Rhizobacterium

Phytochemistry, 2004 Jun, 65(11), 1651 - 9
Impact of sewage sludges on Medicago truncatula symbiotic proteome; Bestel-Corre G et al.; The effects of sewage sludges were investigated on the symbiotic interactions between the model plant Medicago truncatula and the arbuscular mycorrhizal fungus Glomus mosseae or the rhizobial bacteria Sinorhizobium meliloti . By comparison to a control sludge showing positive effects on plant growth and root symbioses, sludges enriched with polycylic aromatic hydrocarbons or heavy metals were deleterious . Symbiosis-related proteins were detected and identified by two-dimensional electrophoresis and matrix-assisted laser desorption ionization mass spectrometry, and image analysis was used to study the effects of sewage sludges on M . truncatula symbiotic proteome.

Plant J, 2004 Aug, 39(4), 587 - 98
From pollen tubes to infection threads: recruitment of Medicago floral pectic genes for symbiosis; Rodriguez-Llorente ID et al.; While the biology of nitrogen-fixing root nodules has been extensively studied, little is known about the evolutionary events that predisposed legume plants to form symbiosis with rhizobia . We have studied the presence and the expression of two pectic gene families in Medicago, polygalacturonases (PGs) and pectin methyl esterases (PMEs) during the early steps of the Sinorhizobium meliloti-Medicago interaction and compared them with related pollen-specific genes . First, we have compared the expression of MsPG3, a PG gene specifically expressed during the symbiotic interaction, with the expression of MsPG11, a highly homologous pollen-specific gene, using promoter-gus fusions in transgenic M . truncatula and tobacco plants . These results demonstrated that the symbiotic promoter functions as a pollen-specific promoter in the non-legume host . Second, we have identified the presence of a gene family of at least eight differentially expressed PMEs in Medicago . One subfamily is represented by one symbiotic gene (MtPER) and two pollen-expressed genes (MtPEF1 and MtPEF2) that are clustered in the M . truncatula genome . The promoter-gus studies presented in this work and the homology between plant PGs, together with the analysis of the PME locus structure and MtPER expression studies, suggest that the symbiotic MsPG3 and MtPER could have as ancestors pollen-expressed genes involved in polar tip growth processes during pollen tube elongation . Moreover, they could have been recruited after gene duplication in the symbiotic interaction to facilitate polar tip growth during infection thread formation.

J Environ Sci (China), 2004, 16(3), 458 - 61
Effect of inoculation with arbuscular mycorrhizal fungi on the degradation of DEHP in soil; Wang SG et al.; The effect of inoculation with arbuscular mycorrhizal (AM) fungi (Acaulospora lavis) on the degradation of di(2-ethylhexyl) phthalate (DEHP) in soil was studies . Cowpea plants (Pigna sinensis) were used as host plants and grown in a specially designed rhizobox . The experimental results indicated that, both in sterile and non-sterile soil, mycorrhizal colonization rates were much higher in the mycorrhizal plants than in the non-mycorrhizal plants . Addition of 4 mg/kg DEHP slightly affected mycorrhizal colonization, but the addition of 100 mg/kg DEHP significantly decreased mycorrhizal colonization . DEHP degradation in the mycorrhizosphere (Ms) and hyphosphere (Hs), especially in the Hs, increased after inoculation with Acaulospora lavis . It is concluded that mycorrhizal hyphae play an important role in the plant uptake, degradation and translocation of DEHP . The mechanism might be attributed to increased numbers of bacteria and actinomycetes and activity of dehydrogenase, urease and acid phosphatase in the Ms and Hs by mycorrhizal fungi.

Chemosphere, 2004 Sep, 56(10), 957 - 65
Implications of rhizospheric heavy metals and nutrients for the growth of alfalfa in sludge amended soil; Su DC et al.; Rhizospheric distribution of nutrients and heavy metals in sludge amended soil was investigated using the rhizobag technique to give an indication of the release of metals from wastewater sludge . DTPA-extractable Zn, Cd, Ni and Mn, and available P, K and NH4+-N in the rhizosphere were markedly depleted when soil was amended with sludge . There was no conspicuous depletion or accumulation of DTPA-extractable Cu in the rhizosphere when the soil was amended with sewage sludge but DTPA-extractable Fe accumulated in the rhizosphere when the soil was amended with increasing amounts of sludge . The pH value in the rhizosphere increased with distance from the roots when soil was amended with larger amounts of sludge . The exchangeable fraction of Cu in the rhizosphere was depleted whether or not the soil was treated with sludge . Carbonate, oxide, organic and residual fractions of Cu and Zn were depleted in the rhizosphere at a distance of 0-2 mm from the roots when soil was amended with 50% sludge . Application of sewage sludge had a positive effect on alfalfa growth . With an increase in sludge amounts, the concentrations of Fe, Cu and Zn in alfalfa shoots did not change . Soil amendments with less than 25% sludge did not increase the availability or mobility of heavy metals . The depletion in rhizospheric DTPA-extractable Zn, Cd and Ni indicates that with the sole exception of Cu, release of metals from sludge amended soil was very limited.

Indian J Exp Biol, 2003 Jun, 41(6), 627 - 31
Rhizoctonia wilt suppression of brinjal (Solanum melongena L) and plant growth activity by Bacillus BS2; Boruah HP et al.; An antibiotic-producing and hydrogen-cyanide-producing rhizobacteria strain Bacillus BS2 showed a wide range of antifungal activity against many Fusarium sp . and brinjal wilt disease pathogen Rhizoctonia solani . Seed bacterization with the strain BS2 promoted seed germination and plant growth in leguminous plants Phaseolus vulgaris and non-leguminous plants Solanum melongena L, Brassica oleracea var . capitata, B . oleraceae var . gongylodes and Lycopersicon esculentum Mill in terms of relative growth rate, shoot height, root length, total biomass production and total chlorophyll content of leaves . Yield of bacterized plants were increased by 10 to 49% compared to uninoculated control plants . Brinjal sapling raised through seed bacterization by the strain BS2 showed a significantly reduced wilt syndrome of brinjal caused by Rhizoctonia solani . Control of wilt disease by the bacterium was clue to the production of antibiotic-like substances, whereas plant growth-promotion was due to the activity of hydrogen cyanide . Root colonization study confirmed that the introduced bacteria colonized the roots and occupied 23-25% of total aerobic bacteria, which was confirmed using dual antibiotic (nalidixic acid and streptomycin sulphate) resistant mutant strain . The results obtained through this investigation suggested the potentiality of the strain BS2 to be used as a plant growth promoter and suppressor of wilt pathogen.

J Mol Microbiol Biotechnol, 2004, 7(3), 133 - 9
Probing for pH-regulated genes in Sinorhizobium medicae using transcriptional analysis; Tiwari RP et al.; The low pH sensitivity of Sinorhizobium species is one of the major causes of reduced productivity of Medicago species (such as lucerne) sown in acidic soils . To investigate the pH response of an acid-tolerant Sinorhizobium medicae strain, a pool of random promoter fusions to gusA was created using minitransposon insertional mutagenesis . Acid-activated expression was identified in 11 mutants; rhizobial DNA flanking insertions in 10 mutants could be cloned and the DNA sequences obtained were used to interrogate the genome database of Sinorhizobium meliloti strain 1021 . Acid activated expression was detected for fixNO, kdpC, lpiA, and phrR and for genes encoding a putative lipoprotein, two ABC-transporter components, a putative DNA ligase and a MPA1-family protein . These findings implicate cytochrome synthesis, potassium ion cycling, lipid biosynthesis and transport processes as key components of pH response in S . medicae .

Indian J Exp Biol, 2004 Jun, 42(6), 611 - 5
Nodulation competitiveness between contrasting phage phenotypes of pigeonpea rhizobial strains; Mishra A et al.; Competitiveness between (I) lysogenic vs . phage-indicator strains, (II) phage-resistant vs phage-sensitive strains, and (III) large plaque vs . small plaque developing strains was examined under laboratory and field conditions in order to study the involvement of these crucial phage sensitivity patterns in the competition for nodule occupancy of pigeonpea rhizobia . The phage-indicator strain (A039) exhibited higher competitiveness over the lysogenic strain (A025 Sm(r)); the phage sensitive strain (IHP-195) over the phage resistant strain (IHP 195 Sm(r)V(r)); and the large plaque developing strain (A059) over the small plaque developing strain (IHP195 Sm(r)) in association with pigeonpea cv . bahar both under laboratory and field conditions . Dual inoculation of A025 Sm(r) + A039 and A059 + IHP195 Sm(r) (mixed in equal proportion just before treatment) improved the nodule occupancy by inoculant strains against native rhizobia and resulted into higher plant dry weight and yield as compared to their application as single inoculum . The phage-resistant mutant IHP195 Sm(r)V(r) showed reduced competitiveness against native rhizobia, compared to its parental strain . The dual inoculation of parental strain and phage-resistant mutant gave the same result as the inoculation of parental strain alone.

Biometals, 2004 Aug, 17(4), 457 - 66
Monitoring of cobalt(II) uptake and transformation in cells of the plant-associated soil bacterium Azospirillum brasilense using emission Mössbauer spectroscopy; Kamnev AA et al.; Interaction of cobalt(II) at micromolar concentrations with live cells of the plant-growth-promoting rhizobacterium Azospirillum brasilense (strain Sp245) and further transformations of the metal cation were monitored using 57Co emission Mossbauer spectroscopy (EMS) . Cell suspensions of the bacterial culture (2.4 x 10(8) cells ml(-1)) were doped with radioactive 57CoCl2 (1 mCi; final concentration 2 x 10(-6) M 57Co2+), kept under physiological conditions for various periods of time (from 2 min up to 1 hour) and then rapidly frozen in liquid nitrogen . Analysis of emission Mossbauer spectra of the frozen aqueous suspensions of the bacterial cell samples shows that the primary absorption of cobalt(II) at micromolar concentrations by the bacterial cells is rapid and virtually complete, giving at least two major forms of cobalt(II) species bound to the cells . Within an hour, the metal is involved in further metabolic transformations reflected by changes occurring in the spectra . The Mossbauer parameters calculated from the EMS data by statistical treatment were different for suspensions of live and dead (thermally killed) bacterial cells that had been in contact with 57Co2+ for 1 h, as well as for the cell-free culture medium containing the same concentration of 57Co2+ . Chemical after-effects of the nuclear transition (57Co --> 5 7Fe), which provide additional information on the chemical environment of metal ions, are also considered . The data presented demonstrate that EMS is a valuable tool for monitoring the chemical state of cobalt species in biological matter providing information at the atomic level in the course of its uptake and/or metabolic transformations.

