6 days later. Phosphorus utilization (40-80 kgP2O5 ha-1) increased nodulation (nodule number, total and individual dry weight) and favoured nodule red colour appearance; also, incremented shoot dry matter weight, height, leaves number and nitrogen concentration. Values were similar with both strain types (similar effectiveness) and to the two doses (40-80 kgP2O5 ha-1), with lower estimations to 0 and 20 kgP2O5 ha-1. Accordingly with the results, the doses of 40 and 80 kgP2O5 ha-1 were the most favourable to cowpea growth and nodulation.

Lotus japonicus symRK-14 uncouples the cortical and epidermal symbiotic program.

SYMRK is a leucine-rich-repeat (LRR)-receptor kinase that mediates intracellular symbioses of legumes with rhizobia and arbuscular mycorrhizal fungi. It participates in signalling events that lead to epidermal calcium spiking, an early cellular response that is typically considered as central for intracellular accommodation and nodule organogenesis. Here, we describe the Lotus japonicus symRK-14 mutation that alters a conserved GDPC amino-acid sequence in the SYMRK extracellular domain. Normal infection of the epidermis by fungal or bacterial symbionts was aborted in symRK-14. Likewise, epidermal responses of symRK-14 to bacterial signalling, including calcium spiking, NIN gene expression and infection thread formation, were significantly reduced. In contrast, no major negative effects on the formation of nodule primordia and cortical infection were detected. Cumulatively, our data show that the symRK-14 mutation uncouples the epidermal and cortical symbiotic program, while indicating that the SYMRK extracellular domain participates in transduction of non-equivalent signalling events. The GDPC sequence was found to be highly conserved in LRR-receptor kinases in legumes and non-legumes, including the evolutionarily distant bryophytes. Conservation of the GDPC sequence in nearly one-fourth of LRR-receptor-like kinases in the genome of Arabidopsis thaliana suggests, however, that this sequence might also play an important non-symbiotic function in this plant.

Role of symbiotic auxotrophy in the Rhizobium-legume symbioses.

BACKGROUND: Rhizobium leguminosarum bv. viciae mutants unable to transport branched-chain amino acids via the two main amino acid ABC transport complexes AapJQMP and BraDEFGC produce a nitrogen starvation phenotype when inoculated on pea (Pisum sativum) plants [1], [2]. Bacteroids in indeterminate pea nodules have reduced abundance and a lower chromosome number. They reduce transcription of pathways for branched-chain amino acid biosynthesis and become dependent on their provision by the host. This has been called "symbiotic auxotrophy". METHODOLOGY/PRINCIPAL FINDINGS: A region important in solute specificity was identified in AapQ and changing P144D in this region reduced branched-chain amino acid transport to a very low rate. Strains carrying P144D were still fully effective for N(2) fixation on peas demonstrating that a low rate of branched amino acid transport in R. leguminosarum bv. viciae supports wild-type rates of nitrogen fixation. The importance of branched-chain amino acid transport was then examined in other legume-Rhizobium symbioses. An aap bra mutant of R. leguminosarum bv. phaseoli also showed nitrogen starvation symptoms when inoculated on French bean (Phaseolus vulgaris), a plant producing determinate nodules. The phenotype is different from that observed on pea and is accompanied by reduced nodule numbers and nitrogen fixation per nodule. However, an aap bra double mutant of Sinorhizobium meliloti 2011 showed no phenotype on alfalfa (Medicago sativa). CONCLUSIONS/SIGNIFICANCE: Symbiotic auxotrophy occurs in both determinate pea and indeterminate bean nodules demonstrating its importance for bacteroid formation and nodule function in legumes with different developmental programmes. However, only small quantities of branched chain amino acids are needed and symbiotic auxotrophy did not occur in the Sinorhizobium meliloti-alfalfa symbiosis under the conditions measured. The contrasting symbiotic phenotypes of aap bra mutants inoculated on different legumes probably reflects altered timing of amino acid availability, development of symbiotic auxotrophy and nodule developmental programmes.

Nodulation of Sesbania species by Rhizobium (Agrobacterium) strain IRBG74 and other rhizobia.

