What Is Rhizobia?

Rhizobia (from the Greek words Riza = Root and Bios = Life) are soil bacteria that fix nitrogen (diazotrophy) after becoming established inside root nodules of legumes (Fabaceae). Although much of the nitrogen is removed when protein-rich grain or hay is harvested, significant amounts can remain in the soil for future crops. This is especially important when nitrogen fertilizer is not used, as in organic rotation schemes or some less-industrialized countries.

There are several different genera of rhizobia. All of them belong to the Rhizobiales, a probably-monophyletic group of proteobacteria.

The plant pathogen Agrobacterium is a closer relative of Rhizobium than the rhizobia that nodulate soybean (and may not really be a separate genus). The genes responsible for the symbiosis with plants, however, may be closer than the organisms themselves, acquired by horizontal transfer rather than from a common ancestor.

The legume-rhizobium symbiosis is a classic example of mutualism, but its evolutionary persistence is actually somewhat surprising. Because several unrelated strains infect each individual plant, any one strain could redirect resources from nitrogen fixation to its own reproduction without killing the host plant upon which they all depend. But this form of cheating should be equally tempting for all strains, a classic tragedy of the commons. It turns out that legume plants guide the evolution of rhizobia towards greater mutualism by reducing the oxygen supply to nodules that fix less nitrogen, thereby reducing the frequency of cheaters in the next generation.

Frankia and Azospirillum, are genera of similar bacteria that do not live on legumes.

Soil microbiota communities have demonstrated their crucial role in maintaining the soil ecological balance and therefore the sustainability of either natural ecosystems or agroecosystems. Rhizospheric microbe-plant interactions have a great influence on plant health and soil quality since these root-associated microorganisms are able to help the host plant to deal with drought, nutritional and soil-borne pathogen stress conditions. Plant growth-promoting rhizobacteria (PGPR) can be considered among rhizosphere-beneficial microorganisms. In a micropropagated plant system, bacterial inoculation at the beginning of the acclimatisation phase must also be observed from the perspective of the establishment of the soil microbiota rhizosphere.

Crit Rev Microbiol, 2004, 30(4), 205 - 40
Rhizosphere bacterial signalling: a love parade beneath our feet; Somers E et al.; Plant roots support the growth and activities of a wide variety of microorganisms that may have a profound effect on the growth and/or health of plants . Among these microorganisms, a high diversity of bacteria have been identified and categorized as deleterious, beneficial, or neutral with respect to the plant . The beneficial bacteria, termed plant growth-promoting rhizobacteria (PGPR), are widely studied by microbiologists and agronomists because of their potential in plant production . Azospirillum, a genus of versatile PGPR, is able to enhance the plant growth and yield of a wide range of economically important crops in different soils and climatic regions . Plant beneficial effects of Azospirillum have mainly been attributed to the production of phytohormones, nitrate reduction, and nitrogen fixation, which have been subject of extensive research throughout the years . These elaborate studies made Azospirillum one of the best-characterized genera of PGPR . However, the genetic and molecular determinants involved in the initial interaction between Azospirillum and plant roots are not yet fully understood . This review will mainly highlight the current knowledge on Azospirillum plant root interactions, in the context of preceding and ongoing research on the association between plants and plant growth-promoting rhizobacteria.

Can J Microbiol, 2004 Nov, 50(11), 977 - 83
Selection of rhizobia for prairie legumes used in restoration and reconstruction programs in Minnesota; Tlusty B et al.; The inoculation of indigenous legumes used in revegetation and wetland reconstruction activities in Minnesota presents problems not found in more traditional seedings . Few studies have sought inoculant-quality rhizobia for the legumes involved or examined their interaction in an environment that involves a number of different legumes seeded simultaneously under initially adverse climatic conditions . In this note, we identify effective strains for Astragalus canadensis, Chamaecrista fasciculata, Dalea purpurea, Dalea candida, Desmodium canadense, and Lespedeza capitata . We are currently using these strains to examine the contribution of inoculated legumes to prairie productivity in Minnesota.

J Am Coll Nutr, 2004 Dec, 23(6), 751S - 3S
Magnesium-content of Rhizobium nodules in different plants: the importance of magnesium in nitrogen-fixation of nodules; Kiss SA et al.; OBJECTIVE: Rhizobium bacteria induce nodules (tumors) in roots of leguminous crops that fix nitrogen (N2) from the atmosphere . METHODS: Trials were carried on in sterile perlite where two species of peas were grown hydroponically, and in two different soil types (brown forest soil and meadow silt soil), in a field where different leguminous plants were chosen for test plants: lupin, soybean, broad-bean, lentil and bean inoculated by Rhizobium bacteria . The Mg-content of the nutrient solution was higher than that of the control, but in the soil tests 1% Mg leaf fertilization was applied after bacterial inoculation . Number, weight and Mg-content of nodules were evaluated using an AAS method compared with the thick and thin hair/roots of the given plant . Results and CONCLUSIONS: Our results clearly show that the Mg nutrition treatment increased the number of Rhizobium nodules and their Mg-content, resulting in increased N(2)-fixation and yield.

Res Microbiol, 2005 Jan-Feb, 156(1), 57 - 67
Diversity in a promiscuous group of rhizobia from three Sesbania spp . colonizing ecologically distinct habitats of the semi-arid Delhi region; Sharma RS et al.; Sesbania-rhizobia associations have immense significance in soil amelioration programs for diverse habitats . Diversity in symbiotic properties, LPS profiles, Sym plasmid and rhizobiophage sensitivity of 28 root- and stem-nodulating bacterial isolates of three Sesbania species (S . sesban, S . aegyptica and S . rostrata) inhabiting six ecologically distinct sites of semi-arid Delhi region was analyzed . The isolates were highly promiscuous among the symbiotic partners (Sesbania spp.) . The root nodules formed by all the isolates were morphologically similar but they differed in their symbiotic efficiency and effectiveness . 16S rDNA sequence analyses revealed that root nodule isolates of sesbanias belong to diverse rhizobial taxa (Sinorhizobium saheli, S . meliloti, Rhizobium huautlense) whereas stem-nodule isolates were strictly Azorhizobium caulinodans . Sinorhizobium spp . seem to dominate as microsymbiont partner of Sesbania in the Delhi region . The genetic diversity revealed by cluster analyses based on NPC-PCR reflects sorting of isolates across the ecological gradient . Parallel diversity was also observed in the grouping based on LPS profiles and sym plasmid (NPC-PCR) . Segregation of different rhizobial taxa into distinct types/clusters based on LPS and NPC-PCR analyses suggest its significance in the circumscription of the taxa . However, subtypes and subclusters showed their sorting across the ecological gradients . Sesbania rhizobia showed extremely high specificity to rhizobiophages . Enormous diversity in LPS profiles and high specificity of rhizobiophages might be the result of environmental selection pressures operating in ecologically distinct habitats . The ability of sesbanias to enter into effective symbioses with different rhizobial taxa and colonize diverse habitats with various biotic and abiotic stresses appears to contribute to its wide ecological amplitude.

Ying Yong Sheng Tai Xue Bao, 2004 Oct, 15(10), 1963 - 6
{Research advances in plant growth-promoting rhizobacteria and its application prospects}; Hu J et al.; The study of plant growth-promoting rhizobacteria will provide possible methods to overcome the difficulties in controlling soil borne plant diseases . PGPR can colonize in rhizosphere at high population density, inhibit plant pathogens and deleterious microorganisms there, and promote crop growth and its yield . More importantly, some PGPR strains applied as biocontrol agents can make treated plant produce induced systemic resistance (ISR), and thus, increases plant overall health . In recent two decades, the researches of PGPR in abroad are very active, and many PGPR products have been applied successfully . In our contry, more attention should be paid to the study of the basic theory and application of GPPR and the industrialization of PGPR products.

Indian J Exp Biol, 2004 Dec, 42(12), 1186 - 94
Molecular and functional characteristics, growth promoting effect and persistence of selected parent isolates and streptomycin resistant derivatives of rice rhizobacteria; Boro RC et al.; Molecular and functional characteristics of seven azospirilla and five phosphorus solubilizing bacteria (PSB) isolates of rice rhizosphere, growth promotion ability of two efficient strains, Azospirillum amazonense A10 (MTCC4716) and Bacillus megaterium P5 (MTCC4714) and their persistence based on streptomycin resistant derivatives (SRD), were determined . SDS-PAGE and isozyme banding patterns of the isolates were used to arbitrarily group the azospirilla into 4 and PSB into 3 clusters and as markers to ascertain their identity . The azospirilla produced 2.0 to 10.5 ppm of IAA like substances and showed nitrogenase activity of 0.02 to 3.55 nmole C2H4/hr/ml of pure culture . PSB isolates produced 7.8 to 15.0 ppm IAA like substances and 20 to 128 ppm soluble P . Induction of resistance to streptomycin resulted in changes of these properties . Co-inoculation of rice with SRD A10 and SRD P5 and their parental strains in separate treatments enhanced grain yield over control by 31 and 12.4%, respectively . Nitrogenase activity of rice roots under SRD co-inoculated treatment was higher (4.16 nmole C2H4/hr/hill) than that-under parental strains co-inoculated treatment (3.76 nmole C2H4/hr/hill) . SDS-PAGE profile and population count of the strains confirmed their establishment in rice rhizosphere and persistence over a year after inoculation.

Indian J Exp Biol, 2004 Dec, 42(12), 1177 - 85
Azide resistance in Rhizobium ciceri linked with superior symbiotic nitrogen fixation; Bhaskar VV; Isolated azide resistant (AzR) native R . ciceri strain 18-7 was resistant to sodium azide at 10 microg/ml . To find if nif-reiteration is responsible for azide resistance and linked to superior symbiotic nitrogen fixation, transposon (Tn5) induced azide sensitive mutants were generated . Using 4 kb nif-reiterated Sinorhizobium meliloti DNA, a clone C4 that complemented azide sensitivity was isolated by DNA hybridization from genomic library of chickpea Rhizobium strain Rcd301 . EcoRI restriction mapping revealed the presence of 7 recognition sites with a total insert size of 19.17 kb . Restriction analysis of C4 clone and nif-reiterated DNA (pRK 290.7) with EcoRI and XhoI revealed similar banding pattern . Wild type strain 18-7, mutant M126 and complemented mutant M126(C4) were characterized for symbiotic properties (viz., acetylene reduction assay, total nitrogen content, nodule number and fresh and dry weight of the infected plants) and explanta nitrogenase activity . Our results suggested that azide resistance, nif-reiteration, and superior symbiotic effectiveness were interlinked with no correlation between ex-planta nitrogenase activity and azide resistance in R . ciceri.

Mycorrhiza . 2004 Dec 23; {Epub ahead of print}
Ectomycorrhizal symbiosis enhanced the efficiency of inoculation with two Bradyrhizobium strains and Acacia holosericea growth; Andre S et al.; Two strains of Bradyrhizobium sp., Aust 13C and Aust 11C, were dually or singly inoculated with an ectomycorrhizal fungus, Pisolithus albus to assess the interactions between ectomycorrhizal symbiosis and the nodulation process in glasshouse conditions . Sequencing of strains Aust 13C and Aust 11C confirmed their previous placement in the genus Bradyrhizobium . After 4 months' culture, the ectomycorrhizal symbiosis promoted plant growth and the nodulation process of both Bradyrhizobium strains, singly or dually inoculated . PCR/RFLP analysis of the nodules randomly collected in each treatment with Aust 13C and/or Aust 11C: (1) showed that all the nodules exhibited the same patterns as those of the Bradyrhizobium strains, and (2) did not detect contaminant rhizobia . When both Bradyrhizobium isolates were inoculated together, but without P . albus IR100, Aust 11C was recorded in 13% of the treated nodules compared to 87% for Aust 13C, whereas Aust 11C and Aust 13C were represented in 20 and 80% of the treated nodules, respectively, in the ectomycorrhizal treatment . Therefore Aust 13C had a high competitive ability and a great persistence in soil . The presence of the fungus did not significantly influence the frequencies of each Bradyrhizobium sp . root nodules . Although the mechanisms remain unknown, these results showed that the ectomycorrhizal and biological nitrogen-fixing symbioses were very dependent on each other . From a practical point of view, the role of ectomycorrhizal symbiosis is of great importance to N(2) fixation and, consequently, these kinds of symbiosis must be associated in any controlled inoculation.

Syst Appl Microbiol, 2004 Nov, 27(6), 737 - 45
Phenotypic and genotypic characterization of rhizobia from diverse geographical origin that nodulate Pachyrhizus species; Rodriguez-Navarro DN et al.; Legumes from the genus Pachyrhizus, commonly known as yam bean, are cultivated in several countries from the American continent and constitute an alternative source for sustainable starch, oil and protein production . The endosymbionts of these legumes have been poorly studied although it is known that this legume is nodulated by fast and slow growing rhizobia . In this study we have analyzed a collection of strains isolated in several countries using different phenotypic and molecular methods . The results obtained by SDS-PAGE analysis, LPS profiling and TP-RAPD fingerprinting showed the high diversity of the strains analyzed, although all of them presented slow growth in yeast mannitol agar (YMA) medium . These results were confirmed using 16S-23S internal transcribed spacer (ITS) region and complete sequencing of the 16S rRNA gene, showing that most strains analyzed belong to different species of genus Bradyrhizobium . Three strains were closely related to B . elkanii and the rest of the strains were related to the phylogenetic group constituted by B . japonicum, B . liaoningense, B . yuanmingense and B . betae . These results support that the study of rhizobia nodulating unexplored legumes in different geographical locations will allow the discovery of new species able to establish legume symbioses.