Microbiology, 2004 Jul, 150(Pt 7), 2237 - 46
The nitrogen-fixing gene (nifH) of Rhodopseudomonas palustris: a case of lateral gene transfer?
Cantera JJ, Kawasaki H, Seki T.
Nitrogen fixation is catalysed by some photosynthetic bacteria . This paper presents a phylogenetic comparison of a nitrogen fixation gene (nifH) with the aim of elucidating the processes underlying the evolutionary history of Rhodopseudomonas palustris . In the NifH phylogeny, strains of Rps . palustris were placed in close association with Rhodobacter spp . and other phototrophic purple non-sulfur bacteria belonging to the alpha-Proteobacteria, separated from its close relatives Bradyrhizobium japonicum and the phototrophic rhizobia (Bradyrhizobium spp . IRBG 2, IRBG 228, IRBG 230 and BTAi 1) as deduced from the 16S rRNA phylogeny . The close association of the strains of Rps . palustris with those of Rhodobacter and Rhodovulum, as well as Rhodospirillum rubrum, was supported by the mol% G+C of their nifH gene and by the signature sequences found in the sequence alignment . In contrast, comparison of a number of informational and operational genes common to Rps . palustris CGA009, B . japonicum USDA 110 and Rhodobacter sphaeroides 2.4.1 suggested that the genome of Rps . palustris is more related to that of B . japonicum than to the Rba . sphaeroides genome . These results strongly suggest that the nifH of Rps . palustris is highly related to those of the phototrophic purple non-sulfur bacteria included in this study, and might have come from an ancestral gene common to these phototrophic species through lateral gene transfer . Although this finding complicates the use of nifH to infer the phylogenetic relationships among the phototrophic bacteria in molecular diversity studies, it establishes a framework to resolve the origins and diversification of nitrogen fixation among the phototrophic bacteria in the alpha-Proteobacteria.

Plant J, 2004 Aug, 39(3), 381 - 92
Plant growth-promoting rhizobacteria systemically protect Arabidopsis thaliana against Cucumber mosaic virus by a salicylic acid and NPR1-independent and jasmonic acid-dependent signaling pathway; Ryu CM et al.; Arabidopsis thaliana ecotype Columbia plants (Col-0) treated with plant growth-promoting rhizobacteria (PGPR) Serattia marcescens strain 90-166 and Bacillus pumilus strain SE34 had significantly reduced symptom severity by Cucumber mosaic virus (CMV) . In some cases, CMV accumulation was also significantly reduced in systemically infected leaves . The signal transduction pathway(s) associated with induced resistance against CMV by strain 90-166 was determined using mutant strains and transgenic and mutant Arabidopsis lines . NahG plants treated with strains 90-166 and SE34 had reduced symptom severity indicating that the resistance did not require salicylic acid (SA) . Strain 90-166 naturally produces SA under iron-limited conditions . Col-0 and NahG plants treated with the SA-deficient mutant, 90-166-1441, had significantly reduced CMV symptom severity with reduced virus accumulation in Col-0 plants . Another PGPR mutant, 90-166-2882, caused reduced disease severity in Col-0 and NahG plants . In a time course study, strain 90-166 reduced virus accumulation at 7 but not at 14 and 21 days post-inoculation (dpi) on the non-inoculated leaves of Col-0 plants . NahG and npr1-1 plants treated with strain 90-166 had reduced amounts of virus at 7 and 14 dpi but not at 21 dpi . In contrast, no decrease in CMV accumulation occurred in strain 90-166-treated fad3-2 fad7-2 fad8 plants . These data indicate that the protection of Arabidopsis against CMV by strain 90-166 follows a signaling pathway for virus protection that is independent of SA and NPR1, but dependent on jasmonic acid.

Indian J Exp Biol, 2003 Apr, 41(4), 341 - 5
Associative diazotrophs of pearl millet (Pennisetum glaucum) from semi arid region--isolation and characterization; Tiwari M et al.; Diversity of the native diazotrophs associated with the rhizosphere of pearl millet (P . glaucumn), grown in nutritionally poor soils of semi-arid regions was studied with a view to isolate effective nitrogen fixing and plant growth stimulating bacteria with root associative characteristics . The native population varied from 10(3)-10(4) g(-1) of rhizosphere soil after 40 d growth and belonged to genera Azospirillum, Azotobacter and Klebsiella . Another non-diazotrophic root associative group was Pseudomonas sp., which also produced IAA and enhanced plant growth . Some of these rhizobacteria showed high in vitro acetylene reduction activity along with production of indole acetic acid . Out of 11 selected diazotrophs used as seed inoculants, M10B (Azospirillum sp.), M11E (Azotobacter sp.) and M12D4 (Klebsiella sp.) resulted in significant increase in total root and shoot nitrogen at 45 and 60 days of plant growth under pot culture conditions.

C R Biol, 2004 May, 327(5), 445 - 53
Performance of phaseolus bean rhizobia in soils from the major production sites in the Nile Delta; Moawad H et al.; The symbiotic and competitive performances of two highly effective rhizobia nodulating French bean P . vulgaris were studied in silty loam and clayey soils . The experiments were carried out to address the performance of two rhizobia strains (CE3 and Ph . 163} and the mixture thereof with the two major cultivated bean cultivars in two soil types from major growing French bean areas in Egypt . Clay and silty loam soils from Menoufia and Ismailia respectively were planted with Bronco and Giza 6 phaseolus bean cultivars . The data obtained from this study indicated that rhizobial inoculation of Giza 6 cultivar in clayey soil showed a positive response to inoculation in terms of nodule numbers and dry weight . This response was also positive in dry matter and biomass accumulation by the plants . The inoculant of strain CE3 enhanced plant growth and N-uptake relative to Ph . 163 . However, the mixed inoculant strains were not always as good as single strain inoculants . The competition for nodulation was assessed using two techniques namely fluorescent antibody testing (FA) and REP-PCR fingerprinting . The nodule occupancy by inoculant strain Ph . 163 in both soils occupied 30-40% and 38-50 of nodules of cultivar Bronco . The mixed inocula resulted in higher proportions of nodules containing CE3 in silty loam soil and Ph . 163 in clayey soil . The native rhizobia occupied at least 50% of the nodules on the Bronco cultivar . For cultivar Giza 6, the native rhizobia were more competitive with the inoculant strains . Therefore, we suggest using the studied strains as commercial inocula for phaseolus bean.

FEMS Microbiol Lett, 2004 Jul 15, 236(2), 291 - 9
Construction of a recA mutant of Azospirillum lipoferum and involvement of recA in phase variation; Vial L et al.; The plant-growth promoting rhizobacterium Azospirillum lipoferum strain 4B generates in vitro a stable phase variant designated 4VI at frequencies of 10(-4) to 10(-3) per cell per generation . Variant 4VI displays pleitropic modifications, such as the loss of swimming motility and the inability to assimilate certain sugars compared to the wild type . The mechanism underlying phase variation is unknown . To determine whether RecA-mediated processes are involved in phase variation, the recA gene of A . lipoferum 4B was cloned and sequenced and a recA mutant (termed 4BrecA) was constructed by allelic exchange . Strain 4BrecA showed increased sensitivity to UV and MMS compared with 4B and impaired recombinase activity . The ability to generate variants in vitro was not altered; the variants from 4BrecA exhibited all morphological and biochemical features characteristic of the variant generated by strain 4B . However, the frequency of variants generated by 4BrecA was increased by up to 10-fold . So, in contrast with many studies showing the abolition or a large reduction of the frequency of phase variation in recA mutants, this study describes an enhancement of phase variation in the absence of a functional recA.

Indian J Exp Biol, 2003 Aug, 41(8), 912 - 4
Curing of symbiotic plasmid of Mesorhizobium huakuii subsp . rengei isolated from Astragalus sinicus; Balachandar D et al.; Astragalus sinicus (Chinese Milk vetch), a green manure leguminous plant, harbors Mesorhizobium huakuii subsp . rengei strain B3 in the root nodules . The visualization of symbiotic plasmid of strain B3 showed the presence of one sym plasmid of about 425 kbp . Curing of sym plasmid by temperature and acrydine orange was studied . Growing rhizobial cells at high temperature (37 degrees C) or treating the cells with acrydine orange at 50 mg/l eliminated sym plasmid of M . huakuii strain B3, which was confirmed by sym plasmid visualization and plant infection test of cured strains.

Mol Ecol, 2004 Aug, 13(8), 2435 - 44
Diversity and specificity of Rhizobium leguminosarum biovar viciae on wild and cultivated legumes; Mutch LA et al.; The symbiotic partnerships between legumes and their root-nodule bacteria (rhizobia) vary widely in their degree of specificity, but the underlying reasons are not understood . To assess the potential for host-range evolution, we have investigated microheterogeneity among the shared symbionts of a group of related legume species . Host specificity and genetic diversity were characterized for a soil population of Rhizobium leguminosarum biovar viciae (Rlv) sampled using six wild Vicia and Lathyrus species and the crop plants pea (Pisum sativum) and broad bean (Vicia faba) . Genetic variation among 625 isolates was assessed by restriction fragment length polymorphism (RFLP) of loci on the chromosome (ribosomal gene spacer) and symbiosis plasmid (nodD region) . Broad bean strongly favoured a particular symbiotic genotype that formed a distinct phylogenetic subgroup of Rlv nodulation genotypes but was associated with a range of chromosomal backgrounds . Host range tests of 80 isolates demonstrated that only 34% of isolates were able to nodulate V . faba . By contrast, 89% were able to nodulate all the local wild hosts tested, so high genetic diversity of the rhizobial population cannot be ascribed directly to the diversity of host species at the site . Overall the picture is of a population of symbionts that is diversified by plasmid transfer and shared fairly indiscriminately by local wild legume hosts . The crop species are less promiscuous in their interaction with symbionts than the wild legumes .

J Appl Microbiol, 1998 Jan, 84(1), 81 - 9
Metabolic properties, stress tolerance and macromolecular profiles of rhizobia nodulating Hedysarum coronarium; Struffi P et al.; The drought-tolerant legume Hedysarum coronarium is a Mediterranean species valued as a forage crop for its high performance in stressful conditions . The plant shows peculiar capabilities of nodulating above pH 9 and thriving in highly calcareous soils . With the aim of providing an adequate characterization of its bacterial symbiotic partner, a study was undertaken, approaching from several viewpoints the physiology and structural features of bacteria isolated from nodules of H . coronarium . Tests involved trophic capabilities on different carbon and nitrogen sources, vitamin requirements, and resistance to factors including antibiotics, heavy metals, salinity, pH, and temperature . Enzyme activities, including those of cellulase, pectinase, urease, beta-galactosidase, nitrate and nitrite reductase, were evaluated . The DNA G + C percentage content was determined . Species-specific bacteriophages were isolated and a strain-typing grid established . In order to characterize further and fingerprint the different Rhizobium 'hedysari' isolates, electrophoretic pattern of proteins, plasmid DNA, and digested genomic DNA (in pulsed-field gel separation) were compared . Adansonian taxonomy yielded similarity clusters of the different isolates.

Indian J Exp Biol, 2003 Oct, 41(10), 1205 - 8
11th International Congress on Molecular Plant-Microbe Interactions held at St . Petersburg--a report; Rolfe BG et al.; The report is a short summary of the most interesting presentations at the 11th International Congress on Molecular Plant-Microbe Interactions held during July 18-27, 2003 at St . Petersburg, Russia . The key elements from several sessions on the legume-Rhizobium interactions have been discussed.