Concatenated sequence analysis with 16S rRNA, rpoB and fusA genes identified a bacterial strain (IRBG74) isolated from root nodules of the aquatic legume Sesbania cannabina as a close relative of the plant pathogen Rhizobium radiobacter (syn. Agrobacterium tumefaciens). However, DNA:DNA hybridization with R. radiobacter, R. rubi, R. vitis and R. huautlense gave only 44%, 5%, 8% and 8% similarity respectively, suggesting that IRBG74 is potentially a new species. Additionally, it contained no vir genes and lacked tumour-forming ability, but harboured a sym-plasmid containing nifH and nodA genes similar to those in other Sesbania symbionts. Indeed, IRBG74 effectively nodulated S. cannabina and seven other Sesbania spp. that nodulate with Ensifer (Sinorhizobium)/Rhizobium strains with similar nodA genes to IRBG74, but not species that nodulate with Azorhizobium or Mesorhizobium. Light and electron microscopy revealed that IRBG74 infected Sesbania spp. via lateral root junctions under flooded conditions, but via root hairs under non-flooded conditions. Thus, IRBG74 is the first confirmed legume-nodulating symbiont from the Rhizobium (Agrobacterium) clade. Cross-inoculation studies with various Sesbania symbionts showed that S. cannabina could form fully effective symbioses with strains in the genera Rhizobium and Ensifer, only ineffective ones with Azorhizobium strains, and either partially effective (Mesorhizobium huakii) or ineffective (Mesorhizobium plurifarium) symbioses with Mesorhizobium. These data are discussed in terms of the molecular phylogeny of Sesbania and its symbionts.

Scanning electron microscopic observations on micro-organisms in the root nodules of Tribulus terrestris L. (Zygophyllaceae).

Scanning electron microscopic observations were made on the micro-organisms of root nodules of Tribulus terrestris L. The results showed that nodules of T. terrestris contained dual infection consisting of Rhizobium sp. and Newmania karachiensis. Based on these observations, T. terrestris should be grouped with nonlegume Parasponia-type bacterial nodules.

Ascending migration of endophytic rhizobia, from roots to leaves, inside rice plants and assessment of benefits to rice growth physiology.

Rhizobia, the root-nodule endosymbionts of leguminous plants, also form natural endophytic associations with roots of important cereal plants. Despite its widespread occurrence, much remains unknown about colonization of cereals by rhizobia. We examined the infection, dissemination, and colonization of healthy rice plant tissues by four species of gfp-tagged rhizobia and their influence on the growth physiology of rice. The results indicated a dynamic infection process beginning with surface colonization of the rhizoplane (especially at lateral root emergence), followed by endophytic colonization within roots, and then ascending endophytic migration into the stem base, leaf sheath, and leaves where they developed high populations. In situ CMEIAS image analysis indicated local endophytic population densities reaching as high as 9 x 10(10) rhizobia per cm3 of infected host tissues, whereas plating experiments indicated rapid, transient or persistent growth depending on the rhizobial strain and rice tissue examined. Rice plants inoculated with certain test strains of gfp-tagged rhizobia produced significantly higher root and shoot biomass; increased their photosynthetic rate, stomatal conductance, transpiration velocity, water utilization efficiency, and flag leaf area (considered to possess the highest photosynthetic activity); and accumulated higher levels of indoleacetic acid and gibberellin growth-regulating phytohormones. Considered collectively, the results indicate that this endophytic plant-bacterium association is far more inclusive, invasive, and dynamic than previously thought, including dissemination in both below-ground and above-ground tissues and enhancement of growth physiology by several rhizobial species, therefore heightening its interest and potential value as a biofertilizer strategy for sustainable agriculture to produce the world's most important cereal crops.

NopA is associated with cell surface appendages produced by the type III secretion system of Rhizobium sp. strain NGR234.

Rhizobium sp. strain NGR234, which is capable of interacting with a large number of legumes, utilizes a variety of signaling molecules to establish nitrogen-fixing symbioses. Among these are nodulation outer proteins (Nops) that transit through a type III secretion system (TTSS). Abolition of Nop secretion affects nodulation of certain legumes. Under free-living conditions, the secretion of Nops can be induced by the addition of flavonoids. Here, we show that an in-frame deletion of nopA abolishes secretion of all other Nops and has the same impact on nodule formation as mutations that lead to a nonfunctional TTSS. This secretion-minus phenotype of the nopA mutant, as well as bioinformatics analysis of NopA itself, suggests that NopA could be an external component of the TTSS. Electron microscopy showed that NGR234 synthesizes fibrillar structures on the cell surface in a flavonoid-inducible and NopA-dependent manner. Purification of the macromolecular surface appendages revealed that NopA is a major component of these structures.