Syst Appl Microbiol, 2004 Nov, 27(6), 707 - 15
Phenotypic and genomic characteristics of rhizobia isolated from Genista tinctoria root nodules; Kalita M et al.; Forty three rhizobial strains isolated from root nodules of Genista tinctoria growing in England, Ukraine, and Poland were compared with 21 representatives of the recognized rhizobial species and two unclassified Bradyrhizobium sp . (Lupinus) strains by performing a numerical analysis of 102 phenotypic features and with the reference bradyrhizobia by simplified AFLP analysis with one restriction enzyme PstI and one selective primer PstI-A . All Genista tinctoria microsymbionts were slow-growing bradyrhizobia with generation time of 10-14 h, acid tolerant, salt sensitive, and antibiotic resistant . Cluster analysis based on the phenotypic properties of all bacteria included, grouped dyer's broom rhizobia together with Bradyrhizobium strains, and classified them into three major phena according to their geographic origin . Genista tinctoria nodule isolates were separated into three clusters with the strain composition as in a phenogrouping by AFLP patterns . The presented results, suggest the relationship of G . tincoria microsymbionts to Bradyrhizobium species and show the usefulness of AFLP analysis for differentiation and classification of the studied rhizobia.

Syst Appl Microbiol, 2004 Nov, 27(6), 703 - 6
Phylogenetic relationships of rhizobia based on citrate synthase gene sequences; Hernandez-Lucas I et al.; Partial nucleotide sequences of the citrate synthase (gltA) gene from different rhizobia genera were determined . Tree topologies based on this housekeeping gene were similar to that obtained using 16S rRNA sequences . However gltA appeared to be more reliable at determining phylogenetic relationships of closely related taxa . We propose gltA sequences as an additional tool to be used in molecular phylogenetic studies.

J Appl Microbiol, 2005, 98(1), 145 - 54
Characterization of antifungal metabolite produced by a new strain Pseudomonas aeruginosa PUPa3 that exhibits broad-spectrum antifungal activity and biofertilizing traits; Kumar RS et al.; Abstract r . sunish kumar, n . ayyadurai, p . pandiaraja, a.v . reddy, y . venkateswarlu, o . prakash and n . sakthivel . 2004.Aim: To study the antifungal activity and plant beneficial traits of a broad-spectrum antagonistic fluorescent pseudomonad strain, PUPa3 . Methods and Results: Strain PUPa3 was isolated from the rhizosphere soil of rice and identified as Pseudomonas aeruginosa on the basis of biochemical tests and by comparison of 16S rDNA sequences . This bacterium exhibits a broad-spectrum antifungal activity towards phytopathogenic fungi . The antifungal metabolite by PUPa3 was extracted, purified and characterized using nuclear magnetic resonance (NMR) and mass spectroscopy (MS) . Production of indole-3-acetic acid (IAA), siderophores, phosphatase and protease in PUPa3 was determined . Strain PUPa3 did not produce hydrogen cyanide, cellulase and pectinase . Conclusion: The antifungal metabolite produced by PUPa3 has been identified as phenazine-1-carboxamide (PCN) on the basis of NMR and MS data . Strain PUPa3 showed a broad-spectrum antifungal activity towards a range of phytopathogenic fungi . This bacterium also showed several plant growth-promoting traits but did not show the traits attributed to deleterious rhizobacteria . Significance and Impact of the Study: Present study reports the production of PCN as well as IAA for the first time by a saprophytic P . aeruginosa strain PUPa3 . Because of the production of siderophore, growth hormone, protease and phosphatase and its innate fungicidal potential, this strain can be used as biofertilizer and antagonist against a range of phytopathogenic fungi that infect rice, groundnut, tobacco, chili, mango, sugarcane, tea, cotton and banana.

Planta, 2004 Oct, 219(6), 993 - 1002 Epub 2004 Jun 16.
Nodulin 22 from Phaseolus vulgaris protects Escherichia coli cells from oxidative stress; Mohammad A et al.; Plant genes that are induced during the formation and function of a root nodule are called nodulin genes . Cloning and functional analysis of nodule-specific gene products are of valuable help in establishing the role and requirements of the host plant for the specificity and effectiveness of the symbiosis . A cDNA clone (nod22) was isolated from Phaseolus vulgaris L . (common bean) cDNA library derived from Rhizobium-infected roots . Nodulin 22 (Nod22) transcripts are accumulated from early to late stages in root nodule development . RT-PCR in situ studies indicated that Nod22 transcripts are highly accumulated in cortical, vascular bundle and infected cells . The deduced Nod22 protein contains a highly hydrophobic N-terminus, with signal peptide characteristics, and a C-terminal extension with high identity to the alpha-crystallin domains found in alpha-crystallin lens chaperone, and other small heat-shock proteins . These domains have not been previously described in other known nodulins, but have been observed in small heat-shock proteins found in plant tissues exposed to elevated temperature and oxidative stress . Nod22, when it is over-expressed in Escherichia coli, cells confers protection against oxidative stress suggesting its possible role in plant host protection from oxidative toxicity during the Rhizobium-legume symbiosis.

J Bacteriol, 2005 Jan, 187(1), 168 - 74
Glutathione plays a fundamental role in growth and symbiotic capacity of Sinorhizobium meliloti; Harrison J et al.; Rhizobia form a symbiotic relationship with plants of the legume family to produce nitrogen-fixing root nodules under nitrogen-limiting conditions . We have examined the importance of glutathione (GSH) during free-living growth and symbiosis of Sinorhizobium meliloti . An S . meliloti mutant strain (SmgshA) which is unable to synthesize GSH due to a gene disruption in gshA, encoding the enzyme for the first step in the biosynthesis of GSH, was unable to grow under nonstress conditions, precluding any nodulation . In contrast, an S . meliloti strain (SmgshB) with gshB, encoding the enzyme involved in the second step in GSH synthesis, deleted was able to grow, indicating that gamma-glutamylcysteine, the dipeptide intermediate, can partially substitute for GSH . However, the SmgshB strain showed a delayed-nodulation phenotype coupled to a 75% reduction in the nitrogen fixation capacity . This phenotype was linked to abnormal nodule development . Both the SmgshA and SmgshB mutant strains exhibited higher catalase activity than the wild-type S . meliloti strain, suggesting that both mutant strains are under oxidative stress . Taken together, these results show that GSH plays a critical role in the growth of S . meliloti and during its interaction with the plant partner.

Mol Phylogenet Evol, 2005 Jan, 34(1), 29 - 54
Population genetics and phylogenetic inference in bacterial molecular systematics: the roles of migration and recombination in Bradyrhizobium species cohesion and delineation; Vinuesa P et al.; A combination of population genetics and phylogenetic inference methods was used to delineate Bradyrhizobium species and to uncover the evolutionary forces acting at the population-species interface of this bacterial genus . Maximum-likelihood gene trees for atpD, glnII, recA, and nifH loci were estimated for diverse strains from all but one of the named Bradyrhizobium species, and three unnamed "genospecies," including photosynthetic isolates . Topological congruence and split decomposition analyses of the three housekeeping loci are consistent with a model of frequent homologous recombination within but not across lineages, whereas strong evidence was found for the consistent lateral gene transfer across lineages of the symbiotic (auxiliary) nifH locus, which grouped strains according to their hosts and not by their species assignation . A well resolved Bayesian species phylogeny was estimated from partially congruent glnII+recA sequences, which is highly consistent with the actual taxonomic scheme of the genus . Population-level analyses of isolates from endemic Canarian genistoid legumes based on REP-PCR genomic fingerprints, allozyme and DNA polymorphism analyses revealed a non-clonal and slightly epidemic population structure for B . canariense isolates of Canarian and Moroccan origin, uncovered recombination and migration as significant evolutionary forces providing the species with internal cohesiveness, and demonstrated its significant genetic differentiation from B . japonicum, its sister species, despite their sympatry and partially overlapped ecological niches . This finding provides strong evidence for the existence of well delineated species in the bacterial world . The results and approaches used herein are discussed in the context of bacterial species concepts and the evolutionary ecology of (brady)rhizobia.

J Bacteriol, 2004 Dec, 186(24), 8433 - 42
A genetic locus necessary for rhamnose uptake and catabolism in Rhizobium leguminosarum bv . trifolii; Richardson JS et al.; Rhizobium leguminosarum bv . trifolii mutants unable to catabolize the methyl-pentose rhamnose are unable to compete effectively for nodule occupancy . In this work we show that the locus responsible for the transport and catabolism of rhamnose spans 10,959 bp . Mutations in this region were generated by transposon mutagenesis, and representative mutants were characterized . The locus contains genes coding for an ABC-type transporter, a putative dehydrogenase, a probable isomerase, and a sugar kinase necessary for the transport and subsequent catabolism of rhamnose . The regulation of these genes, which are inducible by rhamnose, is carried out in part by a DeoR-type negative regulator (RhaR) that is encoded within the same transcript as the ABC-type transporter but is separated from the structural genes encoding the transporter by a terminator-like sequence . RNA dot blot analysis demonstrated that this terminator-like sequence is correlated with transcript attenuation only under noninducing conditions . Transport assays utilizing tritiated rhamnose demonstrated that uptake of rhamnose was inducible and dependent upon the presence of the ABC transporter at this locus . Phenotypic analyses of representative mutants from this locus provide genetic evidence that the catabolism of rhamnose differs from previously described methyl-pentose catabolic pathways.

J Biol Chem . 2004 Nov 30; {Epub ahead of print}
Regulation of plant symbiosis receptor kinase (SYMRK) through serine and threonine phosphorylation; Yoshida S et al.; We studied the biochemical properties of a plant receptor-like kinase (RLK) to gain insights into the yet unknown regulatory mechanism of this largest class of plant kinases . SYMRK (SYMBIOSIS RECEPTOR KINASE) is required for early signal transduction leading to plant root symbioses with nitrogen fixing Rhizobia, and phosphate acquiring arbuscular mycorrhizal fungi . Amino acid substitutions in positions critical for activity of other related kinases cause a non-symbiotic plant phenotype, suggesting that SYMRK kinase activity is required for symbiosis . SYMRK is capable of intermolecular autophosphorylation . Non-phosphorylated SYMRK is less active than the phosphorylated version, suggesting the phosphorylation status of SYMRK determines its activity . Three Ser/Thr residues were identified as regulatory residues required for full kinase activation through targeted mutagenesis . Using Q-ToF analysis, two of these were confirmed to be phosphorylated in vitro . These crucial phosphorylation sites are conserved among various plant receptor-like kinases as well as animal Pelle/IRAK kinases . Despite the distinct domain architecture of RLKs versus Pelle/IRAK, our results suggest the existence of conserved activation mechanisms in these evolutionary related protein kinase families.

BMC Evol Biol . 2004 Nov 19;4(1):47.
Widespread presence of "bacterial-like" PPP phosphatases in eukaryotes; Andreeva AV et al.; BACKGROUND: In eukaryotes, PPP (protein phosphatase P) family is one of the two known protein phosphatase families specific for Ser and Thr . The role of PPP phosphatases in multiple signaling pathways in eukaryotic cell has been extensively studied . Unlike eukaryotic PPP phosphatases, bacterial members of the family have broad substrate specificity or may even be Tyr-specific . Moreover, one group of bacterial PPPs are diadenosine tetraphosphatases, indicating that bacterial PPP phosphatases may not necessarily function as protein phosphatases . RESULTS: We describe the presence in eukaryotes of three groups of expressed genes encoding "non-conventional" phosphatases of the PPP family . These enzymes are more closely related to bacterial PPP phosphatases than to the known eukaryotic members of the family . One group, found exclusively in land plants, is most closely related to PPP phosphatases from some alpha-Proteobacteria, including Rhizobiales, Rhodobacterales and Rhodospirillaceae . This group is therefore termed Rhizobiales / Rhodobacterales / Rhodospirillaceae-like phosphatases, or Rhilphs . Phosphatases of the other group are found in Viridiplantae, Rhodophyta, Trypanosomatidae, Plasmodium and some fungi . They are structurally related to phosphatases from psychrophilic bacteria Shewanella and Colwellia, and are termed Shewanella-like phosphatases, or Shelphs . Phosphatases of the third group are distantly related to ApaH, bacterial diadenosine tetraphosphatases, and are termed ApaH-like phosphatases, or Alphs . Patchy distribution of Alphs in animals, plants, fungi, diatoms and kinetoplasts suggests that these phosphatases were present in the common ancestor of eukaryotes but were independently lost in many lineages . Rhilphs, Shelphs and Alphs form PPP clades, as divergent from "conventional" eukaryotic PPP phosphatases as they are from each other and from major bacterial clades . In addition, comparison of primary structures revealed a previously unrecognised (I/L/V)D(S/T)G motif, conserved in all bacterial and "bacterial-like" eukaryotic PPPs, but not in "conventional" eukaryotic and archaeal PPPs . CONCLUSIONS: Our findings demonstrate that many eukaryotes possess diverse "bacterial-like" PPP phosphatases, the enzymatic characteristics, physiological roles and precise evolutionary history of which have yet to be determined.