Indian J Exp Biol, 2003 Oct, 41(10), 1198 - 204
Isolation and symbiotic characterization of transposon Tn5-induced arginine auxotrophs of Sinorhizobium meliloti; Kumar A et al.; Seventeen arginine auxotrophic mutants of Sinorhizobium meliloti Rmd201 were isolated by random transposon Tn5 mutagenesis using Tn5 delivery vector pGS9 . Based on intermediate feeding studies, these mutants were designated as argA/argB/argC/argD/argE (ornithine auxotrophs), argF/argI, argG and argH mutants . The ornithine auxotrophs induced ineffective nodules whereas all other arginine auxotrophs induced fully effective nodules on alfalfa plants . In comparison to the parental strain induced nodule, only a few nodule cells infected with rhizobia were seen in the nitrogen fixation zone of the nodule induced by the ornithine auxotroph . TEM studies showed that the bacteroids in the nitrogen fixation zone of ornithine auxotroph induced nodule were mostly spherical or oval unlike the elongated bacteroids in the nitrogen fixation zone of the parental strain induced nodule . These results indicate that ornithine or an intermediate of ornithine biosynthesis, or a chemical factor derived from one of these compounds is required for the normal development of nitrogen fixation zone and transformation of rhizobial bacteria into bacteroids during symbiosis of S . meliloti with alfalfa plants.

Indian J Exp Biol, 2003 Oct, 41(10), 1184 - 97
Recent advances in Rhizobium-legume symbiosis; Randhawa GS et al.; The research findings in the field of Rhizobium-legume symbiosis reported worldwide during the years 2002 and 2003 (up to September) have been summarized . The information is presented under the various topics, viz., isolation and characterization of rhizobial strains, physiological aspects of nitrogen fixation, rhizosphere interactions and root surface signals, genomics and proteomics, plant genes involved in nodule formation, bioremediation and biocontrol, and review articles and conference reports . The postal and e-mail addresses of the concerned scientists have also been included.

Indian J Exp Biol, 2003 Oct, 41(10), 1165 - 83
Symbiosis between Frankia and actinorhizal plants: root nodules of non-legumes; Pawlowski K et al.; In actinorhizal symbioses, filamentous nitrogen-fixing soil bacteria of the genus Frankia induce the formation of nodules on the roots of a diverse group of dicotyledonous plants representing trees or woody shrubs, with one exception, Datisca glomerata . In the nodules, Frankia fixes nitrogen and exports the products to the plant cytoplasm, while being supplied with carbon sources by the host . Possibly due to the diversity of the host plants, actinorhizal nodules show considerable variability with regard to structure, oxygen protection mechanisms and physiology . Actinorhizal and legume-rhizobia symbioses are evolutionary related and share several features.

Indian J Exp Biol, 2003 Oct, 41(10), 1160 - 4
Rhizobia as a biological control agent against soil borne plant pathogenic fungi; Deshwal VK et al.; Rhizobia promote the growth of plants either directly through N2 fixation, supply of nutrients, synthesis of phytohormones and solubilization of minerals, or indirectly as a biocontrol agent by inhibiting the growth of pathogens . The biocontrol effect of rhizobia is due to the secretion of secondary metabolites such as antibiotics and HCN . Siderophore production in iron stress conditions provides rhizobia an added advantage, resulting in exclusion of pathogens due to iron starvation.

Indian J Exp Biol, 2003 Oct, 41(10), 1142 - 59
Effects of biotic and abiotic constraints on the symbiosis between rhizobia and the tropical leguminous trees Acacia and Prosopis; Rasanen LA et al.; N2-fixing, drought tolerant and multipurpose Acacia and Prosopis species are appropriate trees for reforestation of degraded areas in arid and semiarid regions of the tropics and subtropics . Acacia and Prosopis trees form N2-fixing nodules with a wide range of rhizobia, for example African acacias mainly with Sinorhizobium sp . and Mesorhizobium sp., and Australian acacias with Bradyrhizobium sp . Although dry and hot seasons restrict formation of N2-fixing nodules on Acacia and Prosopis spp., fully grown trees and their symbiotic partners are well adapted to survive in harsh growth conditions . This review on one hand deals with major constraints of arid and semiarid soils, i.e . drought, salinity and high soil temperature, which affect growth of trees and rhizobia, and on the other hand with adaptation mechanisms by which both organisms survive through unfavourable periods . In addition, defects in infection and nodulation processes due to various abiotic and biotic constraints are reviewed . This knowledge is important when Acacia and Prosopis seedlings are used for forestation of degraded areas in arid and semiarid tropics.

Indian J Exp Biol, 2003 Oct, 41(10), 1136 - 41
Effects of drought stress on legume symbiotic nitrogen fixation: physiological mechanisms; Serraj R; Drought stress is one of the major factors affecting nitrogen fixation by legume-rhizobium symbiosis . Several mechanisms have been previously reported to be involved in the physiological response of symbiotic nitrogen fixation to drought stress, i.e . carbon shortage and nodule carbon metabolism, oxygen limitation, and feedback regulation by the accumulation of N fixation products . The carbon shortage hypothesis was previously investigated by studying the combined effects of CO2 enrichment and water deficits on nodulation and N2 fixation in soybean . Under drought, in a genotype with drought tolerant N2 fixation, approximately four times the amount of 14C was allocated to nodules compared to a drought sensitive genotype . It was found that an important effect of CO2 enrichment of soybean under drought was an enhancement of photo assimilation, an increased partitioning of carbon to nodules, whose main effect was to sustain nodule growth, which helped sustain N2 rates under soil water deficits . The interaction of nodule permeability to O2 and drought stress with N2 fixation was examined in soybean nodules and led to the overall conclusion that O2 limitation seems to be involved only in the initial stages of water deficit stresses in decreasing nodule activity . The involvement of ureides in the drought response of N2 fixation was initially suspected by an increased ureide concentration in shoots and nodules under drought leading to a negative feedback response between ureides and nodule activity . Direct evidence for inhibition of nitrogenase activity by its products, ureides and amides, supported this hypothesis . The overall conclusion was that all three physiological mechanisms are important in understanding the regulation of N2 fixation and its response of to soil drying.

Indian J Exp Biol, 2003 Oct, 41(10), 1133 - 5
Proteomics: a novel approach to explore signal exchanges in Rhizobium-legume symbiosis; Vij N; Recent developments and future strategies on the proteomics approach to explore the signal exchanges in Rhizobium-legume symbiosis have been discussed . It is expected that this approach will provide new possibilities for investigating the complex interactions of rhizobia and legumes.

Indian J Exp Biol, 2003 Oct, 41(10), 1101 - 13
Regulation of expression of symbiotic genes in Rhizobium sp . NGR234; Perret X et al.; Research in the field of Rhizobium-legume symbiosis faces a new challenge: integrate the wealth of information generated by genomic projects . The goal: apprehend the complexity of the molecular mechanisms involved in symbiotic associations . At the time of writing, the genomes of three micro-symbionts (Bradyrhizobium japonicum, Mesorhizobium loti and Sinorhizobium meliloti) have been sequenced, and two more (those of Rhizobium leguminosarum and Rhizobium etli) will be completed in the near future . Together, completed rhizobial genomes represent already 23,393,822 bp of DNA sequence and 21,797 predicted open reading frames (ORFs) . To identify candidate-symbiotic genes in such a broad database, predict their function and dissect the regulatory networks that govern their expression are no simple tasks . One way to confront this problem is to combine different datasets, in particular genetic and transcriptional maps as well as predicted promoters from bioinformatics analyses . Here, we would like to illustrate this type of approach with the analysis of the symbiotic plasmid (pNGR234a) of the broad host-range Rhizobium sp . NGR234.

Indian J Exp Biol, 2003 Oct, 41(10), 1095 - 100
Milestones in the genetical research on rhizobia; Randhawa GS et al.; The first isolation of the rhizobial bacteria from the legume roots was done in 1888 . Since then a large number of scientists have made efforts to understand the molecular basis of Rhizobium-legume symbiosis . The important developments of 115 years of genetical research on rhizobia have been listed in this article.

Mol Plant Microbe Interact, 2004 Jul, 17(7), 816 - 23
Accumulation of lipochitin oligosaccharides and NodD-activating compounds in an efficient plant--Rhizobium nodulation assay; Tak T et al.; During legume plant--Rhizobium spp . interactions, leading to the formation of nitrogen-fixing root nodules, the two major determinants of host plant-specificity are plant-produced nod gene inducers (NodD protein activating compounds) and bacterial lipochitin oligosaccharides (LCOs or Nod factors) . In a time course, we describe the accumulation of LCOs in an efficient nodulation assay with Vicia sativa subsp . nigra and Rhizobium leguminosarum, in connection with the presence of NodD-activating compounds in the exudate of V . sativa roots . Relatively small amounts of both LCOs and NodD-activating compounds were found to be required for initiation of nodulation during the first days after inoculation . A strong increase in the amount of NodRlv-V{18:4,Ac} LCOs preceded root infection and nodule primordium formation . In contrast to the situation with non-nodulating rhizobia and nonmitogenic LCOs, the amount of NodD-activating compounds in the culture medium remained small after addition of nodulating rhizobia or mitogenic LCOs . Furthermore, addition of nodulating rhizobia or mitogenic LCOs resulted in nearly complete inhibition of root hair formation and elongation, whereas nonmitogenic LCOs stimulated root hair growth . Retention of NodD-activating compounds in the root may inhibit root hair growth.

Mol Plant Microbe Interact, 2004 Jul, 17(7), 720 - 8
Glutamine utilization by Rhizobium etli; Tate R et al.; We undertook the study of the use of glutamine (Gln) as the source of carbon and energy by Rhizobium etli . Tn5-induced mutagenesis allowed us to identify several genes required for Gln utilization, including those coding for two broad-range amino acid transporters and a glutamate dehydrogenase . The isolated mutants were characterized by the analysis of their capacity i) to grow on different media, ii) to transport Gln (uptake assays), and iii) to utilize Gln as the C energy source (CO2 production from Gln) . We show that Gln is degraded through the citric acid cycle and that its utilization as the sole C source is related to a change in the bacterial cell shape (from bacillary to coccoid form) and a high susceptibility to a thiol oxidative insult . Both these data and the analysis of ntr-dependent promoters suggested that Gln-grown bacteria are under a condition of C starvation and N sufficiency, and as expected, the addition of glucose counteracted the morphological change and increased both the bacterial growth rate and their resistance to oxidative stress . Finally, a nodulation analysis indicates that the genes involved in Gln transport and degradation are dispensable for the bacterial ability to induce and invade developing nodules, whereas those involved in gluconeogenesis and nucleotide biosynthesis are strictly required.

J Appl Microbiol, 2004, 97(2), 256 - 61
Degradation of trehalose by rhizobia and characteristics of a trehalose-degrading enzyme isolated from Rhizobium species NGR234; Boboye B; AIMS: This study was designed to examine the breakdown of trehalose by rhizobia and to characterize the trehalose-degrading enzyme isolated from Rhizobium sp . NGR234 . METHODS AND RESULTS: Rhizobium sp . NGR234, Rhizobium fredii USDA257, R . phaseoli RCR3622, R . tropici CIAT899 and R . etli CE3 showed good growth in the presence of carbohydrate . Validamycin A did not prevent the growth of NGR234 on trehalose . The expression of a trehalose-degrading enzyme by NGR234 was intracellular and inducible by trehalose . The isolated enzyme digested other disaccharides, p-nitrophenyl-alpha-d-glucopyranoside and the substrate . The enzyme showed optimum activities at pH 7.0 and 30 degrees C . Its pI was 4.75 and the V(max) of the enzyme occurred at 35.7 micromol s(-1) mg(-1) protein with the K(m) of 23 mmol when trehalose was hydrolysed . CONCLUSIONS: An enzyme capable of breaking down trehalose was produced . Some of the properties of the trehalose-degrading enzyme are similar to those isolated from other organisms but, this enzyme was validamycin resistant . These rhizobia like other trehalose-degrading microbes use trehalose by enzymatic catabolic action . SIGNIFICANCE AND IMPACT OF THE STUDY: Trehalose which accumulates during legume-rhizobia symbiosis is toxic to plants . Detoxification by trehalose-degrading enzymes is important for the progress of symbiosis.