NopB, a type III secreted protein of Rhizobium sp. strain NGR234, is associated with pilus-like surface appendages.

Rhizobium sp. strain NGR234 possesses a functional type three secretion system (TTSS), through which a number of proteins, called nodulation outer proteins (Nops), are delivered to the outside of the cell. A major constraint to the identification of Nops is their low abundance in the supernatants of NGR234 strains grown in culture. To overcome this limitation, a more sensitive proteomics-based strategy was developed. Secreted proteins from wild-type NGR234 were separated by two-dimensional gel electrophoresis, and the gel was compared to similar gels containing the proteins from a TTSS mutant (NGROmegarhcN). To identify the proteins, spots unique to the NGR234 gels were analyzed by matrix-assisted laser desorption ionization-time of flight mass spectrometry and the data were compared to the sequence of the symbiotic plasmid of NGR234. A nonpolar mutant of one of these proteins was generated called NopB. NopB is required for Nop secretion but inhibits the interaction with Pachyrhizus tuberosus and augments nodulation of Tephrosia vogelii. Flavonoids and a functional TTSS are required for the formation of some surface appendages on NGR234. In situ immunogold labeling and isolation of these pili showed that they contain NopB.

[Changes and distribution of ATPase activity in the paranodules of Nicotiana tabacum].

Lead phosphate deposition technique was used to investigate the changes and characteristic distribution of ATPase activity in the paranodules of tabacoo. We found that ATPase activity was closely associated with cell type and cell developmental state, thus it was different in various cells and rhizobia. No lead phosphate granules were located in the menstematic cells, although there were a small number of lead phosphate granules in the cytoplasm and organelles of the cells without rhizobia, they did not exist in the young and mature rhizobia. On the contrary, upon the senscence of the cells and rhizobia, a large number of lead phosphate granules appeared on the plasmalemmas and in the cell walls and the inside surfaces of the rhizobia. When the cells and rhizobia progressively senesced, lead phosphate granules increased in number, and they were widely distributed on the tonoplasts, plasmalemmas, cell walls, intercellular layers and in the intercellular spaces. At the same time, they also appeared on the surfaces and in the cytoplasm and nucleoids of the rhizobia. Due to cell disintegration, lead phosphategranules obviously decreased in number, they were only located on the plasmalemmas and membrane-vesicular structures which came from disintegrated organelles.

Screening, identification and kinetic characterization of a bacterium for Mn(II) uptake and oxidation.

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.

Effect of cadmium on nodulation and N2-fixation of soybean in contaminated soils.

The effects of cadmium stress on nodulation, N2-fixation capabilities of the root nodule, the change in ultrastructure of the root nodule, soybean growth, and the distribution of cadmium in plants were studied. The results obtained show that the nodulation of soybean roots was greatly inhibited by the addition of Cd, especially at the addition level of 10 and 20 mg kg(-1) soil. The inhibition of plant growth, especially the root growth, increased as the cadmium concentration increased, with deleterious effects observed for the roots. The weight ratio of soybean root/leaf decreased as the Cd concentration increased, which might explain the reason for nodulation decreases. The results also indicate that N2-fixation of root nodule was stimulated to some extent at the low levels of Cd addition, but decreased sharply with further increase of the Cd concentration. High Cd levels were also associated with changes in the ultrastructure of root nodule, in which the effective N2-fixing area was reduced and the N2-fixing cells in the area also reduced. In addition, the results also reveal that the content of Cd in different parts of the plants was as follows: roots >> stems > seeds, indicating that the accumulation of Cd by roots is much larger than that by any other part of the soybean plant, and might cause deleterious effects to root systems.

Auxotrophic mutant strains of Rhizobium etli reveal new nodule development phenotypes.