Mol Microbiol, 2004 Dec, 54(5), 1431 - 44
Two discrete elements are required for the replication of a repABC plasmid: an antisense RNA and a stem-loop structure; Venkova-Canova T et al.; The repABC replicons contain an operon encoding the initiator protein (RepC) and partitioning proteins (RepA and RepB) . The latter two proteins negatively regulate the transcription of the operon . In this article we have identified two novel regulatory elements, located within the conserved repB-repC intergenic sequence, which negatively modulate the expression of repC, in plasmid p42d of Rhizobium etli . One of them is a small antisense RNA and the other is a stem-loop structure in the repABC mRNA that occludes the Shine-Dalgarno sequence of repC . According to in vivo and in vitro analyses, the small antisense RNA (57-59 nt) resembles canonical negative regulators of replication because: (i) it is transcribed from a strong constitutive promoter (P2), (ii) the transcript overlaps untranslated region upstream of the RepC coding sequences, (iii) the RNA forms one secondary structure acting as a rho-independent terminator, (iv) the antisense RNA is a strong trans-incompatibility factor and (v) its presence reduces the level of repC expression . Surprisingly, both of these seemingly negative regulators are required for efficient plasmid replication.

Prikl Biokhim Mikrobiol, 2004 Sep-Oct, 40(5), 567 - 70
{The significance of exometabolites in the formation and operation of the soybean-rhizobium symbiosis}; The recombination genes addAB are not restricted to gram-positive bacteria: genetic analysis of the recombination initiation enzymes RecF and AddAB in Rhizobium etli; Programa de Genetica Molecular de Plasmidos Bacterianos, Centro de Investigacion sobre Fijacion de Nitrogeno-UNAM, 62210 Cuernavaca, Morelos, MexicoSingle-strand gaps (SSGs) and double-strand breaks (DSBs) are the major initiation sites for recombination . In bacteria, the SSGs are repaired by RecFOR, while the DSBs are processed by RecBCD in gram-negative bacteria and AddAB in gram-positive bacteria . Unexpectedly, instead of recBCD genes, the addAB genes were found in members of the alpha-proteobacteria group (gram negative) . Taking Rhizobium etli as a model, the role of recF and addAB genes in homologous recombination and repair of damaged DNA was evaluated . Inactivation of either recF or addA provoked strong sensitivity to UV radiation and mitomycin C, while an additive effect was observed in the recF-addA mutant . The DSBs generated by nalidixic acid caused low viability only in the addA mutant . The recombination frequency of large and small plasmids was reduced in the recF mutant (24- and 36-fold, respectively), whereas a slight decrease (threefold) in the addA mutant was observed . Moreover, an additive effect (47- and 90-fold, respectively) was observed in the double mutant, but it was not as dramatic as that in a recA mutant . Interestingly, the frequency of deletion and Campbell-type recombination was slightly affected in either single or double mutants . These results suggest that another pathway exists that allows plasmid and Campbell-type recombination in the absence of recF and addA genes.

Biochem Biophys Res Commun, 2004 Dec 17, 325(3), 864 - 70
Modification of soybean sucrose synthase by S-thiolation with ENOD40 peptide A; Rohrig H et al.; The gene ENOD40 is expressed at an early stage of root nodule organogenesis and has been postulated to play a central regulatory role in the Rhizobium-legume interaction . In vitro translation of soybean ENOD40 mRNA showed that the gene encodes two peptides of 12 and 24aa residues (peptides A and B) that bind to sucrose synthase . Here we show that the small Cys-containing peptide A binds to sucrose synthase by disulfide bond formation, which may represent a novel form of posttranslational modification of this important metabolic enzyme . Assays using nanomolar concentrations of peptide A revealed that the monomeric reduced form of this peptide binds to purified sucrose synthase . Using a cysteinyl capture strategy combined with MALDI-TOF MS analysis we identified the Cys residue C264 of soybean sucrose synthase as the binding site of peptide A . Modification of sucrose synthase with ENOD40 peptide A activates sucrose cleavage activity whereas the synthesis activity of the enzyme is unaffected. , . The results are discussed in relation to the role of sucrose synthase in the control of sucrose utilization in nitrogen-fixing nodules.

J Environ Biol, 2004 Apr, 25(2), 151 - 6
Effect of some herbicides used in Nigeria on Rhizobium phaseoli, Azotobacter vinelandii and Bacillus subtilis; Adeleye IA et al.; The effect of three herbicides namely Agroxone, Atranex 50SC and 2,4-Damine on Azotobacter vinelandii, Rhizobium phaseoli and Bacillus subtilis were studied . These bacteria were isolated from a bean-garden in Lagos . The results revealed that 2, 4-Damine was the most toxic of the three herbicides studied and Azotobacter vinelandii was found to be most sensitive to the herbicides . There was a reduction in LC50 of herbicides with increased number of days . The percentage survival decreased with increased concentration of herbicides and days for Rhizobium phaseoli and Azotobacter vinelandii while an initial reduction in population was followed by increased percentage survival of organisms for Bacillus subtilis.

Microbiology, 2004 Nov, 150(Pt 11), 3703 - 13
The C-terminal receiver domain of the Rhizobium leguminosarum bv . viciae FixL protein is required for free-living microaerobic induction of the fnrN promoter; Boesten B et al.; The Rhizobium leguminosarum bv . viciae VF39 FixL protein belongs to a distinct group of hybrid regulatory sensor proteins that bear a covalently linked C-terminal receiver domain . FixL has an unorthodox histidine kinase domain, which is shared with many other hybrid regulators . The purified FixL protein had autophosphorylation activity . A truncated protein, lacking the receiver domain, had a much-reduced autophosphorylation activity . However, this truncated protein still efficiently phosphorylated the purified receiver domain in trans . This indicates that, in the full-length FixL protein, the conserved histidine residue in the kinase domain is phosphorylated only transiently and that most of the phosphoryl label accumulates in the C-terminal receiver domain . Gene-fusion studies showed that the fixL gene is required for free-living microaerobic induction of the fnrN promoter . The presence of a functional fixK gene is not required . An R . leguminosarum strain lacking fixL could not be complemented with a truncated copy of the gene lacking the receiver domain . This indicates that the C-terminal receiver domain is an intermediate in the signal transduction pathway that links oxygen limitation to induction of the fnrN promoter in R . leguminosarum bv . viciae VF39.

Sci China C Life Sci, 2004 Jun, 47(3), 268 - 78
Phylogenetic clusters of rhizobia revealed by genome structures; Zheng J et al.; Rhizobia, bacteria that fix atmospheric nitrogen, are important agricultural resources . In order to establish the evolutionary relationships among rhizobia isolated from different geographic regions and different plant hosts for systematic studies, we evaluated the use of physical structure of the rhizobial genomes as a phylogenetic marker to categorize these bacteria . In this work, we analyzed the features of genome structures of 64 rhizobial strains . These rhizobial strains were divided into 21 phylogenetic clusters according to the features of genome structures evaluated by the endonuclease I-CeuI . These clusters were supported by 16S rRNA comparisons and genomic sequences of four rhizobial strains, but they are largely different from those based on the current taxonomic scheme (except 16S rRNA).

Mikrobiologiia, 2004 Jul-Aug, 73(4), 461 - 4
{The effect of the carbohydrate components of pea roots on the enzymatic activity of the surface agglutinins of Rhizobium leguminosarum bv . viciae 252}; Transfer of the symbiotic plasmid of Rhizobium etli CFN42 requires cointegration with p42a et al.; Programa de Genetica Molecular de Plasmidos Bacterianos, Centro de Investigacion sobre Fijacion de Nitrogeno, UNAM, Cuernavaca, Morelos, Mexico . sbrom@cifn.unam.mx

Plasmid p42a from Rhizobium etli CFN42 is self-transmissible and indispensable for conjugative transfer of the symbiotic plasmid (pSym) . Most pSym transconjugants also inherit p42a . pSym transconjugants that lack p42a always contain recombinant pSyms, which we designated RpSyms* . RpSyms* do not contain some pSym segments and instead have p42a sequences, including the replication and transfer regions . These novel recombinant plasmids are compatible with wild-type pSym, incompatible with p42a, and self-transmissible . The symbiotic features of derivatives simultaneously containing a wild-type pSym and an RpSym* were analyzed . Structural analysis of 10 RpSyms* showed that 7 shared one of the two pSym-p42a junctions . Sequencing of this common junction revealed a 53-bp region that was 90% identical in pSym and p42a, including a 5-bp central region flanked by 9- to 11-bp inverted repeats reminiscent of bacterial and phage attachment sites . A gene encoding an integrase-like protein (intA) was localized downstream of the attachment site on p42a . Mutation or the absence of intA abolished pSym transfer from a recA mutant donor . Complementation with the wild-type intA gene restored transfer of pSym . We propose that pSym-p42a cointegration is required for pSym transfer; cointegration may be achieved either through homologous recombination among large reiterated sequences or through IntA-mediated site-specific recombination between the attachment sites . Cointegrates formed through the site-specific system but resolved through RecA-dependent recombination or vice versa generate RpSyms* . A site-specific recombination system for plasmid cointegration is a novel feature of these large plasmids and implies that there is unique regulation which affects the distribution of pSym in nature due to the role of the cointegrate in conjugative transfer.

Plant Physiol, 2004 Nov, 136(3), 3682 - 91 Epub 2004 Oct 29.
LIN, a Medicago truncatula gene required for nodule differentiation and persistence of rhizobial infections; Kuppusamy KT et al.; Ethyl methanesulfonate mutagenesis of the model legume Medicago truncatula has previously identified several genes required for early steps in nodulation . Here, we describe a new mutant that is defective in intermediate steps of nodule differentiation . The lin (lumpy infections) mutant is characterized by a 4-fold reduction in the number of infections, all of which arrest in the root epidermis, and by nodule primordia that initiate normally but fail to mature . Genetic analyses indicate that the symbiotic phenotype is conferred by a single gene that maps to the lower arm of linkage group 1 . Transcriptional markers for early Nod factor responses (RIP1 and ENOD40) are induced in lin, as is another early nodulin, ENOD20, a gene expressed during the differentiation of nodule primordia . By contrast, other markers correlated with primordium differentiation (CCS52A), infection progression (MtN6), or nodule morphogenesis (ENOD2 and ENOD8) show reduced or no induction in homozygous lin individuals . Taken together, these results suggest that LIN functions in maintenance of rhizobial infections and differentiation of nodules from nodule primordia.

Plant Physiol, 2004 Nov, 136(3), 3692 - 702 Epub 2004 Oct 29.
nip, a symbiotic Medicago truncatula mutant that forms root nodules with aberrant infection threads and plant defense-like response; Veereshlingam H et al.; To investigate the legume-Rhizobium symbiosis, we isolated and studied a novel symbiotic mutant of the model legume Medicago truncatula, designated nip (numerous infections and polyphenolics) . When grown on nitrogen-free media in the presence of the compatible bacterium Sinorhizobium meliloti, the nip mutant showed nitrogen deficiency symptoms . The mutant failed to form pink nitrogen-fixing nodules that occur in the wild-type symbiosis, but instead developed small bump-like nodules on its roots that were blocked at an early stage of development . Examination of the nip nodules by light microscopy after staining with X-Gal for S . meliloti expressing a constitutive GUS gene, by confocal microscopy following staining with SYTO-13, and by electron microscopy revealed that nip initiated symbiotic interactions and formed nodule primordia and infection threads . The infection threads in nip proliferated abnormally and very rarely deposited rhizobia into plant host cells; rhizobia failed to differentiate further in these cases . nip nodules contained autofluorescent cells and accumulated a brown pigment . Histochemical staining of nip nodules revealed this pigment to be polyphenolic accumulation . RNA blot analyses demonstrated that nip nodules expressed only a subset of genes associated with nodule organogenesis, as well as elevated expression of a host defense-associated phenylalanine ammonia lyase gene . nip plants were observed to have abnormal lateral roots . nip plant root growth and nodulation responded normally to ethylene inhibitors and precursors . Allelism tests showed that nip complements 14 other M . truncatula nodulation mutants but not latd, a mutant with a more severe nodulation phenotype as well as primary and lateral root defects . Thus, the nip mutant defines a new locus, NIP, required for appropriate infection thread development during invasion of the nascent nodule by rhizobia, normal lateral root elongation, and normal regulation of host defense-like responses during symbiotic interactions.