Q Rev Biol, 2004 Jun, 79(2), 135 - 60
The evolution of cooperation; Sachs JL et al.; Darwin recognized that natural selection could not favor a trait in one species solely for the benefit of another species . The modern, selfish-gene view of the world suggests that cooperation between individuals, whether of the same species or different species, should be especially vulnerable to the evolution of noncooperators . Yet, cooperation is prevalent in nature both within and between species . What special circumstances or mechanisms thus favor cooperation? Currently, evolutionary biology offers a set of disparate explanations, and a general framework for this breadth of models has not emerged . Here, we offer a tripartite structure that links previously disconnected views of cooperation . We distinguish three general models by which cooperation can evolve and be maintained: (i) directed reciprocation--cooperation with individuals who give in return; (ii) shared genes--cooperation with relatives (e.g., kin selection); and (iii) byproduct benefits--cooperation as an incidental consequence of selfish action . Each general model is further subdivided . Several renowned examples of cooperation that have lacked explanation until recently--plant-rhizobium symbioses and bacteria-squid light organs--fit squarely within this framework . Natural systems of cooperation often involve more than one model, and a fruitful direction for future research is to understand how these models interact to maintain cooperation in the long term.

J Bacteriol, 2004 Jul, 186(14), 4774 - 80
Characterization of NopP, a type III secreted effector of Rhizobium sp . strain NGR234; Ausmees N et al.; The type three secretion system (TTSS) encoded by pNGR234a, the symbiotic plasmid of Rhizobium sp . strain NGR234, is responsible for the flavonoid- and NodD1-dependent secretion of nodulation outer proteins (Nops) . Abolition of secretion of all or specific Nops significantly alters the nodulation ability of NGR234 on many of its hosts . In the closely related strain Rhizobium fredii USDA257, inactivation of the TTSS modifies the host range of the mutant so that it includes the improved Glycine max variety McCall . To assess the impact of individual TTSS-secreted proteins on symbioses with legumes, various attempts were made to identify nop genes . Amino-terminal sequencing of peptides purified from gels was used to characterize NopA, NopL, and NopX, but it failed to identify SR3, a TTSS-dependent product of USDA257 . By using phage display and antibodies that recognize SR3, the corresponding protein of NGR234 was identified as NopP . NopP, like NopL, is an effector secreted by the TTSS of NGR234, and depending on the legume host, it may have a deleterious or beneficial effect on nodulation or it may have little effect .

Curr Opin Plant Biol, 2004 Aug, 7(4), 408 - 13
Genetic and genomic analysis in model legumes bring Nod-factor signaling to center stage; Riely BK et al.; The control of host-specificity in the Rhizobium-legume symbiosis has been a topic of long-standing interest to plant biologists . By the early 1990s, biologists had deciphered the chemical signals that trigger early symbiotic responses . Flavonoids from the plant root trigger bacterial gene expression and the production of lipo-chitooligosaccharide signals (called Nod factors) that are recognized by the plant host . Genetic differences between bacterial strains modify the oligosaccharide backbone, for example by the addition of sulfate, acetate or fucose, and simultaneously alter the host-specificity of the purified Nod factor and the bacterium . Recent studies have begun to reveal the genetic and molecular basis of Nod-factor perception in legumes, a signaling system that also controls plant interactions with mycorrhizal fungi.

J Environ Qual, 2004 May-Jun, 33(3), 976 - 83
Short- and longer-term effects of the willow root system on metal extractability in contaminated dredged sediment; Vervaeke P et al.; Willow (Salix spp.) stands are often proposed as vegetation covers for the restoration and stabilization of contaminated and derelict land . Planting willows on dredged sediment disposal sites for biomass production can be an alternative to traditional capping techniques . However, with the introduction of willow stands on dredged sediment disposal sites, the possibility of increased contaminant availability in the root zone must be acknowledged as it can increase the risk of leaching . Two trials investigated the availability of Cd, Zn, Cu, and Pb in the root zones of willows grown on contaminated sediment . To assess the effects of willow root growth on metal extractability and mobility, bulk and rhizosphere sediment samples were extracted with deionized water, ammonium acetate at pH 7, and ammonium acetate-EDTA at pH 4.65 . A rhizobox experiment was used to investigate the short-term effect of willow roots on metal availability in oxic and anoxic sediment . Longer-term effects were assessed in a field trial . The rhizobox trial showed that Cd, Zn, and Cu extractability in the rhizosphere increased while the opposite was observed for Pb . This was attributed to the increased willow-induced oxidation rate in the root zone as a result of aeration and evapotranspiration, which masked the direct chemical and biological influences of the willow roots . The field trial showed that Cu and Pb, but not Cd, were more available in the root zone after water and ammonium acetate (pH 7) extraction compared with the bulk sediment . Sediment in the root zone was better structured and aggregated and thus more permeable for downward water flows, causing leaching of a fraction of the metals and significantly lower total contents of Cd, Cu, and Pb . These findings indicate that a vegetation cover strategy to stabilize sediments can increase metal availability in the root zone and that potential metal losses to the environment should be considered.

Int J Phytoremediation, 2004, 6(1), 49 - 61
Hyperaccumulation of metals by Thlaspi caerulescens as affected by root development and Cd-Zn/Ca-Mg interactions; Saison C et al.; The aim of this work was to study, in a rhizobox experiment, the phytoextraction of metals by the hyperaccumulator plant Thlaspi caerulescens in relation to the heterogeneity of metal pollution . Six treatments were designed with soils containing various levels of metals . Homogeneous soils and inclusions of soils in other soil matrices were prepared in order to vary metal concentration and localization . Growth parameters of the plant (rosette diameter and shoot biomass) and localization of roots and shoot uptake of Zn, Cd, Ca, and Mg were determined after 10 weeks of growth . The plants grown on the polluted industrial soils provided a larger biomass and had lower mortality rates than those grown on the agricultural soil . Moreover, these plants accumulated more Zn and Cd (up to 17,516 and 375 mg kg(-1) DM, respectively) than plants grown on the agricultural soil (up to 7300 mg Zn kg(-1) and 83 mg Cd kg(-1) DM) . The roots preferentially explored metal-contaminated areas . The exploration of polluted soil inclusions by the roots was associated with a higher extraction of metals . Zinc and Cd in the shoots of Thlaspi caerulescens were negatively correlated with Ca and Mg concentrations; however, the soil supply for these two elements was identical . This suggests that there is competition for the uptake of these elements and that Zn is preferentially accumulated.

Proc Natl Acad Sci U S A, 2004 Jul 6, 101(27), 10217 - 22 Epub 2004 Jun 25.
Six nonnodulating plant mutants defective for Nod factor-induced transcriptional changes associated with the legume-rhizobia symbiosis; Mitra RM et al.; As the legume-rhizobia symbiosis is established, the plant recognizes bacterial-signaling molecules, Nod factors (NFs), and initiates transcriptional and developmental changes within the root to allow bacterial invasion and the construction of a novel organ, the nodule . Plant mutants defective in nodule initiation (Nod(-)) are thought to have defects in NF-signal transduction . However, it is unknown whether WT plants respond to NF-independent bacterial-derived signals or whether Nod(-) plant mutants show defects in global symbiosis-associated gene expression . To characterize plant gene expression in the establishment of the symbiosis, we used an Affymetrix oligonucleotide microarray representing 9,935 Medicago truncatula expressed sequences . We identified 46 sequences that are differentially expressed in plants exposed for 24 h to WT Sinorhizobium meliloti or to the invasion defective S . meliloti mutant, exoA . Eight of these genes encode nucleolar proteins, which are implicated in ribosome biogenesis . We also identified differentially expressed transcription factors, signaling components, defense response proteins, stress response proteins, and several previously uncharacterized genes . NF appears both necessary and sufficient to induce most changes . Six of seven Nod(-) M . truncatula mutants (nfp, dmi1, dmi2, dmi3, nsp1, and nsp2) showed no transcriptional response to S . meliloti, suggesting that the encoded proteins are required for initiating new transcription . The Nod(-) mutant hcl, however, exhibits a reduced transcriptional response to S . meliloti, indicating that the machinery responsible for initiating new transcription is at least partially functional in this mutant.

Fungal Genet Biol, 2004 Aug, 41(8), 794 - 804
Recognition events in AM symbiosis: analysis of fungal gene expression at the early appressorium stage; Breuninger M et al.; Arbuscular mycorrhizal symbiosis is induced upon a series of recognition events involving the reorganization of both plant and fungal cellular programs culminating in the formation of appressoria on the epidermal root cells . In this work we monitored for the first time the genetic changes occurring in the fungal partner during early appressorium development . We established an in vitro system of Glomus mosseae and Petroselinum crispum for studying appressorium formation and found that after 120 h first appressoria developed in the root epidermis . We have constructed a fungal subtractive suppressive library enriched in genes up-regulated at this stage . Our aim was to identify early signaling events during plant recognition leading to appressoria formation . The library contains 375 clones with an average size of 500 bp . From these, 200 clones were sequenced and most of them represent gene fragments with no known homologues (63%) and therefore putative new genes specific to the mycorrhiza symbiosis . Reverse-Northern blot and RT-PCR analyses confirmed that ca . 30% of the genes present in the library were up-regulated upon plant induction after 120 h . Among the genes with homologues in other organisms we found several genes common to other plant-microbe interactions including some genes related to Ca2+-dependent signaling . The up-regulation of these genes opens the possibility that Ca2+ plays a role in the early stages of mycorrhiza formation as it has been found in other plant-microbe interactions such as the Rhizobium symbiosis or the Magnaporthe grisea/rice pathogenic interaction.

Lett Appl Microbiol, 2004, 38(4), 296 - 300
Cloning and characterization of ce/8A gene from Rhizobium leguminosarum bv . trifolii 1536; An JM et al.; AIMS: To isolate the cellulase gene from Rhizobium leguminosarum bv . trifolii 1536 . METHODS AND RESULTS: By the shot-gun method a clone (cel8A) harbouring 3.1 kb genomic DNA fragment from R . leguminosarum bv . trifolii 1536 was obtained . The cel8A gene coded 348 amino acids and it belongs to the glycosyl hydrolase family 8 . The molecular mass of Cel8A protein induced from Escherichia coli DH5alpha, appeared to be 35 kDa . The optimum pH and optimum temperature was 7.0, and about 30 degrees C for its enzymatic activity respectively . CONCLUSIONS: R . leguminosarum bv . trifolii 1536 had cel8A gene having an open reading frame of 1047 bp coded for the activity of hydrolyzation of carboxymethyl cellulose . SIGNIFICANCE AND IMPACT OF THE STUDY: The production of celluloytic enzyme by R . leguminosarum bv . trifolii was confirmed, which would play specific roles in rhizobia . Future study should focus on its role in the infection and nodulation phenomena.