We report here the isolation and characterization of amino acid-requiring mutant strains of Rhizobium etli. We observe that the phenotype of most mutations, even when causing a strict auxotrophy, is overcome by cross-feeding from the host plant Phaseolus vulgaris, thereby allowing bacterial production of Nod factors and, consequently, nodule induction. Conversely, light and electron microscopy analysis reveals that the nodules induced by all mutants, including those with normal external morphology, are halted or strongly altered at intermediate or late stages of development. Moreover, some mutants induce nodules that display novel symbiotic phenotypes, such as specific alterations of the invaded cells or the presence of a reduced number of abnormally shaped uninvaded cells. Other mutants induce nodules showing an early and vast necrosis of the central tissue, a phenotype not previously observed in bean nodules, not even in nodules induced by a Fix- mutant. These observations indicate that amino acid auxotrophs represent a powerful tool to study the development of globose determinate-type nodules and emphasize the importance of establishing their histology and cytology before considerations of metabolic exchange are made.

Morphological changes of rhizobia in peat cultures.

Morphological changes that take place in peat cultures of several species of rhizobia were examined. These changes seemed to be associated with enhanced survival of cells in peat and after inoculation onto plastic beads, which were used as a model system for seeds. Cell wall changes, in which the periplasmic space appeared to be occluded with electron-dense material, were observed in Rhizobium sp. strain SU343 and Bradyrhizobium lupini WU425 cells after 7 and 14 days in peat, respectively. Nutrient limitation and low O(2) concentration in peat are suggested to be factors involved in the induction of the morphological changes. Polyhydroxybutyrate reserves, which were present in broth-cultured cells of both species of rhizobia, were mobilized after transfer into peat but did not appear to influence survival after inoculation onto beads. Enhanced expression of an iron-manganese superoxide dismutase was also observed after the cells were transferred into peat. We conclude that cell wall thickening in rhizobia after transfer from broth cultures into peat is an adaptive response for long-term survival under nutrient-limited conditions in peat. Cells with thickened walls may also be more resistant to other types of stress, such as that encountered on a seed surface.

Role of programmed cell death in development.

Programmed cell death (PCD) is an integral part of both animal and plant development. In animals, model systems such as Caenorhabditis elegans, Drosophila melanogaster, and mice have shown a general cell death profile of induction, caspase mediation, cell death, and phagocytosis. Tremendous strides have been made in cell death research in animals in the past decade. The ordering of the C. elegans genes Ced-3, 4 and 9, identification of caspase-activated DNase that degrades nuclear DNA during PCD, identification of signal transduction modules involving caspases as well as the caspase-independent pathway, and the involvement of mitochondria are some of the findings of immense value in understanding animal PCDs. Similarly, the caspase inactivation mechanisms of infecting viruses to stall host cell death give a new dimension to the viral infection process. However, plant cell death profiles provide an entirely different scenario. The presence of a cell wall that cannot be phagocytosed, absence of the hallmarks of animal PCDs such as DNA laddering, formation of apoptotic bodies, a cell-death-specific nuclease, a biochemical machinery of killer enzymes such as caspases all point to novel ways of cell elimination. Large gaps in our understanding of plant cell death have prompted speculative inferences and comparisons with animal cell death mechanisms. This paper deals with both animals and plants for a holistic view on cell death in eukaryotes.

Microbiological and electron microscopic studies of urea treated Rhizobium sp. cells.

Microbiological investigation of urea treated Rhizobium sp. cells showed a gradual decrease of colony forming unit from initial 100% to 2.13% value. Maximum effect was reached at 90 min onwards. The liquid holding recovery in phosphate buffer saline at pH 7.0 also was studied. Electron microscopic studies revealed important structural changes in treated cells.

Localization of VirB1 protein on the agrobacterial cell surface.

Using colloidal gold-labelled VirB1-specific antibodies, it was found that VirB1 proteins are included into the composition of short pilus-like structures, which emerge at the poles of acetosyringone (AS)-induced agrobacterial cells.

Localization of the protein VirB1 involved in contact formation during conjugation among Agrobacterium cells.

Supramembrane structures of Agrobacterium, which link cells during mating, were for the first time visualized using transmission electron microscopy. The initial cell contact was found to be mediated by long pili. Using colloidal gold-labeled, VirB1-specific antibodies, it was established that VirB1 proteins enter into the composition of short pilus-like structures, which emerge at the poles of acetosyringone (AS)-induced agrobacterial cells. Labeling of non-centrifuged agrobacterial cells on a nitrocellulose membrane using colloidal gold-conjugated antibodies to VirB1 showed that the labeled complex could bind to AS-induced cells, but failed to form red stains during incubation with cells of the Ti plasmidless A. tumefaciens strains LBA288 and UBAPF-2.