Indian J Exp Biol, 2004 Oct, 42(10), 1028 - 31
Mini transposon vector mediated foreign gene expression in Mesorhizobium huakuii subsp . rengei; Balachandar D et al.; Among the transposable elements, mini-Tn5 transposon vector has proven to be of greater utility for insertion mutagenesis of variety of Gram negative bacteria . The mini-Tn5 vector containing promoter less egfp gene and gentamycin resistant gene was used for the present study . The transposon vector was introduced to M . huakuii from E . coli S17 by conjugation . The conjugants were screened for stable expression of egfp both in free-living and in nodules of Astragalus sinicus . The result showed that the conjugant #3 showed stable expression of green fluorescent both in free-living and bacteroid stage . The visualization of sym plasmid of wild strain and conjugants showed that conjugant #3 had a fragmentation of large sized plasmid into two but without affecting the nodulating ability. , . These results clearly indicated that mini-Tn5 vectors (Transposon vectors) the best alternate tools for plasmid vectors for integration of foreign genes in chromosomal DNA or symbiotic plasmid and expression, both in free-living and bacteroid stage of Rhizobium.

Arch Microbiol, 2004 Dec, 182(6), 505 - 13 Epub 2004 Dec.
Rhizobium leguminosarum methyl-accepting chemotaxis protein genes are down-regulated in the pea nodule; Yost CK et al.; Regulation of methyl-accepting chemotaxis protein (MCP) genes of Rhizobium leguminosarum was studied under symbiotic conditions . Transcriptional fusions using both beta-galactosidase and beta-glucuronidase genes within two different mcp genes demonstrated that mcp expression decreased significantly during nodulation . Immunoblots using an anti-MCP antibody detected MCPs in free-living cells but not in bacteroids . Down-regulation during nodulation was not dependent upon known regulatory proteins involved in induction of expression of genes involved in nitrogen fixation . Environmental conditions found in the bacteroid that may trigger down-regulation were investigated by growing free-living cultures under a variety of growth conditions . Growth under low oxygen concentration or using succinate as a sole carbon source did not lower expression of the mcp gene fusions.

Res Microbiol, 2004 Nov, 155(9), 770 - 80
A novel genetic locus outside the symbiotic island is required for effective symbiosis of Bradyrhizobium japonicum with soybean Glycine max; Becker BU et al.; In order to investigate the symbiotic interaction between soybean and Bradyrhizobium japonicum, TnphoA mutagenesis of the microsymbiont was performed . Mutant strain 2-10 was found to induce a strongly reduced number of ineffective nodules . Ultrastructural analysis of the soybean nodule central tissue revealed the presence of numerous starch granules and vacuoles in the infected cells . In addition, the number of symbiosomes was extremely low, indicating an impaired interaction between the plant and invading bacteria . Cloning and sequencing of the mutated DNA region uncovered four open reading frames (ORFs) lacking any data base similarities . ORFs srrA1 and srrA2, the 2-10 TnphoA insertion site, are encoded in the same reading frame . A 35-kDa expression product in Escherichia coli indicated the presence of a common protein, called SrrA (symbiotically relevant region) in B . japonicum 110spc4, encoded by combined srrA1 and srrA2 genes . The analysis of gene disruption mutants revealed that srrB and srrC were also required for effective symbiosis with soybeans . Further downstream the gene for a putative inner membrane protein (pipA) of unknown function was encoded on the opposite strand . Primer extension studies led to the conclusion that the organization of genes differed from the RhizoBase annotation in this particular region of B . japonicum USDA110.

Res Microbiol, 2004 Nov, 155(9), 747 - 54
Screening procedures for selecting rhizobacteria with biocontrol effects upon Fusarium verticillioides growth and fumonisin B1 production; Cavaglieri L et al.; Screening is a critical step in the discovery of microbial agents that can exert biological control of Fusarium verticillioides at the root level . The objectives of this research were to determine the utility of a niche overlap index to realise the first screening of maize rhizobacterial isolates during different water activities . Studies were conducted to evaluate various methods for second screening with different modes of action . The antifungal activity of bacterial isolates through antibiosis assay was checked and the influence of different isolates on Fusarium verticilliodes growth and fumonisin B(1) was studied . Eleven competitive rhizobacterial isolates (Arthrobacter globiformis RC1, Azotobacter armeniacus RC2, A . armeniacus RC3, A . globiformis RC4, A . globiformis RC5, A . armeniacus RC6, Pseudomonas solanacearum RC7, Bacillus subtilis RC8, B . subtilis RC9, P . solanacearum RC10, B . subtilis RC11) were selected for the studies which followed . All bacteria were able to utilise the widest range of carbon sources and showed the highest niche overlap indices at the water activities tested . All bacterial antagonists reduced fumonisin B(1) production at all levels tested . Isolates belonging to Pseudomonas and Bacillus genera significantly inhibited fumonisin B(1) production, which ranged between 70 and 100% . Also, A . armeniacus RC2 caused important fumonisin B(1) reduction . The results of the present work suggest that A . armeniacus RC2, A . armeniacus RC3, B . subtilis RC8, B . subtilis RC9, B . subtilis RC11, P . solanacearum RC7, and P . solanacearum RC10 could have practical value in the control of F . verticillioides root colonisation . This paper is part of an on-going study to determine their application at the field level.

Trends Plant Sci, 2004 Nov, 9(11), 518 - 22
Rhizobium infection: lessons from the versatile nodulation behaviour of water-tolerant legumes; Goormachtig S et al.; Water-tolerant legumes provide bacteria with special ways of invading roots to establish N(2)-fixing symbiosis upon flooding . On well-aerated roots, root hair curling (RHC) invasion is used, whereas, under hydroponic conditions, rhizobia enter the cortex through cracks at lateral root bases (LRBs) . Here, we compare the physiological and anatomical traits of these invasions . During waterlogging, accumulating ethylene inhibits the epidermal stages of RHC invasion . LRB invasion circumvents this step by direct colonization of the cortical tissue . By avoiding the epidermis for bacterial entry under hydroponic conditions, the stringent nodulation (Nod) factor perception systems that are active within the epidermis are not needed . Consequently, LRB invasion might be useful for analysing the requirement for Nod factor perception and other signal transduction systems downstream of the epidermis.

Plant J, 2004 Nov, 40(4), 622 - 31
Virus-induced gene silencing as a tool for functional genomics in a legume species; Constantin GD et al.; Virus-induced gene silencing (VIGS) is an attractive reverse-genetics tool for studies of gene function . However, efficient VIGS has only been accomplished in a few plant species . In order to extend the application of VIGS, we examined whether a VIGS vector based on Pea early browning virus (PEBV) would produce recognizable phenotypes in Pisum sativum . A plasmid vector of PEBV was modified to allow agro-inoculation and insertion of heterologous sequences . cDNA fragments of the P . sativum phytoene desaturase (PDS), LEAFY (LFY) and KORRIGAN1 (KOR1) homologues were inserted into the PEBV RNA2 vector, replacing the genes required for nematode transmission . Pisum sativum inoculated with PEBV carrying a fragment of PsPDS developed characteristic photo-bleached leaves and this phenotype was associated with a significant reduction in PsPDS mRNA . The P . sativum homologue of LFY is known as UNIFOLIATA (UNI) . Plants inoculated with PEBV carrying a fragment of UNI developed distorted flowers and leaves with modified architecture, which are also observed in UNI-mutants . In Arabidopsis thaliana, the KOR1-mutant is characterized by an extreme dwarf phenotype . Pisum sativum plants inoculated with PEBV carrying a fragment of PsKOR1 displayed a significant reduction in height and inhibition of root growth . The PEBV VIGS vector did not affect the ability of P . sativum to flower, set seeds, and form nodules characteristic of symbiosis with rhizobium . These results suggest that the PEBV vector can be applied to functional genomics in a legume species to study genes involved in a wide range of biological processes.

DNA Res, 2004 Aug 31, 11(4), 263 - 74
Large-scale analysis of gene expression profiles during early stages of root nodlule formation in a model legume, Lotus japonicus; Kouchi H et al.; Gene expression profiles during early stages of formation of symbiotic nitrogen-fixing nodules in a model legume Lotus japonicus were analyzed by means of a cDNA array of 18,144 non-redundant expressed sequence tags (ESTs) isolated from L . japonicus . Expression of a total of 1,076 genes was significantly accelerated during the successive stages that represent infection of Mesorhizobium loti, nodule primordium initiation, nodule organogenesis, and the onset of nitrogen fixation . These include 32 nodulin and nodulinhomolog genes as well as a number of genes involved in the catabolism of photosynthates and assimilation of fixed nitrogen that were previously known to be abundantly expressed in root nodules of many legumes . We also identified a large number of novel nodule-specific or enhanced genes, which include genes involved in many cellular processes such as membrane transport, defense responses, phytohormone synthesis and responses, signal transduction, cell wall synthesis, and transcriptional regulation . Notably, our data indicate that the gene expression profile in early steps of Rhizobium-legume interactions is considerably different from that in subsequent stages of nodule development . A number of genes involved in the defense responses to pathogens and other stresses were induced abundantly in the infection process, but their expression was suppressed during subsequent nodule formation . The results provide a comprehensive data source for investigation of molecular mechanisms underlying nodulation and symbiotic nitrogen fixation.

Mol Plant Microbe Interact, 2004 Oct, 17(10), 1153 - 61
Flavonoids, NodD1, NodD2, and nod-box NB15 modulate expression of the y4wEFG locus that is required for indole-3-acetic acid synthesis in Rhizobium sp . strain NGR234; Theunis M et al.; Flavonoids secreted by host plants activate, in conjunction with the transcriptional activator NodD, nod gene expression of rhizobia resulting in the synthesis of Nod factors, which trigger nodule organogenesis . Interestingly, addition of inducing flavonoids also stimulates the production of the phytohormone indole-3-acetic acid (IAA) in several rhizobia . Here, the molecular basis of IAA synthesis in Rhizobium sp . NGR234 was investigated . Mass spectrometric analysis of culture supernatants indicated that NGR234 is capable of synthesizing IAA via three different pathways . The production of IAA is increased strongly by exposure of NGR234 to daidzein in a NodD1-, NodD2-, and SyrM2-dependent manner . This suggests that the y4wEFG locus that is downstream of nod-box NB15 encodes proteins involved in IAA synthesis . Knockout mutations in y4wE and y4wF abolished flavonoid-inducible IAA synthesis and a functional y4wF was required for constitutive IAA production . The promoter activity of NB15 and IAA production both were enhanced by introduction of a multicopy plasmid carrying nodD2 into NGR234. , . Surprisingly, the y4wE mutant still nodulated Vigna unguiculata and Tephrosia vogelii, although the nodules contained less IAA and IAA conjugates than those formed by the wild-type bacterium.

Mol Plant Microbe Interact, 2004 Oct, 17(10), 1043 - 50
Development of ectopic roots from abortive nodule primordia; Ferraioli S et al.; The symbiotic phenotype of five Tn5-induced mutants of Rhizobium etli affected in different anabolic pathways (namely, gluconeogenesis and biosynthesis of lysine, purine, or pyrimidine) was analyzed . These mutants induced, on the root of Phaseolus vulgaris, a normal early sequence of morphogenetics events, including root hair deformation and development of nodule primordia . Later on, however, from the resulting root outgrowths, instead of nodules, one or more ectopic roots (spaced closely related and agravitropic) emerged . Therefore, this group of mutant was collectively called "root inducer" (RIND) . It was observed that the RIND-induced infection threads aborted early inside the invaded root hair, and that the resulting abortive nodules lack induction of late nodulin genes . Moreover, experiments performed using a conditional mutant (a methionine-requiring invader) revealed that bacterial invasion plays a key role in the maintenance of the program of nodule development and, in particular, in the differentiation of the most specific symbiotic tissue of globose nodules, the central tissue . These data indicate that, in P . vulgaris, the nodule primordium is a root-specified pro-meristematic tissue.

Plant J, 1998 Apr, 14(1), 23 - 34
Auxin transport inhibition precedes root nodule formation in white clover roots and is regulated by flavonoids and derivatives of chitin oligosaccharides; Mathesius U et al.; Summary The expression of the auxin responsive reporter construct, GH3:gusA, was examined in transgenic white clover plants to assess changes in the auxin balance during the earliest stages of root nodule formation . Reporter gene expression was monitored at marked locations after the application of bacteria or signal molecules using two precise inoculation techniques: spot-inoculation and a novel method for ballistic microtargeting . Changes in GH3:gusA expression were monitored after the inoculation of Rhizobium leguminosarum biovar trifolii, non-host rhizobia, lipo-chitin oligosaccharides (LCOs), chitin oligosaccharides, a synthetic auxin transport inhibitor (naphthylphthalamic acid; NPA), auxin, the ENOD40-1 peptide or different flavonoids . The results show that clover-nodulating rhizobia induce a rapid, transient and local downregulation of GH3:gusA expression during nodule initiation followed by an upregulation of reporter gene expression at the site of nodule initiation . Microtargeting of auxin caused a local and acropetal upregulation of GH3:gusA expression, whereas NPA caused local and acropetal downregulation of expression . Both spot-inoculation and microtargeting of R . l . bv . trifolii LCOs or flavonoid aglycones induced similar changes to GH3:gusA expression as NPA . O-acetylated chitin oligosaccharides caused similar changes to GH3:gusA expression as R . l . bv . trifolii spot-inoculation, but only after delivery by microtargeting . Non-O-acetylated chitin oligosaccharides, flavonoid glucosides or the ENOD40-1 peptide failed to induce any detectable changes in GH3:gusA expression . GH3:gusA expression patterns during the later stages of nodule and lateral root development were similar . These results support the hypothesis that LCOs and chitin oligosaccharides act by perturbing the auxin flow in the root during the earliest stages of nodule formation, and that endogenous flavonoids could mediate this response.