Syst Appl Microbiol, 2004 May, 27(3), 380 - 95
Characterisation of wild legume nodulating bacteria (LNB) in the infra-arid zone of Tunisia; Zakhia F et al.; We report on the isolation and the characterization of nitrogen-fixing root nodule bacteria isolated from natural legumes in a region of South Tunisia corresponding to the infra-arid climatic zone . A collection of 60 new bacterial root nodule isolates were obtained from 19 legume species belonging to the genera Acacia, Anthyllis, Argyrolobium, Astragalus, Calycotome, Coronilla, Ebenus, Genista, Hedysarum, Hippocrepis, Lathyrus, Lotus, Medicago, Ononis . The isolates were characterised by (1) comparative 16S ARDRA using 7 enzymes, (2) total cell protein SDS-PAGE analysis and (3) 16S rDNA sequencing . The results show that these isolates are diverse and belong to the genera Rhizobium, Sinorhizobium, Mesorhizobium and Bradyrhizobium . Bradyrhizobium were further characterised by 16S-23S rDNA IGS sequencing . Surprisingly strains nodulating Astragalus cruciatus, Lotus creticus and Anthyllis henoniana were identified as Rhizobium galegae, a species recorded only as endosymbiont of Galega officinalis and G . orientalis in northern regions so far.

Can J Microbiol, 2004 May, 50(5), 313 - 22
Phylogeny based on 16S rDNA and nifH sequences of Ralstonia taiwanensis strains isolated from nitrogen-fixing nodules of Mimosa pudica, in India; Verma SC et al.; Bacterial symbionts present in the indeterminate-type nitrogen (N)-fixing nodules of Mimosa pudica grown in North and South India showed maximum similarity to Ralstonia taiwanensis on the basis of carbon-source utilization patterns and 16S rDNA sequence . Isolates from the nodules of M . pudica from North India and South India showed identical ARDRA (Amplified Ribosomal DNA Restriction Analysis) patterns with Sau3AI and RsaI, but AluI revealed dimorphy between the North Indian and South Indian isolates . Alignment of 16S rDNA sequences revealed similarity of North Indian isolates with an R . taiwanensis strain isolated from M . pudica in Taiwan, whereas South Indian isolates showed closer relatedness with the isolates from Mimosa diplotricha . Alignment of nifH sequences from both North Indian and South Indian isolates with that of the related isolates revealed their closer affinity to alpha-rhizobia, suggesting that nif genes in the beta-rhizobia might have been acquired from alpha-rhizobia via lateral transfer during co-occupancy of nodules by alpha-rhizobia and progenitors of R . taiwanensis, members of the beta-subclass of Proteobacteria . Immunological cross-reaction of the bacteroid preparation of M . pudica nodules showed strong a positive signal with anti-dinitrogenase reductase antibody, whereas a weak positive cross-reaction was observed with free-living R . taiwanensis grown microaerobically in minimal medium with and without NH4Cl . In spite of the expression of dinitrogenase reductase under free-living conditions, acetylene reduction was not observed under N-free conditions even after prolonged incubation.

Ann Bot (Lond), 2004 Aug, 94(2), 251 - 8 Epub 2004 Jun 17.
The influence of Rhizobium and arbuscular mycorrhizal fungi on nitrogen and phosphorus accumulation by Vicia faba; Jia Y et al.; BACKGROUND AND AIMS: The aim of this study was to investigate the effects of the interactions between the microbial symbionts, Rhizobium and arbuscular mycorrhizal fungi (AMF) on N and P accumulation by broad bean (Vicia faba) and how increased N and P content influence biomass production, leaf area and net photosynthetic rate . METHODS: A multi-factorial experiment consisting of four different legume-microbial symbiotic associations and two nitrogen treatments was used to investigate the influence of the different microbial symbiotic associations on P accumulation, total N accumulation, biomass, leaf area and net photosynthesis in broad bean grown under low P conditions . KEY RESULTS: AMF promoted biomass production and photosynthetic rates by increasing the ratio of P to N accumulation . An increase in P was consistently associated with an increase in N accumulation and N productivity, expressed in terms of biomass and leaf area . Photosynthetic N use efficiency, irrespective of the inorganic source of N (e.g . NO3- or N2), was enhanced by increased P supply due to AMF . The presence of Rhizobium resulted in a significant decline in AMF colonization levels irrespective of N supply . Without Rhizobium, AMF colonization levels were higher in low N treatments . Presence or absence of AMF did not have a significant effect on nodule mass but high N with or without AMF led to a significant decline in nodule biomass . Plants with the Rhizobium and AMF symbiotic associations had higher photosynthetic rates per unit leaf area . CONCLUSIONS: The results indicated that the synergistic or additive interactions among the components of the tripartite symbiotic association (Rhizobium-AMF-broad bean) increased plant productivity.

Mikrobiologiia, 2004 Mar-Apr, 73(2), 195 - 8
{The effect of tryptophan of plant root metabolites on the phyto stimulating activity of rhizobacteria }; Kravchenko LV et al.; Aseptic tomato and radish roots were found to exude 2.8-5.3 and 290-390 ng tryptophan per seedling per day . The inoculation of radish plants with rhizosphere pseudomonads increased the root biomass by 1.4 times . The inoculation of tomato plants with the same pseudomonads was ineffective . The beneficial effect of bacterial inoculation on the radish plants can be explained by the fact that the introduced rhizobacteria produce the plant growth-stimulating hormone indole-3-acetic acid . In pot experiments, the addition of this phytohormone to the soil increased the mass of radish roots by 36% . The phytohormonal action of the rhizosphere microflora was found to be efficient provided that the concentration of tryptophan in the rhizosphere is sufficiently high.

Biotechnol Lett, 2004 May, 26(9), 723 - 7
Expression in sugar beet of the introduced cercosporin toxin export (CFP) gene from Cercospora kikuchii, the causative organism of purple seed stain in soybean; Kuykendall LD et al.; The Cercospora kikuchii cercosporin export gene, CFP, introduced into Beta vulgaris L . by conjugation with Rhizobium radiobacter, was stably maintained during vegetative propagation as verified by PCR using primers specific for the CFP gene . Transcriptional expression of the CFP gene in leaves was determined by RT-PCR using CFP-specific primers . CFP protein was detected using Western analysis with an affinity-purified polypeptide-specifc antibody . Analysis of the relative susceptibility of CFP-transgenic and non-transgenic sugar beet plants is planned but will probably take several years to complete.

Mol Plant Microbe Interact, 2004 Jun, 17(6), 613 - 22
The expression of MaEXP1, a Melilotus alba expansin gene, is upregulated during the sweetclover-Sinorhizobium meliloti interaction; Giordano W et al.; Expansins are a highly conserved group of cell wall-localized proteins that appear to mediate changes in cell wall plasticity during cell expansion or differentiation . The accumulation of expansin protein or the mRNA for specific expansin gene family members has been correlated with the growth of various plant organs . Because expansin proteins are closely associated with plant cell wall expansion, and as part of a larger study to determine the role of different gene products in the legume-Rhizobium spp . symbiosis, we investigated whether a Melilotus alba (white sweetclover) expansin gene is expressed during nodule development . A cDNA fragment encoding an expansin gene (EXP) was isolated from Sinorhizobium meliloti-inoculated sweetclover root RNA by reverse-transcriptase polymerase chain reaction using degenerate primers, and a full-length sweetclover expansin sequence (MaEXP1) was obtained using 5' and 3' rapid amplification of cDNA end cloning . The predicted amino acid of the sweetclover expansin is highly conserved with the various alpha-expansins in the GenBank database . MaEXP1 contains a series of eight cysteines and four tryptophans that are conserved in the alpha-expansin protein family . Northern analysis and whole-mount in situ hybridization analyses indicate that MaEXP1 mRNA expression is enhanced in roots within hours after inoculation with S . meliloti and in nodules . Western and immunolocalization studies using a cucumber expansin antibody demonstrated that a cross-reacting protein accumulated in the expanding cells of the nodule.

Microbiol Mol Biol Rev, 2004 Jun, 68(2), 280 - 300
Infection and invasion of roots by symbiotic, nitrogen-fixing rhizobia during nodulation of temperate legumes; Gage DJ; Bacteria belonging to the genera Rhizobium, Mesorhizobium, Sinorhizobium, Bradyrhizobium, and Azorhizobium (collectively referred to as rhizobia) grow in the soil as free-living organisms but can also live as nitrogen-fixing symbionts inside root nodule cells of legume plants . The interactions between several rhizobial species and their host plants have become models for this type of nitrogen-fixing symbiosis . Temperate legumes such as alfalfa, pea, and vetch form indeterminate nodules that arise from root inner and middle cortical cells and grow out from the root via a persistent meristem . During the formation of functional indeterminate nodules, symbiotic bacteria must gain access to the interior of the host root . To get from the outside to the inside, rhizobia grow and divide in tubules called infection threads, which are composite structures derived from the two symbiotic partners . This review focuses on symbiotic infection and invasion during the formation of indeterminate nodules . It summarizes root hair growth, how root hair growth is influenced by rhizobial signaling molecules, infection of root hairs, infection thread extension down root hairs, infection thread growth into root tissue, and the plant and bacterial contributions necessary for infection thread formation and growth . The review also summarizes recent advances concerning the growth dynamics of rhizobial populations in infection threads.

Appl Environ Microbiol, 2004 Jun, 70(6), 3272 - 81
Engineering the nifH promoter region and abolishing poly-beta-hydroxybutyrate accumulation in Rhizobium etli enhance nitrogen fixation in symbiosis with Phaseolus vulgaris; Peralta H et al.; Rhizobium etli, as well as some other rhizobia, presents nitrogenase reductase (nifH) gene reiterations . Several R . etli strains studied in this laboratory showed a unique organization and contained two complete nifHDK operons (copies a and b) and a truncated nifHD operon (copy c) . Expression analysis of lacZ fusion demonstrated that copies a and b in strain CFN42 are transcribed at lower levels than copy c, although this copy has no discernible role during nitrogen fixation . To increase nitrogenase production, we constructed a chimeric nifHDK operon regulated by the strong nifHc promoter sequence and expressed it in symbiosis with the common bean plant (Phaseolus vulgaris), either cloned on a stably inherited plasmid or incorporated into the symbiotic plasmid (pSym) . Compared with the wild-type strain, strains with the nitrogenase overexpression construction assayed in greenhouse experiments had, increased nitrogenase activity (58% on average), increased plant weight (32% on average), increased nitrogen content in plants (15% at 32 days postinoculation), and most importantly, higher seed yield (36% on average), higher nitrogen content (25%), and higher nitrogen yield (72% on average) in seeds . Additionally, expression of the chimeric nifHDK operon in a poly-beta-hydroxybutyrate-negative R . etli strain produced an additive effect in enhancing symbiosis . To our knowledge, this is the first report of increased seed yield and nutritional content in the common bean obtained by using only the genetic material already present in Rhizobium.

Environ Pollut, 2004 Aug, 130(3), 465 - 76
A multi-process phytoremediation system for removal of polycyclic aromatic hydrocarbons from contaminated soils; Huang XD et al.; To improve phytoremediation processes, multiple techniques that comprise different aspects of contaminant removal from soils have been combined . Using creosote as a test contaminant, a multi-process phytoremediation system composed of physical (volatilization), photochemical (photooxidation) and microbial remediation, and phytoremediation (plant-assisted remediation) processes was developed . The techniques applied to realize these processes were land-farming (aeration and light exposure), introduction of contaminant degrading bacteria, plant growth promoting rhizobacteria (PGPR), and plant growth of contaminant-tolerant tall fescue (Festuca arundinacea) . Over a 4-month period, the average efficiency of removal of 16 priority PAHs by the multi-process remediation system was twice that of land-farming, 50% more than bioremediation alone, and 45% more than phytoremediation by itself . Importantly, the multi-process system was capable of removing most of the highly hydrophobic, soil-bound PAHs from soil . The key elements for successful phytoremediation were the use of plant species that have the ability to proliferate in the presence of high levels of contaminants and strains of PGPR that increase plant tolerance to contaminants and accelerate plant growth in heavily contaminated soils . The synergistic use of these approaches resulted in rapid and massive biomass accumulation of plant tissue in contaminated soil, putatively providing more active metabolic processes, leading to more rapid and more complete removal of PAHs.

Environ Pollut, 2004 Aug, 130(3), 453 - 63
Responses of three grass species to creosote during phytoremediation; Huang XD et al.; Phytoremediation of creosote-contaminated soil was monitored in the presence of Tall fescue, Kentucky blue grass, or Wild rye . For all three grass species, plant growth promoting rhizobacteria (PGPR) were evaluated for plant growth promotion and protection of plants from contaminant toxicity . A number of parameters were monitored including plant tissue water content, root growth, plant chlorophyll content and the chlorophyll a/b ratio . The observed physiological data indicate that some plants mitigated the toxic effects of contaminants . In addition, in agreement with our previous experiments reported in the accompanying paper (Huang, X.-D., El-Alawi, Y., Penrose, D.M., Glick, B.R., Greenberg, B.M., 2004 . A multi-process phytoremediation system for removal of polycyclic aromatic hydrocarbons from contaminated soil . Environ . Poll . doi: 10.1016/j.envpol.2003.09.031), PGPR were able to greatly enhance phytoremediation . PGPR accelerated plant growth, especially roots, in heavily contaminated soils, diminishing the toxic effects of contaminants to plants . Thus, the increased root biomass in PGPR-treated plants led to more effective remediation.

Eur J Biochem, 2004 Jun, 271(12), 2494 - 503
Temperature and concentration-controlled dynamics of rhizobial small heat shock proteins; Lentze N et al.; A hallmark of alpha-crystallin-type small heat shock proteins (sHsps) is their highly dynamic oligomeric structure which promotes intermolecular interactions involved in subunit exchange and substrate binding (chaperone-like activity) . We studied the oligomeric features of two classes of bacterial sHsps by size exclusion chromatography and nanoelectrospray mass spectrometry . Proteins of both classes formed large complexes that rapidly dissociated upon dilution and at physiologically relevant heat shock temperatures . As the secondary structure was not perturbed, temperature- and concentration-dependent dissociations were fully reversible . Complexes formed between sHsps and the model substrate citrate synthase were stable and exceeded the size of sHsp oligomers . Small Hsps, mutated in a highly conserved glycine residue at the C-terminal end of the alpha-crystallin domain, formed labile complexes that disassembled more readily than the corresponding wild-type proteins . Reduced complex stability coincided with reduced chaperone activity.

Genetika, 2004 Apr, 40(4), 454 - 61
{Instability of a cryptic plasmid in Sinorhizobium meliloti P108 during symbiosis of it with alfalfa Medicago sativa}; Rumiantseva ML et al.; Instability of cryptic plasmids in Sinorhizobium meliloti laboratory strains SKhM1-188, DM7-R, and P108 as well as in their clones isolated from nodules of alfalfa grown during a long-term microvegetation experiment (120 days) was studied . The isolated clones of strains SKhM1-188 and DM7-R manifested stable inheritance of plasmids, whereas 12.7-14.0% of clones with changed plasmid profile were detected in a population of clones from strain P108 . These segregants were designated as P108c . Segregants P108c exhibited significantly decreased symbiotic effectiveness, nitrogenase activity, and the competitiveness with respect to alfalfa, compared to the original strain P108 . It was established that a 80-kb deletion occurred in a larger of two cryptic plasmids (240 and 230 kb) of segregants P108c . It was concluded that genetic rearrangements are possible in rhizobial clones that did not undergo structural transformation and retained viability in the nodule during the natural vegetation period of alfalfa.

J Biol Chem, 2004 Aug 13, 279(33), 34624 - 30 Epub 2004 May 24.
The galactolipid digalactosyldiacylglycerol accumulates in the peribacteroid membrane of nitrogen-fixing nodules of soybean and Lotus; Gaude N et al.; The peribacteroid membrane (PBM) surrounding nitrogen fixing rhizobia in the nodules of legumes is crucial for the exchange of ammonium and nutrients between the bacteria and the host cell . Digalactosyldiacylglycerol (DGDG), a galactolipid abundant in chloroplasts, was detected in the PBM of soybean (Glycine max) and Lotus japonicus . Analyses of membrane marker proteins and of fatty acid composition confirmed that DGDG represents an authentic PBM lipid of plant origin and is not derived from the bacteria or from plastid contamination . In Arabidopsis, DGDG is known to accumulate in extraplastidic membranes during phosphate deprivation . However, the presence of DGDG in soybean PBM was not restricted to phosphate limiting conditions . Complementary DNA sequences corresponding to the two DGDG synthases, DGD1 and DGD2 from Arabidopsis, were isolated from soybean and Lotus . The two genes were expressed during later stages of nodule development in infected cells and in cortical tissue . Because nodule development depends on the presence of high amounts of phosphate in the growth medium, the accumulation of the non-phosphorus galactolipid DGDG in the PBM might be important to save phosphate for other essential processes, i.e . nucleic acid synthesis in bacteroids and host cells.

Infect Immun, 2004 Jun, 72(6), 3398 - 409
Characterization of the icmH and icmF genes required for Legionella pneumophila intracellular growth, genes that are present in many bacteria associated with eukaryotic cells; Zusman T et al.; Legionella pneumophila, the causative agent of Legionnaires' disease, replicates intracellularly within a specialized phagosome of mammalian and protozoan host cells, and the Icm/Dot type IV secretion system has been shown to be essential for this process . Unlike all the other known Icm/Dot proteins, the IcmF protein, which was described before, and the IcmH protein, which is characterized here, have homologous proteins in many bacteria (such as Yersinia pestis, Salmonella enterica, Rhizobium leguminosarum, and Vibrio cholerae), all of which associate with eukaryotic cells . Here, we have characterized the L . pneumophila icmH and icmF genes and found that both genes are present in 16 different Legionella species examined . The icmH and icmF genes were found to be absolutely required for intracellular multiplication in Acanthamoeba castellanii and partially required for intracellular growth in HL-60-derived human macrophages, for immediate cytotoxicity, and for salt sensitivity . Mutagenesis of the predicted ATP/GTP binding site of IcmF revealed that the site is partially required for intracellular growth in A . castellanii . Analysis of the regulatory region of the icmH and icmF genes, which were found to be cotranscribed, revealed that it contains at least two regulatory elements . In addition, an icmH::lacZ fusion was shown to be activated during stationary phase in a LetA- and RelA-dependent manner . Our results indicate that although the icmH and icmF genes probably have a different evolutionary origin than the rest of the icm/dot genes, they are part of the icm/dot system and are required for L . pneumophila pathogenesis.

Commun Agric Appl Biol Sci, 2003, 68(4 Pt A), 129 - 34
Managing the mycorrhizosphere--an approach to sustainable agriculture after the phaseout of methyl bromide?
Reimann S, Sikora RA.
Plant Health Promoting Rhizobacteria (PHPR) with known biocontrol activity toward the root knot nematode, Meloidogyne incognito, were tested for their ability to promote root colonization of arbuscular mycorrhizal fungi on tomato under greenhouse conditions . In addition, bacteria isolated from mycorrhizal spores were tested for their direct biological control activity on nematode early root penetration . The nematode antagonistic rhizobacteria Rhizobium etli G12 significantly increased mycorrhizal establishment . In addition, two of nine bacteria isolated from the mycorrhizosphere were shown to significantly reduce M . incognita early root penetration . The experiments produced important data on the interaction between rhizobacteria and the symbiotic fungus that in some combinations work synergistically in controlling the root-knot nematode . The potential of using such multitrophic treatment combinations in integrated management targeted first at reducing nematode early root penetration in young transplants and then later for sustainable biocontrol of root-knot under field conditions are discussed.

DNA Res, 2004 Feb 29, 11(1), 57 - 65
Genome-wide comparison of the His-to-Asp phosphorelay signaling components of three symbiotic genera of Rhizobia; Hagiwara D et al.; Histidine-to-aspartate (His-Asp) phosphorelay (or two-component) systems are very common signal transduction mechanisms that are implicated in a wide variety of cellular responses to environmental stimuli . The His-Asp phosphorelay components include "sensor histidine kinase (HK)", "phosphotransfer intermediate (HPt)", and "response regulator (RR)" . With special reference to three bacterial species (Mesorhizobium loti, Bradyrhizobium japonicum, Sinorhizobium meliloti), each of which belongs to a different genera of Rhizobia, here we attempted to compile all of the His-Asp phosphorelay components in order to reveal a comparative genome-wide overview as to the His-Asp phosphorelay . It was revealed that M . loti has 47 HKs, 1 HPts, and 58 RRs; B . japonicum has 80 HKs, 3 HPts, and 91 RRs; whereas S . meliloti has 40 HKs, 1 HPt, and 58 RRs . These His-Asp phosphorelay components were extensively compiled and characterized . The resulting overview as to the His-Asp phosphorelay of Rhizobia will provide us with a basis for understanding of the fundamental mechanisms underlying interactions between plants and microorganisms (including symbiosis), as well as nitrogen fixation.

Ying Yong Sheng Tai Xue Bao, 2004 Jan, 15(1), 44 - 8
{Diversity and resistance of rhizobia isolated from Caragana intermedia in Maowusu sandland}; Gao L et al.; Fifteen rhizobia strains were isolated from wild shrubby legume Caragana intermedia in Maowusu sandland . A dendrogram was constructed based on esterase profiles, showing a rich diversity of these rhizobia . Many biochemical characteristics were detected, including acid or alkali production, catalase activity, utilization of sole carbon sources, and resistance to salt, acid-alkali and temperature variation . The results indicated that all the rhizobia strains isolated from Caragana intermedia could excrete H+ on YMA agar and produce catalase . 73.3% strains could tolerate NaCl stress at 3% concentration, and 80% strains could grow at 50 degrees C . Except the difference in lactose and starch utilization, rhizobia strains had no bias on the rest carbon sources . However, the difference in resistance to stress existed among strains, which might be related to the adaptation of rhizobia to diverse landscapes in Maowusu sandland . It was revealed that rhizobia nodulating Caragana intermedia could be used as a new germplasm to fix nitrogen under severe environment.