Plant Physiol, 2004 Nov, 136(3), 3582 - 93 Epub 2004 Oct 15.
Pharmacological evidence that multiple phospholipid signaling pathways link Rhizobium nodulation factor perception in Medicago truncatula root hairs to intracellular responses, including Ca2+ spiking and specific ENOD gene expression; Charron D et al.; Rhizobium nodulation (Nod) factors are specific lipochito-oligosaccharide signals essential for initiating in root hairs of the host legume developmental responses that are required for controlled entry of the microsymbiont . In this article, we focus on the Nod factor signal transduction pathway leading to specific and cell autonomous gene activation in Medicago truncatula cv Jemalong in a study making use of the Nod factor-inducible MtENOD11 gene . First, we show that pharmacological antagonists that interfere with intracellular ion channel and Ca2+ pump activities are efficient blockers of Nod factor-elicited pMtENOD11-beta-glucuronidase (GUS) expression in root hairs of transgenic M . truncatula . These results indicate that intracellular Ca2+ release and recycling activities, essential for Ca2+ spiking, are also required for specific gene activation . Second, pharmacological effectors that inhibit phospholipase D and phosphoinositide-dependent phospholipase C activities are also able to block pMtENOD11-GUS activation, thus underlining a central role for multiple phospholipid signaling pathways in Nod factor signal transduction . Finally, pMtENOD11-GUS was introduced into all three Nod-/Myc- dmi M . truncatula mutant backgrounds, and gene expression was evaluated in response to the mastoparan peptide agonist Mas7 . We found that Mas7 elicits root hair MtENOD11 expression in dmi1 and dmi2 mutants, but not in the dmi3 mutant, suggesting that the agonist acts downstream of DMI1/DMI2 and upstream of DMI3 . In light of these results and the recently discovered identities of the DMI gene products, we propose an integrated cellular model for Nod factor signaling in legume root hairs in which phospholipids play a key role in linking the Nod factor perception apparatus to downstream components such as Ca2+ spiking and ENOD gene expression.

Microbes Infect, 2004 Nov, 6(13), 1235 - 9
Why are most rhizobia beneficial to their plant hosts, rather than parasitic?
Denison RF, Toby Kiers E.
Multiple strains per plant and root-to-root (not seed-borne) transmission should favor rhizobia that invest in their own reproduction, rather than symbiotic N2 fixation, as analogous factors may favor pathogen virulence . But legumes can select for greater mutualism, controlling nodule O2 supply and reducing reproduction of rhizobia that fix less N2.

Biochem Biophys Res Commun, 2004 Nov 12, 324(2), 822 - 8
Three GroEL homologues from Rhizobium leguminosarum have distinct in vitro properties; George R et al.; The GroEL molecular chaperone of Escherichia coli and its cofactor GroES are highly conserved, and are required for the folding of many proteins . Most but not all bacteria express single GroEL and GroES proteins . Rhizobium leguminosarum strain A34 encodes three complete operons encoding homologues to GroEL and GroES . We have used circular dichroism and measurement of ATPase activity to compare the stabilities of these chaperonins after expression in and purification from E . coli . Significant differences in the stabilities of the proteins with respect to denaturant and temperature were found . The proteins also differed in their ability to refold denatured lactate dehydrogenase . This study, the first to compare the properties of three different GroEL homologues from the same organism, shows that despite the high degree of similarity between different homologues, they can display distinct properties in vitro.

Biochem Biophys Res Commun, 2004 Nov 12, 324(2), 611 - 20
Reversible and nonoxidative gamma-resorcylic acid decarboxylase: characterization and gene cloning of a novel enzyme catalyzing carboxylation of resorcinol, 1,3-dihydroxybenzene, from Rhizobium radiobacter; Ishii Y et al.; We found a gamma-resorcylic acid (gamma-RA, 2,6-dihydroxybenzoic acid) decarboxylase, as a novel enzyme applicable to carboxylation of resorcinol (RE, 1,3-dihydroxybenzene) to form gamma-RA, in a bacterial strain Rhizobium radiobacter WU-0108 isolated through the screening of gamma-RA degrading microorganisms . The activities for carboxylation of RE and decarboxylation of gamma-RA were detected in the cell-free extracts of R . radiobacter WU-0108 grown aerobically with gamma-RA . The enzyme, gamma-RA decarboxylase, was purified to homogeneity on SDS-PAGE through the steps of one ion-exchange chromatography and two kinds of hydrophobic chromatography . The molecular weight of the enzyme was estimated to be 130 kDa by gel-filtration, and that of the subunit was determined to be 34 kDa by SDS-PAGE, suggesting that the enzyme is a homotetrameric structure . The enzyme catalyzed the decarboxylation of gamma-RA, but not alpha-RA or beta-RA . Without addition of any cofactors, the enzyme catalyzed the regio-selective carboxylation of RE to form gamma-RA, without formation of alpha-RA and beta-RA, and of catechol to 2,3-dihydroxybenzoic acid . In the presence of oxygen, this gamma-RA decarboxylase showed no decrease in both of the activities as for decarboxylation of gamma-RA and carboxylation of RE, different from other decarboxylases reported so far . The gene, rdc, encoding the gamma-RA decarboxylase was cloned into Escherichia coli, sequenced, and subjected to over-expression . The deduced amino acid sequence of the rdc gene consists of 327 amino acid residues corresponding to 34 kDa protein, and shows 42% and 30% identity to those of a 2,3-dihydroxybenzoic acid decarboxylase from Aspergillus niger and a 5- carboxyvanillate decarboxylase from Sphingomonas paucimobilis SYK-6 . A site-directed mutagenesis study revealed the two histidine residues at positions of 164 and 218 in Rdc to be essential for the catalytic activities of decarboxylation of gamma-RA and carboxylation of RE.

Plant Physiol Biochem, 2004 Sep, 42(9), 723 - 9
Gas chromatography-mass spectrometry analysis of indoleacetic acid and tryptophan following aqueous chloroformate derivatisation of Rhizobium exudates; Perrine FM et al.; A new method for preparing alkyl esters of indole-3-acetic acid (IAA) in aqueous solution is adapted from the chloroformate method originally described by Husek for the analysis of amino acids . This method has the significant advantage of avoiding the generation and use of diazomethane, and is done in aqueous solution without the need to dry the sample with concomitant non-specific losses of IAA . The effectiveness of this method is demonstrated by its use in an isotope dilution gas chromatography-mass spectrometry (GC-MS) assay of IAA and L-tryptophan (Trp) in the culture supernatant of a series of Sinorhizobium meliloti and Rhizobium leguminosarum bv . trifolii strains that can interact with rice to either enhance or inhibit rice plant growth . We were testing the hypothesis that the rice growth inhibition was related to the biosynthesis of IAA . It was found that S . meliloti and Rhizobium strains produced high amounts of IAA in Trp supplemented BIII minimal medium compared to BIII media. , . All the strains produced more than the minimum amount of IAA required to inhibit rice growth and thus IAA is not the major inhibitory factor of rice seedling growth from S . meliloti and Rhizobium strains.

Plant Physiol Biochem, 2004 Sep, 42(9), 717 - 22
Rhizobial strain involvement in plant growth, nodule protein composition and antioxidant enzyme activities of chickpea-rhizobia symbioses: modulation by salt stress; Mhadhbi H et al.; Mesorhizobium ciceri, Mesorhizobium mediterraneum and Sinorhizobium medicae strains showed different symbiotic performances when inoculated to chickpea (Cicer arietinum L., cv . chetoui) at unstressed conditions and under salt stress . The analysis of nodular proteic composition and antioxidant enzyme activities revealed a polymorphism of patterns on SDS and native PAGE suggesting a potential dependence on the bacterial partner . Salt effect was analysed on plant growth, nitrogen fixation and antioxidant enzymes . M . ciceri, the most efficient strain, seemed to allow a best tolerance to chickpea plants under salt stress . This constraint did not affect the nodular superoxide dismutase (SOD, E.C . 1.15.1.1) activity of the symbiosis implicating the latter strain . This symbiosis showed the least decrease for the nodule protein level and the catalase (CAT, E.C . 1.11.1.6) activity, and the highest increase of peroxidase (POX, E.C . 1.11.1.7) activity that seemed to be related with the tolerance to salt.

Microbiology, 2004 Oct, 150(Pt 10), 3473 - 82
Regulation of exopolysaccharide synthesis in Rhizobium sp . strain TAL1145 involves an alternative sigma factor gene, rpoH2; Kaufusi PH et al.; Exopolysaccharide (EPS) produced by Rhizobium sp . strain TAL1145 has been shown to be essential for effective nodulation on Leucaena leucocephala (leucaena) . This paper reports the isolation and characterization of an alternative sigma factor gene, rpoH2, involved in the regulation of EPS synthesis in TAL1145 . Disruption of this gene in TAL1145 resulted in a Calcofluor-dim mutant RUH102 that produced approximately 18 % of the amount of EPS made by TAL1145 . This mutation did not affect the normal growth of RUH102 in free-living state . RUH102 induced few nitrogen-fixing nodules, resulting in a significant reduction in total nitrogen content in leucaena . It was complemented for EPS production and nodulation by a 2.0 kb HindIII fragment of TAL1145 . Sequence analysis of this fragment revealed the rpoH2 ORF of 870 bp that encoded a protein of 32 kDa . Expression of the rpoH2 ORF in Escherichia coli also revealed a 32 kDa protein . A PCR-constructed clone of 1263 bp, containing the rpoH2 ORF and its upstream putative regulatory region, complemented RUH102 for EPS defects . Comparison of the RpoH2 sequence to proteins in the databases showed significant similarity to RpoH-like sigma factors of other Gram-negative bacteria . By constructing several exo : : Tn3Hogus fusions and transferring them to the backgrounds of TAL1145 and RUH102, it was demonstrated that RpoH2 positively regulates the transcription of some exo genes.

Appl Environ Microbiol, 2004 Oct, 70(10), 6240 - 6
Nonlegumes, legumes, and root nodules harbor different arbuscular mycorrhizal fungal communities; Scheublin TR et al.; Legumes are an important plant functional group since they can form a tripartite symbiosis with nitrogen-fixing Rhizobium bacteria and phosphorus-acquiring arbuscular mycorrhizal fungi (AMF) . However, not much is known about AMF community composition in legumes and their root nodules . In this study, we analyzed the AMF community composition in the roots of three nonlegumes and in the roots and root nodules of three legumes growing in a natural dune grassland . We amplified a portion of the small-subunit ribosomal DNA and analyzed it by using restriction fragment length polymorphism and direct sequencing . We found differences in AMF communities between legumes and nonlegumes and between legume roots and root nodules . Different plant species also contained different AMF communities, with different AMF diversity . One AMF sequence type was much more abundant in legumes than in nonlegumes (39 and 13%, respectively) . Root nodules contained characteristic AMF communities that were different from those in legume roots, even though the communities were similar in nodules from different legume species . One AMF sequence type was found almost exclusively in root nodules . Legumes and root nodules have relatively high nitrogen concentrations and high phosphorus demands . Accordingly, the presence of legume- and nodule-related AMF can be explained by the specific nutritional requirements of legumes or by host-specific interactions among legumes, root nodules, and AMF . In summary, we found that AMF communities vary between plant functional groups (legumes and nonlegumes), between plant species, and between parts of a root system (roots and root nodules).

Appl Environ Microbiol, 2004 Oct, 70(10), 5980 - 7
Unexpectedly diverse Mesorhizobium strains and Rhizobium leguminosarum nodulate native legume genera of New Zealand, while introduced legume weeds are nodulated by Bradyrhizobium species; Weir BS et al.; The New Zealand native legume flora are represented by four genera, Sophora, Carmichaelia, Clianthus, and Montigena . The adventive flora of New Zealand contains several legume species introduced in the 19th century and now established as serious invasive weeds . Until now, nothing has been reported on the identification of the associated rhizobia of native or introduced legumes in New Zealand . The success of the introduced species may be due, at least in part, to the nature of their rhizobial symbioses . This study set out to address this issue by identifying rhizobial strains isolated from species of the four native legume genera and from the introduced weeds: Acacia spp . (wattles), Cytisus scoparius (broom), and Ulex europaeus (gorse) . The identities of the isolates and their relationship to known rhizobia were established by comparative analysis of 16S ribosomal DNA, atpD, glnII, and recA gene sequences . Maximum-likelihood analysis of the resultant data partitioned the bacteria into three genera . Most isolates from native legumes aligned with the genus Mesorhizobium, either as members of named species or as putative novel species . The widespread distribution of strains from individual native legume genera across Mesorhizobium spp . contrasts with previous reports implying that bacterial species are specific to limited numbers of legume genera . In addition, four isolates were identified as Rhizobium leguminosarum . In contrast, all sequences from isolates from introduced weeds aligned with Bradyrhizobium species but formed clusters distinct from existing named species . These results show that native legume genera and these introduced legume genera do not have the same rhizobial populations.