FEMS Microbiol Lett, 2004 May 15, 234(2), 225 - 30
The nitrogen-fixing symbiotic bacterium Mesorhizobium loti has and expresses the gene encoding pyridoxine 4-oxidase involved in the degradation of vitamin B6; Yuan B et al.; The gene product of mll6785 of a nitrogen-fixing symbiotic bacterium Mesorhizobium loti MAFF303099 was identified as pyridoxine 4-oxidase, the first enzyme in the vitamin B6-degradation pathway . The gene was cloned and ligated into pET-21a+ . Escherichia coli BL21(DE3) was co-transformed with the constructed plasmid plus pKY206 containing groESL genes encoding chaperonins . The overexpressed protein was purified to homogeneity by the ammonium sulfate fractionation and three chromatography steps . The enzymatic properties of the purified protein, such as K(m) values for pyridoxine (213+/-19 microM) and oxygen (78+/-10 microM), were compared to those of pyridoxine 4-oxidase from two bacteria with known vitamin B6-degradation pathway . M . loti grown in a Rhizobium medium showed the enzyme activity . The results suggest that M . loti also contains the degradation pathway of vitamin B6 .

Microbiology, 2004 May, 150(Pt 5), 1447 - 56
The Fur-like protein Mur of Rhizobium leguminosarum is a Mn(2+)-responsive transcriptional regulator; Diaz-Mireles E et al.; In wild-type Rhizobium leguminosarum, the sitABCD operon specifies a Mn(2+) transporter whose expression is severely reduced in cells grown in the presence of this metal . Mutations in the R . leguminosarum gene, mur (manganese uptake regulator), whose product resembles the Fur transcriptional regulator, cause high-level expression of sitABCD in the presence of Mn(2+) . In gel-shift mobility assays, purified R . leguminosarum Mur protein bound to at least two regions near the sitABCD promoter region, although this DNA has no conventional consensus Fur-binding sequences (fur boxes) . Thus, in contrast to gamma-proteobacteria, where Fur binds Fe(2+), the R . leguminosarum Fur homologue, Mur, act as a Mn(2)-responsive transcriptional regulator.

Biotechnol Lett, 2004 Mar, 26(6), 487 - 91
Growth promotion of red pepper plug seedlings and the production of gibberellins by Bacillus cereus, Bacillus macroides and Bacillus pumilus; Joo GJ et al.; The growth of red pepper plug seedlings was promoted by Bacillus cereus MJ-1, B . macroides CJ-29, and B . pumilus CJ-69 isolated from the rhizosphere . Gibberellins (GAs), a well-known plant growth-promoting hormone, were detected in the culture broth of their rhizobacteria . Among the GAs, the contents of GA1, GA3, GA4, and GA7, physiologically active GAs, were comparatively higher than those of others, suggesting that the growth promoting effect was originated from the GAs . This isthe first report on the production of GA5, GA8, GA34, GA44, and GA53 by bacteria.

J Bacteriol, 2004 May, 186(10), 2996 - 3005
Identification of rhtX and fptX, novel genes encoding proteins that show homology and function in the utilization of the siderophores rhizobactin 1021 by Sinorhizobium meliloti and pyochelin by Pseudomonas aeruginosa, respectively; Cuiv PO et al.; Rhizobactin 1021 is a hydroxymate siderophore produced by the soil bacterium Sinorhizobium meliloti 2011 . A regulon comprising rhtA, encoding the outer membrane receptor protein for the ferrisiderophore; the biosynthesis operon rhbABCDEF; and rhrA, the Ara-C-like regulator of the receptor and biosynthesis genes has been previously described . We report the discovery of a gene, located upstream of rhbA and named rhtX (for "rhizobactin transport"), which is required, in addition to rhtA, to confer the ability to utilize rhizobactin 1021 on a strain of S . meliloti that does not naturally utilize the siderophore . Rhizobactin 1021 is structurally similar to aerobactin, which is transported in Escherichia coli via the IutA outer membrane receptor and the FhuCDB inner membrane transport system . E . coli expressing iutA and fhuCDB was found to also transport rhizobactin 1021 . We demonstrated that RhtX alone could substitute for FhuCDB to transport rhizobactin 1021 in E . coli . RhtX shows similarity to a number of uncharacterized proteins which are encoded proximal to genes that are either known to be or predicted to be involved in iron acquisition . Among these is PA4218 of Pseudomonas aeruginosa, which is located close to the gene cluster that functions in pyochelin biosynthesis and outer membrane transport . PA4218 was mutated by allelic replacement, and the mutant was found to have a pyochelin utilization-defective phenotype . It is proposed that PA4218 be named fptX (for "ferripyochelin transport") . RhtX and FptX appear to be members of a novel family of permeases that function as single-subunit transporters of siderophores.

Environ Health Perspect, 2004 May, 112(6), 648 - 53
Chemical communication threatened by endocrine-disrupting chemicals; Fox JE; Communication on a cellular level--defined as chemical signaling, sensing, and response--is an essential and universal component of all living organisms and the framework that unites all ecosystems . Evolutionarily conserved signaling "webs," existing both within an organism and between organisms, rely on efficient and accurate interpretation of chemical signals by receptors . Therefore, endocrine-disrupting chemicals (EDCs), which have been shown to disrupt hormone signaling in laboratory animals and exposed wildlife, may have broader implications for disrupting signaling webs that have yet to be identified as possible targets . In this article, I explore common evolutionary themes of chemical signaling (e.g., estrogen signaling in vertebrates and phytoestrogen signaling from plants to symbiotic soil bacteria) and show that such signaling systems are targets of disruption by EDCs . Recent evolutionary phylogenetic data have shown that the estrogen receptor (ER) is the ancestral receptor from which all other steroid receptors have evolved . In addition to binding endogenous estrogens, ERs also bind phytoestrogens, an ability shared in common with nodulation D protein (NodD) receptors found in Rhizobium soil bacteria . Recent data have shown that many of the same synthetic and natural environmental chemicals that disrupt endocrine signaling in vertebrates also disrupt phytoestrogen-NodD receptor signaling in soil bacteria, which is necessary for nitrogen-fixing symbiosis . Bacteria-plant symbiosis is an unexpected target of EDCs, and other unexpected nontarget species may also be vulnerable to EDCs found in the environment.

Plant Cell Physiol, 2004 Apr, 45(4), 427 - 35
Transgenic Lotus japonicus with an ethylene receptor gene Cm-ERS1/H70A enhances formation of infection threads and nodule primordia; Nukui N et al.; Ethylene inhibits the establishment of symbiosis between rhizobia and legumes . To examine how and when endogenous ethylene inhibits rhizobial infection and nodulation, we produced transgenic Lotus japonicus carrying the mutated melon ethylene receptor gene Cm-ERS1/H70A that confers ethylene insensitivity and fixes the transgene in the T(3) generation . The resultant transgenic plants showed reduced ethylene sensitivity because of 1-aminocyclopropane-1-carboxylate resistance and increased flowering duration, probably due to a dominant negative mechanism . When inoculated with Mesorhizobium loti, transgenic plants showed markedly higher numbers of infection threads and nodule primordia on their roots than did either wild-type or azygous plants during the early stage of cultivation period as well as during later stages, when the number of mature nodules had reached a steady state . In addition, transcripts of NIN, a gene governing infection thread formation, increased in the inoculated transgenic plants as compared with the wild-type plants . The infection responses of transgenic plants were similar to those of wild-type plants treated with ethylene inhibitors . These results imply that the endogenous ethylene in L . japonicus roots inhibits rhizobial infection at the primary nodulation, probably via NIN gene, and suggest that ethylene perception assists negative feedback regulation of secondary nodule initiation.

Plasmid, 2004 May, 51(3), 203 - 16
Incompatibility and the partitioning site of the repABC basic replicon of the symbiotic plasmid from Rhizobium etli; Soberon N et al.; The basic replicon of the symbiotic plasmid (p42d) of Rhizobium etli CE3 is constituted by the repABC operon . Whereas RepC is essential for plasmid replication, RepA and RepB are involved in plasmid partitioning . Three incompatibility regions have been previously identified in this plasmid: the first one encodes RepA, a partitioning protein that also down-regulates the repABC transcription . The second region is situated within the repB-repC intergenic sequence (inc(alpha)), and the last one, inc(beta), is located in a 502 bp EcoRI fragment spanning the last 72-bp of the coding region of repC and the following downstream sequence . In this paper we show that: (1) The inc(beta) region is required for plasmid partitioning . (2) A 16-bp palindrome sequence, located 40 bp downstream of the repC gene of plasmid p42d, is necessary and sufficient to induce incompatibility towards the parental plasmid, and accounts for all the incompatibility properties of this region (inc(beta)) . (3) . The palindrome is the DNA target site for RepB binding . With these findings we propose that inc(beta) contains the partitioning site (par site) of the basic replicon of plasmid p42d, and that the 16-bp palindrome is the core sequence to nucleate the RepB binding .

Plasmid, 2004 May, 51(3), 185 - 91
A gfp reporter plasmid to visualize Azorhizobium caulinodans during nodulation of Sesbania rostrata; D'Haeze W et al.; Compared with other labeling techniques, the use of the green fluorescent protein (GFP) is advantageous to visualize bacteria because observations can be performed in real time . This feature is particularly interesting to study invasion events of rhizobia during nodule development on their legume host plant . To investigate the symbiotic interaction between Azorhizobium caulinodans ORS571 and Sesbania rostrata, we constructed two plasmids, pMP220-hem-gfp5 and pBBR5-hem-gfp5-S65T, that carry a modified gfp gene, the expression of which is controlled by the constitutive hem promoter . Introduction of either of these plasmids into A . caulinodans allowed the visualization of single bacteria . Determination of the plasmid stability in cultured bacteria and in nodules demonstrated that pBBR5-hem-gfp5-S65T is more stable than pMP220-hem-gfp5 . The plasmid pBBR5-hem-gfp5-S65T can be used to study early invasion events during nodule development on hydroponic roots of S . rostrata .

Planta, 2004 Aug, 219(4), 626 - 38 Epub 2004 Apr 24.
Sinorhizobium meliloti-induced chitinase gene expression in Medicago truncatula ecotype R108-1: a comparison between symbiosis-specific class V and defence-related class IV chitinases; Salzer P et al.; The Medicago truncatula (Gaertn.) ecotypes Jemalong A17 and R108-1 differ in Sinorhizobium meliloti-induced chitinase gene expression . The pathogen-inducible class IV chitinase gene, Mtchit 4, was strongly induced during nodule formation of the ecotype Jemalong A17 with the S . meliloti wild-type strain 1021 . In the ecotype R108-1, the S . meliloti wild types Sm1021 and Sm41 did not induce Mtchit 4 expression . On the other hand, expression of the putative class V chitinase gene, Mtchit 5, was found in roots of M . truncatula cv . R108-1 nodulated with either of the rhizobial strains . Mtchit 5 expression was specific for interactions with rhizobia . It was not induced in response to fungal pathogen attack, and not induced in roots colonized with arbuscular mycorrhizal (AM) fungi . Elevated Mtchit 5 gene expression was first detectable in roots forming nodule primordia . In contrast to Mtchit 4, expression of Mtchit 5 was stimulated by purified Nod factors . Conversely, Mtchit 4 expression was strongly elevated in nodules formed with the K-antigen-deficient mutant PP699 . Expression levels of Mtchit 5 were similarly increased in nodules formed with PP699 and its parental wild-type strain Sm41 . Phylogenetic analysis of the deduced amino acid sequences of Mtchit 5 (calculated molecular weight = 41,810 Da, isoelectric point pH 7.7) and Mtchit 4 (calculated molecular weight 30,527 Da, isoelectric point pH 4.9) revealed that the putative Mtchit 5 chitinase forms a separate clade within class V chitinases of plants, whereas the Mtchit 4 chitinase clusters with pathogen-induced class IV chitinases from other plants . These findings demonstrate that: (i) Rhizobium-induced chitinase gene expression in M . truncatula occurs in a plant ecotype-specific manner, (ii) Mtchit 5 is a putative chitinase gene that is specifically induced by rhizobia, and (iii) rhizobia-specific and defence-related chitinase genes are differentially influenced by rhizobial Nod factors and K antigens.