Appl Environ Microbiol, 2004 Oct, 70(10), 5916 - 22
Functional expression of Sinorhizobium meliloti BetS, a high-affinity betaine transporter, in Bradyrhizobium japonicum USDA110; Boscari A et al.; Among the Rhizobiaceae, Bradyrhizobium japonicum strain USDA110 appears to be extremely salt sensitive, and the presence of glycine betaine cannot restore its growth in medium with an increased osmolarity (E . Boncompagni, M . Osteras, M . C . Poggi, and D . Le Rudulier, Appl . Environ . Microbiol . 65:2072-2077, 1999) . In order to improve the salt tolerance of B . japonicum, cells were transformed with the betS gene of Sinorhizobium meliloti . This gene encodes a major glycine betaine/proline betaine transporter from the betaine choline carnitine transporter family and is required for early osmotic adjustment . Whereas betaine transport was absent in the USDA110 strain, such transformation induced glycine betaine and proline betaine uptake in an osmotically dependent manner . Salt-treated transformed cells accumulated large amounts of glycine betaine, which was not catabolized . However, the accumulation was reversed through rapid efflux during osmotic downshock . An increased tolerance of transformant cells to a moderate NaCl concentration (80 mM) was also observed in the presence of glycine betaine or proline betaine, whereas the growth of the wild-type strain was totally abolished at 80 mM NaCl . Surprisingly, the deleterious effect due to a higher salt concentration (100 mM) could not be overcome by glycine betaine, despite a significant accumulation of this compound . Cell viability was not significantly affected in the presence of 100 mM NaCl, whereas 75% cell death occurred at 150 mM NaCl . The absence of a potential gene encoding Na(+)/H(+) antiporters in B . japonicum could explain its very high Na(+) sensitivity.

Appl Environ Microbiol, 2004 Oct, 70(10), 5891 - 7
Expression of an exogenous 1-aminocyclopropane-1-carboxylate deaminase gene in Sinorhizobium meliloti increases its ability to nodulate alfalfa; Ma W et al.; 1-Aminocyclopropane-1-carboxylate (ACC) deaminase has been found in various plant growth-promoting rhizobacteria, including rhizobia . This enzyme degrades ACC, the immediate precursor of ethylene, and thus decreases the biosynthesis of ethylene in higher plants . The ACC deaminase of Rhizobium leguminosarum bv . viciae 128C53K was previously reported to be able to enhance nodulation of peas . The ACC deaminase structural gene (acdS) and its upstream regulatory gene, a leucine-responsive regulatory protein (LRP)-like gene (lrpL), from R . leguminosarum bv . viciae 128C53K were introduced into Sinorhizobium meliloti, which does not produce this enzyme, in two different ways: through a plasmid vector and by in situ transposon replacement . The resulting ACC deaminase-producing S . meliloti strains showed 35 to 40% greater efficiency in nodulating Medicago sativa (alfalfa), likely by reducing ethylene production in the host plants . Furthermore, the ACC deaminase-producing S . meliloti strain was more competitive in nodulation than the wild-type strain . We postulate that the increased competitiveness might be related to utilization of ACC as a nutrient within the infection threads.

Plant Physiol, 2004 Oct, 136(2), 3191 - 7 Epub 2004 Oct 01.
Infection-related activation of the cg12 promoter is conserved between actinorhizal and legume-rhizobia root nodule symbiosis; Svistoonoff S et al.; Two nitrogen-fixing root nodule symbioses between soil bacteria and higher plants have been described: the symbiosis between legume and rhizobia and actinorhizal symbioses between plants belonging to eight angiosperm families and the actinomycete Frankia . We have recently shown that the subtilisin-like Ser protease gene cg12 (isolated from the actinorhizal plant Casuarina glauca) is specifically expressed during plant cell infection by Frankia . Here we report on the study of C . glauca cg12 promoter activity in the transgenic legume Medicago truncatula . We found that cg12 promoter activation is associated with plant cell infection by Sinorhizobium meliloti . Furthermore, applications of purified Nod factors and mycorrhizal inoculation failed to trigger expression of the cg12-reporter gene construct . This indicates that at least part of the transcriptional environment in plant cells infected by endosymbiotic nitrogen-fixing bacteria is conserved between legume and actinorhizal plants. , . These results are discussed in view of recent data concerning molecular phylogeny that suggest a common evolutionary origin of all plants entering nitrogen-fixing root nodule symbioses.

Mikrobiol Z, 2004 May-Jun, 66(3), 14 - 21
{Effect of aboriginal populations of soy nodule bacteria on symbiotic activity of introduced strain Bradyrhizobium japonicum 634b}; TtsI et al.; LBMPS, Departement de Biologie Vegetale, Sciences III, 30 quai Ernest-Ansermet, University of Geneva, CH-1211 Geneva 4, SwitzerlandFormation of nitrogen-fixing nodules on legume roots by Rhizobium sp . NGR234 requires an array of bacterial factors, including nodulation outer proteins (Nops) secreted through a type III secretion system (TTSS) . Secretion of Nops is abolished upon inactivation of ttsI (formerly y4xI), a protein with characteristics of two-component response regulators that was predicted to activate transcription of TTSS-related genes . During the symbiotic interaction, the phenotype of NGR omega ttsI differs from that of a mutant with a nonfunctional secretion machine, however . This indicated that TtsI regulates the synthesis of other symbiotic factors as well . Conserved sequences, called tts boxes, proposed to act as binding sites for TtsI, were identified not only within the TTSS cluster but also in the promoter regions of i) genes predicted to encode homologs of virulence factors secreted by pathogenic bacteria, ii) loci involved in the synthesis of a rhamnose-rich component (rhamnan) of the lipopolysaccharides (LPS), and iii) open reading frames that play roles in plasmid partitioning . Transcription studies showed that TtsI and tts boxes are required for the activation of TTSS-related genes and those involved in rhamnose synthesis . Furthermore, extraction of polysaccharides revealed that inactivation of ttsI abolishes the synthesis of the rhamnan component of the LPS . The phenotypes of mutants impaired in TTSS-dependent protein secretion, rhamnan synthesis, or in both functions were compared to assess the roles of some of the TtsI-controlled factors during symbiosis.

Res Microbiol, 2004 Oct, 155(8), 672 - 80
Characterization of phenol and trichloroethene degradation by the rhizobium Ralstonia taiwanensis; Chen WM et al.; Ralstonia taiwanensis is a root nodule bacterium originally isolated from Mimosa sp . in southern Taiwan . Some strains of R . taiwanensis demonstrated the ability to grow on medium containing phenol as the sole carbon source, especially strain TJ86, which was able to survive and grow at phenol concentrations of up to 900 mg/l . The dependence of the phenol degradation rate on the phenol concentration can be described by Haldane's model with a low KS (the apparent half-saturation constant) of 5.46 microM and an extremely high KSI (the apparent inhibition constant) 9075 microM . The optimal phenol degradation rate was 61 micromol/min/g cell, which occurred at a phenol concentration of 228 microM . The phenol-limited growth kinetics of TJ86 by Andrews's model also followed a similar trend to that of phenol degradation, indicating the close links between phenol degradation and cell growth . Strain TJ86 also achieved 100 and 40% degradation for soil samples amended with 500 and 1000 microg phenol/g soil (dry weight) within 9 days, respectively . Moreover, strain TJ86 cometabolically degraded trichloroethene (TCE) after being cultivated with media containing phenol or m-cresol as the carbon substrate . The sequence of the large-subunit phenol hydroxylase (LmPH) gene obtained from TJ86 displayed high homology to that of other phenol-utilizing bacteria . Results from kinetic and phylogenetic analyses suggest that strain TJ86 most likely belongs to group I phenol-degrading bacteria which are considered to be efficient TCE degraders . It is proposed that the symbiotic relationship between rhizobia R . taiwanensis and its host plant Mimosa sp . may have the potential for rhizoremediation of aquatic and soil environments contaminated by phenol and TCE.

Amino Acids, 2004 Oct, 27(2), 169 - 74 Epub 2004 Sep 22.
Production of amino acids by Rhizobium, Mesorhizobium and Sinorhizobium strains in chemically defined media; Salmeron-Lopez V et al.; Five strains of Rhizobium spp, one strain of Mesorhizobium loti and two strains of Sinorhizobium meliloti were tested for their ability to grow in chemically-defined medium lacking growth factor . Qualitative and quantitative production of aspartic acid, serine, glutamic acid, glycine, histidine, threonine, arginine, alanine, proline, cysteine, tyrosine, valine, methionine, lysine, isoleucine, leucine, and phenylalanine was determined by the use of mannitol as sole carbon source.Strains of Rhizobium spp . and Sinorhizobium sp . produced all the amino acids analysed with the exception of cysteine and high biological levels of serine, glycine and alanine were detected after 2 days of culture in mineral medium.Strain U226 of M . loti only produced small amounts of amino acids and glutamic acid, histidine, arginine, cysteine, methionine, lysine and phenylalanine was not liberated into the media.

J Bacteriol, 2004 Oct, 186(19), 6617 - 25
Involvement of exo5 in production of surface polysaccharides in Rhizobium leguminosarum and its role in nodulation of Vicia sativa subsp . nigra; Laus MC et al.; Analysis of two exopolysaccharide-deficient mutants of Rhizobium leguminosarum, RBL5808 and RBL5812, revealed independent Tn5 transposon integrations in a single gene, designated exo5 . As judged from structural and functional homology, this gene encodes a UDP-glucose dehydrogenase responsible for the oxidation of UDP-glucose to UDP-glucuronic acid . A mutation in exo5 affects all glucuronic acid-containing polysaccharides and, consequently, all galacturonic acid-containing polysaccharides . Exo5-deficient rhizobia do not produce extracellular polysaccharide (EPS) or capsular polysaccharide (CPS), both of which contain glucuronic acid . Carbohydrate composition analysis and nuclear magnetic resonance studies demonstrated that EPS and CPS from the parent strain have very similar structures . Lipopolysaccharide (LPS) molecules produced by the mutant strains are deficient in galacturonic acid, which is normally present in the core and lipid A portions of the LPS . The sensitivity of exo5 mutant rhizobia to hydrophobic compounds shows the involvement of the galacturonic acid residues in the outer membrane structure . Nodulation studies with Vicia sativa subsp . nigra showed that exo5 mutant rhizobia are impaired in successful infection thread colonization . This is caused by strong agglutination of EPS-deficient bacteria in the root hair curl . Root infection could be restored by simultaneous inoculation with a Nod factor-defective strain which retained the ability to produce EPS and CPS . However, in this case colonization of the nodule tissue was impaired.

J Bacteriol, 2004 Oct, 186(19), 6586 - 94
Symbiotic autoregulation of nifA expression in Rhizobium leguminosarum bv . viciae; Martinez M et al.; NifA is the general transcriptional activator of nitrogen fixation genes in diazotrophic bacteria . In Rhizobium leguminosarum bv . viciae UPM791, the nifA gene is part of a gene cluster (orf71 orf79 fixW orf5 fixABCX nifAB) separated by 896 bp from an upstream and divergent truncated duplication of nifH (DeltanifH) . Symbiotic expression analysis of genomic nifA::lacZ fusions revealed that in strain UPM791 nifA is expressed mainly from a sigma54-dependent promoter (P(nifA1)) located upstream of orf71 . This promoter contains canonical NifA upstream activating sequences located 91 bp from the transcription initiation site . The transcript initiated in P(nifA1) spans 5.1 kb and includes nifA and nifB genes . NifA from Klebsiella pneumoniae was able to activate transcription from P(nifA1) in a heterologous Escherichia coli system . In R . leguminosarum, the P(nifA1) promoter is essential for effective nitrogen fixation in symbiosis with peas . In its absence, partially efficient nitrogen-fixing nodules were produced, and the corresponding bacteroids exhibited only low levels of nifA gene expression . The basal level of nifA expression resulted from a promoter activity originating upstream of the fixX-nifA intergenic region and probably from an incomplete duplication of P(nifA1) located immediately upstream of fixA.

FEMS Microbiol Rev, 2004 Oct, 28(4), 489 - 501
Dicarboxylate transport by rhizobia; Yurgel SN et al.; Soil bacteria collectively known as rhizobia are able to convert atmospheric dinitrogen to ammonia while participating in a symbiotic association with legume plants . This capability has made the bacteria an attractive research subject at many levels of investigation, especially since physiological and metabolic specialization are central to this ecological niche . Dicarboxylate transport plays an important role in the operation of an effective, nitrogen-fixing symbiosis and considerable evidence suggests that dicarboxylates are a major energy and carbon source for the nitrogen-fixing rhizobia . The dicarboxylate transport (Dct) system responsible for importing these compounds generally consists of a dicarboxylate carrier protein, DctA, and a two component kinase regulatory system, DctB/DctD . DctA and DctB/D differ in the substrates that they recognize and a model for substrate recognition by DctA and DctB is discussed . In some rhizobia, DctA expression can be induced during symbiosis in the absence of DctB/DctD by an alternative, uncharacterized, mechanism . The DctA protein belongs to a subgroup of the glutamate transporter family now thought to have an unusual structure that combines aspects of permeases and ion channels . While the structure of C(4)-dicarboxylate transporters has not been analyzed in detail, mutagenesis of S . meliloti DctA has produced results consistent with the alignment of the rhizobial protein with the more characterized bacterial and eukaryotic glutamate transporters in this family.

Chemistry, 2004 Oct 4, 10(19), 4798 - 807
Synthesis and biological evaluation of a lipid a derivative that contains an aminogluconate moiety; Santhanam B et al.; A highly convergent strategy for the synthesis of several derivatives of the lipid A of Rhizobium sin-1 has been developed . The synthetic derivatives are 2-aminogluconate 3 and 2-aminogluconolactone 4, both of which lack C-3 acylation . These derivatives were obtained by the preparation of disaccharides in which the two amino groups and the C-3' hydroxy group could be modified individually with acyl or beta-hydroxy fatty acyl groups . Detailed NMR spectroscopy and MS analysis of 3 and 4 revealed that, even under neutral conditions, the two compounds equilibrate . The synthetic compounds lack the proinflammatory effects of Escherichia coli lipopolysaccharide (LPS), as indicated by an absence of tumor necrosis factor production . Although 3 and 4 were able to antagonize E . coli LPS, they were significantly less potent than the synthetic compound 2, which is acylated at C-3, and R . sin-1 LPS; these results indicate that the beta-hydroxy fatty acyl group at C-3 contributes to the antagonistic properties of R . sin-1 LPS. , , , . Based on a comparison of the biological responses of the synthetic lipid A derivatives with those of the R . sin-1 LPS and lipid A, the 3-deoxy-D-manno-octulosonic moieties appear to be important for the optimal antagonization of enteric LPS-induced cytokine production.

Syst Appl Microbiol, 2004 Aug, 27(4), 469 - 77
Identification of fast-growing rhizobia nodulating tropical legumes from Puerto Rico as Rhizobium gallicum and Rhizobium tropici; Zurdo-Pineiro JL et al.; Fifteen isolates from several nodulated tropical legumes from Puerto Rico (USA) were characterised by their phenotypic, molecular and symbiotic features . The identification of isolates was based on a polyphasic approach, including phenotypic characteristics, 16S rRNA sequencing, Low molecular weight (LMW) RNA profiles, Two Primers-RAPD patterns, and restriction patterns from 16S rDNA molecules . Despite of the variety of hosts included in this study the 15 isolates were separated into only two groups that corresponded to Rhizobium gallicum and Rhizobium tropici . This work shows that R . gallicum and R . tropici nodulate legume plants, such as Sesbania, Caliandra, Poitea, Piptadenia, Neptunia and Mimosa species, that were not previously considered as hosts for these rhizobia . Moreover, some of these host plants can be nodulated by both species . The results confirm the great promiscuity of R . tropici and also support the hypothesis that the species R . gallicum may be native from America or cosmopolitan and worldwide spread.

J Exp Bot, 2004 Dec, 55(408), 2635 - 40 Epub 2004 Sep 10.
Nod factor-treated Medicago truncatula roots and seeds show an increased number of nodules when inoculated with a limiting population of Sinorhizobium meliloti; Macchiavelli RE et al.; Medicago truncatula is a model legume plant that interacts symbiotically with Sinorhizobium meliloti, the alfalfa symbiont . This process involves a molecular dialogue between the bacterium and the plant . Legume roots exude flavonoids that induce the expression of a set of rhizobial genes, the nod genes, which are essential for nodulation and determination of the host range . In turn, nod genes control the synthesis of lipo-chito-oligosaccharides (LCOs), Nod factors, which are bacteria-to-plant signal molecules mediating recognition and nodule organogenesis . M . truncatula roots or seeds have been treated with Nod factors and hydroponically growing seedlings have been inoculated with a limiting population of S . meliloti . It has been shown that submicromolar concentrations of Nod factors increase the number of nodules per plant on M . truncatula . Compared with roots, this increase is more noticeable when seeds are treated . M . truncatula seeds are receptive to submicromolar concentrations of Nod factors, suggesting the possibility of a high affinity LCO perception system in seeds or embryos as well.

J Exp Bot, 2004 Dec, 55(408), 2641 - 6 Epub 2004 Sep 10.
Perception of Bradyrhizobium japonicum Nod factor by soybean {Glycine max (L.) Merr.} root hairs under abiotic stress conditions; Duzan HM et al.; Suboptimal growth conditions, such as low rhizosphere temperature, high salinity, and low pH can negatively affect the rhizobia-legume symbioses, resulting in poor nodulation and lower amounts of nitrogen fixed . Early stages of the Bradyrhizobium japonicum-soybean {Glycine max (L.) Merr.} symbiosis, such as excretion of genistein (the plant-to-bacteria signal) and infection initiation can be inhibited by abiotic stresses; however, the effect on early events modulated by Nod factors (bacteria-to-plant signalling), particularly root hair deformations is unknown . Thus, the objective of this study was to evaluate the perception of Nod factor by soybean root hairs under three stress conditions: low temperature, low pH, and high salinity . Three experiments were conducted using a 1:1 ratio of Nod Bj-V (C(18:1), MeFuc) and Nod Bj-V (Ac, C(16:0), MeFuc) . Nod factor induced four types of root hair deformation (HAD), wiggling, bulging, curling, and branching . Under optimal experimental conditions root hair response to the three levels of Nod factor tested (10(-6), 10(-8), and 10(-10) M) was dose-dependent . The highest frequency of root hair deformations was elicited by the 10(-6) M level . Root hair deformation decreased with temperature (25, 17, and 15 degrees C), low pH, and high salinity . Nod factor concentration did not interact with either low temperature or pH . However, salinity strongly inhibited HAD responses to increases in Nod factor concentration . Thus, the addition of higher levels of Nod factor is able to overcome the effects of low pH and temperature stress, but not salinity.

Glycobiology, 2005 Jan, 15(1), 101 - 8 Epub 2004 Sep 08.
Sinorhizobium meliloti strain 1021 produces a low-molecular-mass capsular polysaccharide that is a homopolymer of 3-deoxy-D-manno-oct-2-ulosonic acid harboring a phospholipid anchor; Fraysse N et al.; Sinorhizobium meliloti strain 1021 possesses the particularity to synthesize biologically inefficient capsular polysaccharides (KPS) . It has been assumed that this class of compounds is not produced in high-molecular-mass (HMM) forms, even if many genetic analyses show the existence of expression of genes involved in the biosynthesis of capsular polysaccharides . The expression of these genes that are involved in the export of a KPS throughout the membrane and in the attachment of a lipid moiety has never been related to a structurally characterized surface polysaccharide . It is now reported that S . meliloti strain 1021 produces low-molecular-mass polysaccharides (4-4.5 kDa) that are exclusively composed of beta-(2-->7)-linked 3-deoxy-d-manno-oct-2-ulopyranosonic acid (Kdo) residues . These compounds are considered precursor molecules of HMM KPS, whose biosynthesis is arrested in the case of S . meliloti strain 1021 . For the first time, the phospholipid anchor of a rhizobial KPS has been found, and its structure could be partially identified-namely, a phosphoglycerol moiety bearing a hydroxy-octacosanoic acid . When compared to other rhizobial KPS (composed of dimeric hexose-Kdo-like sugar repeating units), the Kdo homopolymer described here may explain why a complementation of S . meliloti strain 1021 Exo B mutant with an effective rkpZ gene restoring an active higher KPS size does not completely lead to the fully effective nitrogen fixing phenotype.

Org Lett, 2004 Sep 16, 6(19), 3333 - 6
Preparation of a lipid a derivative that contains a 27-hydroxyoctacosanoic acid moiety; Santhanam B et al.; {structure: see text} A general synthetic strategy for long-chain omega-1 hydroxy fatty acids has been developed, which employs as a key reaction step a cross metathesis between omega-unsaturated ester and 3-butene-2-ol . The resulting lipids were used for the preparation of lipid A derivatives of Rhizobium sin-1, which have the ability to inhibit the E . coli LPS-dependent synthesis of tumor necrosis factor by human monocytes.

Electrophoresis, 2004 Aug, 25(16), 2671 - 4
Enantioseparation using sulfated cyclosophoraoses as a novel chiral additive in capillary electrophoresis; Park H et al.; Highly sulfated cyclosophoraoses (HS-Cys) were synthesized by the chemical modification of a family of neutral cyclosophoraoses isolated from Rhizobium leguminosarum bv . trifolii . The HS-Cys were then analytically characterized using Fourier transform-infrared spectroscopy and elemental analysis . These HS-Cys were successfully used as a novel chiral additive, in low-pH aqueous background electrolytes, for capillary electrophoretic separation of five basic chiral drugs such as arterenol, atenolol, isoproterenol, propranolol, and metoprolol.

Arch Microbiol, 2004 Oct, 182(4), 331 - 6 Epub 2004 Sep 03.
Complexity of phenotypes and symbiotic behaviour of Rhizobium leguminosarum biovar trifolii exopolysaccharide mutants; Wielbo J et al.; Rhizobium leguminosarum biovar trifolii strain TA1 polysaccharide synthesis (pss) mutants in the pssD, pssP, pssT and pssO genes and altered in exopolysaccharide (EPS) synthesis were investigated . EPS-deficient mutants were also changed in lipopolysaccharide structure . All mutants exhibited varied sensitivities to detergents, ethanol and antibiotics, thus indicating changes in bacterial membrane integrity . Using pss mutants marked with the gusA gene, EPS-deficient mutants were found to have abnormalities in nodule development and to provoke severe plant defence reactions . The pss mutants that produced altered quantities of EPS with a changed degree of polymerisation generally occupied the younger developmental zones of the nodules and elicited moderate plant defence reactions .

J Bacteriol, 2004 Sep, 186(18), 5988 - 96
The Sinorhizobium meliloti ABC transporter Cho is highly specific for choline and expressed in bacteroids from Medicago sativa nodules; Dupont L et al.; In Sinorhizobium meliloti, choline is the direct precursor of phosphatidylcholine, a major lipid membrane component in the Rhizobiaceae family, and glycine betaine, an important osmoprotectant . Moreover, choline is an efficient energy source which supports growth . Using a PCR strategy, we identified three chromosomal genes (choXWV) which encode components of an ABC transporter: ChoX (binding protein), ChoW (permease), and ChoV (ATPase) . Whereas the best homology scores were obtained with components of betaine ProU-like systems, Cho is not involved in betaine transport . Site-directed mutagenesis of choX strongly reduced (60 to 75%) the choline uptake activity, and purification of ChoX, together with analysis of the ligand-binding specificity, showed that ChoX binds choline with a high affinity (KD, 2.7 microM) and acetylcholine with a low affinity (KD, 145 microM) but binds none of the betaines . Uptake competition experiments also revealed that ectoine, various betaines, and choline derivatives were not effective competitors for Cho-mediated choline transport . Thus, Cho is a highly specific high-affinity choline transporter . Choline transport activity and ChoX expression were induced by choline but not by salt stress . Western blotting experiments with antibodies raised against ChoX demonstrated the presence of ChoX in bacteroids isolated from nitrogen-fixing nodules obtained from Medicago sativa roots . The choX mutation did not have an effect on growth under standard conditions, and neither Nod nor Fix phenotypes were impaired in the mutant, suggesting that the remaining choline uptake system(s) still present in the mutant strain can compensate for the lack of Cho transporter.

Proc Natl Acad Sci U S A, 2004 Sep 14, 101(37), 13548 - 53 Epub 2004 Aug 31.
Analysis of Rhizobium etli and of its symbiosis with wild Phaseolus vulgaris supports coevolution in centers of host diversification; Aguilar OM et al.; Common beans (Phaseolus vulgaris) comprise three major geographic genetic pools, one in Mexico, Central America, and Colombia, another in the southern Andes, and a third in Ecuador and northern Peru . Species Rhizobium etli is the predominant rhizobia found symbiotically associated with beans in the Americas . We have found polymorphism in the common nodulation gene nodC among R . etli strains from a wide range of geographical origins, which disclosed three nodC types . The different nodC alleles in American strains show varying predominance in their regional distributions in correlation with the centers of bean genetic diversification (BD centers) . By cross-inoculating wild common beans from the three BD centers with soils from Mexico, Ecuador, Bolivia, and Northwestern Argentina, the R . etli populations from nodules originated from Mexican soil again showed allele predominance that was opposite to those originated from Bolivian and Argentinean soil, whereas populations from Ecuadorian soil were intermediate . These results also indicated that the preferential nodulation of beans by geographically related R . etli lineages was independent of the nodulating environment . Coinoculation of wild common beans from each of the three BD centers with an equicellular mixture of R . etli strains representative of the Mesoamerican and southern Andean lineages revealed a host-dependent distinct competitiveness: beans from the Mesoamerican genetic pool were almost exclusively nodulated by strains from their host region, whereas nodules of beans from the southern Andes were largely occupied by the geographically cognate R . etli lineages . These results suggest coevolution in the centers of host genetic diversification.

Curr Opin Plant Biol, 2004 Oct, 7(5), 547 - 52
NPR1, all things considered; Dong X; Recent work has shown that the Arabidopsis NPR1 protein not only plays an essential role in salicylic acid (SA)-mediated systemic acquired resistance and rhizobacterium-triggered induced systemic resistance, but also is involved in crosstalk inhibition of jasmonic acid (JA)-mediated defense responses . Molecular characterization has revealed that activation of NPR1 and certain TGA transcription factors occurs under the reducing conditions that follow an initial oxidative burst after the induction of defense responses . In addition to NPR1 and TGA, the single-stranded DNA-binding transcription factor AtWhy1 and the WRKY70 transcription factor were recently found to be involved in SA-mediated defense and SA-JA crosstalk, respectively.

FEMS Microbiol Lett, 2004 Sep 1, 238(1), 17 - 22
The interaction of Vatairea macrocarca and Rhizobium tropici: net H+ efflux stimulus and alteration of extracellular Na+ concentration; Martinez CR et al.; We studied the effect of a lectin isolated from seeds of the legume Vatairea macrocarpa on net H+ efflux in Rhizobium tropici, a bacterium capable of nodulating legume Phaseolus vulgaris . V . macrocarpa lectin (VML) was observed to temporarily stimulate the specific net H(+) efflux in R . tropici . When VML was present at 32 microg ml(-1), with or without 2 microM galactose (Gal), a specific net efflux >2.4 pM H+(min)(-1) mg dry biomass(-1) was induced . There was no detectable net H+ efflux when bovine serum albumin (16 microg ml(-1)) was tested . Addition of 16 microgVMLml(-1) resulted in a 700% increase of the extracellular Na+ concentration . The soluble proteins in the supernatant containing VML extract indicate a maximum immobilization of +/-10 microgVMLml(-1), with a minimum of 36,600 dimers or 8500 larger aggregates of VML binding in each bacterium . Our data suggest that VML activates Rhizobium as a bioenergetic substrate molecule, resulting in potential alterations of the external bacterial membrane.

Acta Microbiol Pol, 2004, 53(1), 53 - 60
Possible roles of nitrogen fixation and mineral uptake induced by rhizobacterial inoculation on salt tolerance of maize; El-Komy HM et al.; Pot experiments were conducted to evaluate the possible roles of nitrogen fixation and/or enhanced mineral uptake by Azospirillum lipoferum and Bacillus polymexa inoculation in improving salt tolerance of maize plants . Plants were inoculated and grown under salt stress (osmotic potential: -0.3, -0.6, -0.9 and -1.2 Mpa) . Both microorganisms were able to fix nitrogen up to -0.9 Mpa salinity level accompanied with increased total N-yield compared with the control plants . In order to investigate the role of bacterial inoculation on enhanced mineral uptake, the growth and some physiological parameters of inoculated plants were compared with plants fertilized by K and P foliar application . Plant inoculation with the N2-fixers or plant spraying with KH2PO4 resulted in an increase in fresh and dry matter as well as water content of plants . Treated plants exhibited changed plant mineral content which was associated with increased Mg/K and decreased P/K, Ca/K and Na/K ratios . This was accompanied by accumulation of soluble sugars, amino acids in shoots and roots of plants resulting in a concomitant increase in the osmotic potential of the cell sap as a possible mechanism of adaptation to salinity.

J Microbiol Methods, 2004 Oct, 59(1), 81 - 9
Application of a new PCR primer for terminal restriction fragment length polymorphism analysis of the bacterial communities in plant roots; Sakai M et al.; Contamination with plastid small subunit (SSU) rDNA is a major drawback when analyzing the bacterial communities of plant roots using culture-independent methods . In this study, a polymerase chain reaction (PCR) primer, 783r, was designed and tested to specifically amplify the SSU rDNA of various bacterial species without amplifying the SSU rDNA of plant plastids . To confirm how useful the community analysis of rhizobacteria is using 783r, the terminal restriction fragment length polymorphism (T-RFLP) method was performed with wheat (Triticum aestivum) and spinach (Spinacea oleracea) root samples . Using the standard T-RFLP method, a large T-RF peak of plant plastid SSU rDNA interfered with the bacterial community analysis . In contrast, the T-RFLP method using the 783r primer was able to detect the bacterial DNA while directly eliminating the influence of the plant-derived DNA extracted from the plant roots . Primer 783r might, therefore, be a useful PCR primer for the culture-independent analysis of bacterial communities in plant roots using SSU rDNA.

FEMS Microbiol Lett, 2004 Aug 15, 237(2), 399 - 405
Molecular and functional characterization of the Azorhizobium caulinodans ORS571 hydrogenase gene cluster; Baginsky C et al.; In this work, we report the cloning and sequencing of the Azorhizobium caulinodans ORS571 hydrogenase gene cluster . Sequence analysis revealed the presence of 20 open reading frames hupTUVhypFhupSLCDFGHJK hypABhupRhypCDEhupE . The physical and genetic organization of A . caulinodans ORS571 hydrogenase system suggests a close relatedness to that of Rhodobacter capsulatus . In contrast to the latter species, a gene homologous to Rhizobium leguminosarum hupE was identified downstream of the hyp operon . A hupSL mutation drastically reduced the high levels of hydrogenase activity induced by the A . caulinodans ORS571 wild-type strain in symbiosis with Sesbania rostrata plants . However, no significant effects on dry weight and nitrogen content of S . rostrata plants inoculated with the hupSL mutant were observed in plant growth experiments.

FEMS Microbiol Lett, 2004 Aug 15, 237(2), 195 - 203
Arabinose content of extracellular polysaccharide plays a role in cell aggregation of Azospirillum brasilense; Bahat-Samet E et al.; Extracellular polysaccharides play an important role in aggregation and surface colonization of plant-associated bacteria . In this work, we report the time course production and monomer composition of the exopolysaccharide (EPS) produced by wild type strain and several mutants of the plant growth promoting rhizobacterium (PGPR) Azospirillum brasilense . In a fructose synthetic medium, wild type strain Sp7 produced a glucose-rich EPS during exponential phase growth and an arabinose-rich EPS during stationary and death phase growth . D-glucose or L-arabinose did not support cell growth as sole carbon sources . However, glucose and arabinose-rich EPSs, when used as carbon source, supported bacterial growth . Cell aggregation of Sp7 correlated with the synthesis of arabinose-rich EPS . exoB (UDP-glucose 4'-epimerase), exoC (phosphomannomutase) and phbC (poly-beta-hydroxyburyrate synthase) mutant strains, under tested conditions, produced arabinose-rich EPS and exhibited highly cell aggregation capability . A mutant defective in LPS production (dTDP 4-rhamnose reductase; rmlD) produced glucose-rich EPS and did not aggregate . These results support that arabinose content of EPS plays an important role in cell aggregation. , , . Cell aggregation appears to be a time course phenomenon that takes place during reduced metabolic cell activity . Thus, aggregation could constitute a protected model of growth that allows survival in a hostile environment . The occurrence of exoC and rmlD was detected in several species of Azospirillum.

FEMS Microbiol Lett, 2004 Aug 15, 237(2), 187 - 93
Lifestyle alternatives for rhizobia: mutualism, parasitism, and forgoing symbiosis; Denison RF et al.; Strains of rhizobia within a single species can have three different genetically determined strategies . Mutualistic rhizobia provide their legume hosts with nitrogen . Parasitic rhizobia infect legumes, but fix little or no nitrogen . Nonsymbiotic strains are unable to infect legumes at all . Why have rhizobium strains with one of these three strategies not displaced the others? A symbiotic (mutualistic or parasitic) rhizobium that succeeds in founding a nodule may produce many millions of descendants . The chances of success can be so low, however, that nonsymbiotic rhizobia can have greater reproductive success . Legume sanctions against nodules that fix little or no nitrogen favor more mutualistic strains, but parasitic strains that use plant resources only for their own reproduction may do well when they share nodules with mutualistic strains.

J Bacteriol, 2004 Sep, 186(17), 5753 - 61
Identification of functional mob regions in Rhizobium etli: evidence for self-transmissibility of the symbiotic plasmid pRetCFN42d; Perez-Mendoza D et al.; An approach originally designed to identify functional origins of conjugative transfer (oriT or mob) in a bacterial genome (J . A . Herrera-Cervera, J . M . Sanjuan-Pinilla, J . Olivares, and J . Sanjuan, J . Bacteriol . 180:4583-4590, 1998) was modified to improve its reliability and prevent selection of undesired false mob clones . By following this modified approach, we were able to identify two functional mob regions in the genome of Rhizobium etli CFN42 . One corresponds to the recently characterized transfer region of the nonsymbiotic, self-transmissible plasmid pRetCFN42a (C . Tun-Garrido, P . Bustos, V . Gonzalez, and S . Brom, J . Bacteriol . 185:1681-1692, 2003), whereas the second mob region belongs to the symbiotic plasmid pRetCFN42d . The new transfer region identified contains a putative oriT and a typical conjugative (tra) gene cluster organization . Although pRetCFN42d had not previously been shown to be self-transmissible, mobilization of cosmids containing this tra region required the presence of a wild-type pRetCFN42d in the donor cell; the presence of multiple copies of this mob region in CFN42 also promoted conjugal transfer of the Sym plasmid pRetCFN42d . The overexpression of a small open reading frame, named yp028, located downstream of the putative relaxase gene traA, appeared to be responsible for promoting the conjugal transfer of the R . etli pSym under laboratory conditions . This yp028-dependent conjugal transfer required a wild-type pRetCFN42d traA gene . Our results suggest for the first time that the R . etli symbiotic plasmid is self-transmissible and that its transfer is subject to regulation . In wild-type CFN42, pRetCFN42d tra gene expression appears to be insufficient to promote plasmid transfer under standard laboratory conditions; gene yp028 may play some role in the activation of conjugal transfer in response to as-yet-unknown environmental conditions.

Mikrobiologiia, 2004 May-Jun, 73(3), 416 - 22
{Functional activity of exoglycans from Rhizobium leguminosarum bv . viciae 250a and its nitrogen-resistant mutant M-71 during the formation of legume-rhizobia symbiosis against a high-nitrogen background}; Kosenko LV et al.; The functional activity of the exoglycan complex (EGC) polysaccharides from Rhizobium leguminosarum bv . viciae 250a and its nitrogen-resistant mutant M-71 capable of inducing the formation of nitrogen-fixing nodules on pea roots against a high-nitrogen background (4.8 mM NO3-) was studied in vegetation tests . For this purpose, the bacterial inoculum washed free of its own exoglycans was supplemented with EGC of this or another strain grown in the presence of 6 or 20 mM nitrate . The best symbiotic characteristics (nodule number and nitrogenase activity, mass of the roots and aerial parts of plants) were recorded when the inoculum cells and exoglycans were obtained from strain M-71 grown in the presence of 20 mM nitrate . When the plants were inoculated with the cells (grown at 6 mM nitrate) + EGC (obtained at 6 mM nitrate) of this strain, the nodulation characteristics and the effectiveness of symbiosis decreased 1.5-2-fold . Partial recovery of the symbiotic potential of strain M-71 was observed when EGC (obtained at 20 mM nitrate) was substituted for its exoglycans (obtained at 6 mM nitrate) . In the presence of exoglycans of the parent strain 250a (obtained at 6 or 20 mM nitrate), the mutant formed a substantially lesser number of nodules with a very low nitrogen-fixing activity . In turn, the mutant exoglycans synthesized in medium with either high or low nitrate nitrogen concentration did not recover the fix+ phenotype of strain 250a capable of forming symbiosis with pea plants only against a low-nitrogen background . When studying the relative content of high-molecular-weight exopolysaccharide components and low-molecular-weight glycans in the exoglycan complex, it was established that, in strain 250a (grown at 6 and 20 mM nitrate), as well as in its mutant M-71 (grown at 6 mM nitrate), exopolysaccharides prevailed, accounting for 72-75% of the sum of both types of glycopolymers, while low-molecular-weight glycans accounted for 25-28% . In contrast, in the EGC of strain M-71 obtained at 20 mM nitrate, which was the most active inducer of the formation of the symbiotrophic system by strain M-71 in the presence of a high mineral nitrogen concentration, low-molecular-weight glycans were the main component, accounting for 61% of total glycopolymers, while the polysaccharide content was 39% . Low-molecular-weight exoglycans are supposed to be involved in maintaining the physiological activity and the symbiotic status of rhizobia under unfavorable environmental conditions.

Plant Cell Physiol, 2004 Jul, 45(7), 914 - 22
Control of nodule number by the phytohormone abscisic Acid in the roots of two leguminous species; Suzuki A et al.; The effects of the phytohormone abscisic acid (ABA) on plant growth and root nodule formation were analyzed in Trifolium repense (white clover) and Lotus japonicus, which form indeterminate and determinate nodules, respectively . In T . repense, although the number of nodules formed after inoculation with Rhizobium leguminosarum bv . trifolii strain 4S (wild type) was slightly affected by exogenous ABA, those formed by strain H1(pC4S8), which forms ineffective nodules, were dramatically reduced 28 days after inoculation (DAI) . At 14 and 21 DAI, the number of nodules formed with the wild-type strain was decreased by exogenous ABA . In L . japonicus, the number of nodules was also reduced by ABA treatment . Thus, exogenous ABA inhibits root nodule formation after inoculation with rhizobia . Observation of root hair deformation revealed that ABA blocked the step between root hair swelling and curling . When the ABA concentration in plants was decreased by using abamine, a specific inhibitor of