Can J Microbiol, 2004 Mar, 50(3), 201 - 11
Environmental modulation of the pssTNOP gene expression in Rhizobium leguminosarum bv . trifolii; Wielbo J et al.; Exopolysaccharide production by Rhizobium leguminosarum bv . trifolii is required for successful establishment of nitrogen-fixing symbiosis with clover (Trifolium pratense L.) . Using plasmid-borne transcriptional fusions of promoters of pss genes with promoterless lacZ the effect of root exudate, phosphate, and ammonia on expression of pssT, pssN, pssO, and pssP genes in wild-type strain RtTA1 background was determined . A stimulating effect of these environmental factors on pssO and pssP gene expression was observed . The putative pssO gene promoter was determined to be a strong promoter within which the divergent nod-box element was identified . The pssO promoter was slightly inducible in a flavonoid-dependent manner in wild-type R . leguminosarum bv . trifolii strains RtTA1 and ANU843 and very weakly active in a mutant of strain ANU843 that lacks the regulatory nodD gene . The expression of pssO and pssP genes in planta was investigated using plasmid-borne pssO-gusA and pssP-gusA fusions under different phosphate availability to clover . The level of pssO-gusA fusion expression was shown to be dependent on phosphate concentration in the plant growth medium.

Antonie Van Leeuwenhoek, 2004 Jul, 86(1), 1 - 25
Applications of free living plant growth-promoting rhizobacteria; Lucy M et al.; Free-living plant growth-promoting rhizobacteria (PGPR) can be used in a variety of ways when plant growth enhancements are required . The most intensively researched use of PGPR has been in agriculture and horticulture . Several PGPR formulations are currently available as commercial products for agricultural production . Recently developing areas of PGPR usage include forest regeneration and phytoremediation of contaminated soils . As the mechanisms of plant growth promotion by these bacteria are unravelled, the possibility of more efficient plant-bacteria pairings for novel and practical uses will follow . The progress to date in using PGPR in a variety of applications with different plants is summarized and discussed here.

J Microsc, 2004 May, 214(Pt 2), 104 - 13
Dissection of Nod factor signalling in legumes: cell biology, mutants and pharmacological approaches; Esseling JJ et al.; Nodulation factors (NFs) are lipochito-oligosaccharide signal molecules excreted by soil-living rhizobia . These molecules elicit a range of responses in the legume roots, with which the bacteria can live in symbiosis . In this review we focus on the genetic, pharmacological and cell biological approaches that have been, and are being, undertaken to decipher the signalling pathways that lead to the symbiotic responses in the plant.

Environ Pollut, 1991, 73(1), 11 - 23
Radioactive isotope uptake in a grass-legume association; Douka CE et al.; The radioactive uptake of Medicago sativa and Rye grass in a pasture exposed to the fallout from the Chernobyl reactor accident, was determined in four consecutive harvests covering a period of one year after the accident,In plants of Medicago sativa, inoculated with an effective Rhizobia meliloti strain isolated from Greek soils, a high degree of biological nitrogen fixation was observed at all harvests using N-15 techniques . At the second and third harvests, the percentage nitrogen derived from fixation (%NdfF), the percentage nitrogen derived from soil (%NdfS), as well as the radioactive uptake from the soil remained stable . At the fourth harvest, however, the %NdfF decreased while the %NdfS and the radioactive uptake from soil significantly increased . At the first harvest the radioactivity in both plants, caused mainly by direct fallout contamination, was considerably higher than that observed at the later harvests . Medicago sativa contained significantly less radioactivity than the grass at all harvests, although both plants were grown under the same environmental conditions . Even at the fourth harvest, almost one year after the initial contamination, the radioactivity of grass remained at high levels ( 20 Bq g(-1) of protein) while in Medicago sativa it assumed considerably lower values (3.6 Bq g(-1) of protein) . A possible involvement of biological nitrogen fixation in the reduction of radioactive uptake is discussed . Finally, certain practical conclusions are drawn with respect to a safer management of pastures exposed to radioactivity.

Environ Pollut, 1993, 79(2), 105 - 12
The effect of heavy metals on dinitrogen fixation by Rhizobium-white colover in a range of long-term sewage sludge amended and metal-contaminated soils; Obbard JP et al.; An investigation was conducted to determine whether effective strains of Rhizobium leguminosarum biovar . trifolii capable of symbiotic N(2) fixation with white clover (Trifolium repens) were present in a range of metal-contaminated soils . A number of historically sewage-amended sites (including experimental, pasture grassland and arable sites) were selected and compared with highly contaminated samples from abandoned heavy metal mines . Many sites had metal concentrations above the limits established by the UK Government, based on those developed by the European Commission (EC) for sludge-amended soils . Acetylene reduction activity (ARA) was used to screen the samples for effective N(2) fixation . When the host plant was indigenous to the sward, rhizobia were found in the nodules and in the soil rhizosphere at all the sites tested . They were shown to be capable of effective symbiosis and N(2) fixation, even though metal concentrations greatly exceeded the soil metal limits in some cases . However, nodulation failed to occur in some cases where T . repens was not indigenous to metal-contaminated soils . This indicated either that an ineffective rhizobial population was present, or that effective cells were absent from the soil . The influence of individual metals on ARA could not be determined conclusively because of the confounding effects of soil physicochemical variability and the presence of different metals at high concentrations together in the soil . However, Cd concentrations appeared to be particularly important in determining the presence of effective ARA in soils with no indigenous clover . In contrast to previous studies, the results presented here suggest that heavy metals may have had a quantitative effect on the free-living population of rhizobia, rather than a genetic effect.

Environ Pollut, 1996, 92(1), 55 - 66
Effects of rhizobium, arbuscular mycorrhizal fungi and anion content of simulated rain on subterranean clover; Shafer SR et al.; An experiment was conducted to determine the extent to which rhizobia, mycorrhizal fungi, and anions in simulated rain affect plant growth response to acid deposition . Germinating subterranean clover seeds were planted in steam-pasteurized soil in pots and inoculated with Rhizobium leguminosarum, Glomus intraradices, Glomus etunicatum, R . leguminosarum + G . intraradices, R . leguminosarum + G . etunicatum, or no microbial symbionts . Beginning 3 weeks later, plants and the soil surface were exposed to simulated rain in a greenhouse on 3 days week(-1) for 12 weeks . Rain solutions were deionized water amended with background ions only (pH 5.0) or also adjusted to pH 3.0 with HNO(3) only, H(2)SO(4) only, or a 50/50 mixture of the two acids . Glomus intraradices colonized plant roots poorly, and G . intraradices-inoculated plants responded like nonmycorrhizal plants to rhizobia and rain treatments . Variation in plant biomass attributable to different rain formulations was strongest for G . etunicatum-inoculated plants, and the effect of rain formulation differed with respect to nodulation by rhizobia . The smallest plants at the end of the experiment were noninoculated plants exposed to rains (0.38 g mean dry weight total for 3 plants pot(-1)) . Among nonnodulated plants infected by G . etunicatum, those exposed to HNO(3) rain were largest, followed by plants exposed to HNO(3) + H(2)SO(4), pH 5.0, and H(2)SO(4) rain, in that order . Among plants inoculated with both R . leguminosarum + G . etunicatum, however, the greatest biomass occurred with pH 5.0 rains, resulting in the largest plants in the study (1.00 g/3 plants) . Treatment-related variation among root and shoot biomass data reflected those for whole-plant biomass . Based on quantification of biomass and N concentrations in shoot and root tissues, total N content of plants inoculated with G . etunicatum alone and exposed to the HNO(3) + H(2)SO(4) rains was approximately the same as plants inoculated with R . leguminosarum + G . entunicatum and exposed to pH 5 rains . Thus, the acid-mixture rains and rhizobia under no acid deposition provided approximately equal amounts of N in biomass . The significant interactions among rain formulation and the symbiotic status of the plants suggest that conclusions concerning the impact of acid deposition on plants in the environment cannot be considered reliable because most experiments on which such assessments are based have not tested confounding influences of microorganisms and precipitation characteristics.

BMC Biochem . 2004 Apr 13;5(1):4.
Radiolabeling of lipo-chitooligosaccharides using the NodH sulfotransferase: a two-step enzymatic procedure; Gressent F et al.; BACKGROUND: The NodH sulfotransferase from Sinorhizobium meliloti has been used to radiolabel lipochitooligosaccharidic (LCO) Nod factor signals with 35S from inorganic sulfate in a two-step enzymatic procedure . The first step involved the production of 3'-phosphoadenosine 5'-phosphosulfate (PAPS), a sulphate donor, using enzymes contained in a yeast extract, and the second step used the NodH enzyme . However with this established procedure, only a low incorporation of the initial inorganic sulfate into the Nod factors was obtained (about 7% after purification of the labeled compounds) . The aim of this work was to optimize the radiolabelling of Nod factors with 35S . RESULTS: The limiting step has been shown to be the sulfation of ATP and its subsequent conversion into PAPS (first step), the sulfate donor for the NodH sulfotransferase activity (second step) . By the addition of GTP to the reaction mixture and by manipulating the {ATP}/{Mg2+} ratio the yield of PAPS has been increased from 13% to 80% . Using the radiolabeled PAPS we have shown that the efficiency of sulfate transfer to LCOs, by the recombinant S . meliloti NodH sulfotransferase is strongly influenced by the length of the oligosaccharide chain . Variations in the substitutions on the non-reducing sugar, including the structure of the fatty acyl chain, had little effect and Nod factors from the heterologous bacterium Rhizobium tropici could be sulfated by NodH from S . meliloti . CONCLUSIONS: By characterizing the two steps we have optimized the procedure to radiolabel biologically-important, lipo-chitooligosaccharide (LCO) Nod factors to a specific radioactivity of about 800 Ci x mmol(-1) with an incorporation of 60% of the initial inorganic sulfate . The two-step sulfation procedure may be used to radiolabel a variety of related LCO molecules.

Proc Natl Acad Sci U S A, 2004 Apr 20, 101(16), 6303 - 8 Epub 2004 Apr 12.
Switch from intracellular to intercellular invasion during water stress-tolerant legume nodulation; Goormachtig S et al.; Rhizobia colonize their legume hosts by different modes of entry while initiating symbiotic nitrogen fixation . Most legumes are invaded via growing root hairs by the root hair-curl mechanism, which involves epidermal cell responses . However, invasion of a number of tropical legumes happens through fissures at lateral root bases by cortical, intercellular crack entry . In the semiaquatic Sesbania rostrata, the bacteria entered via root hair curls under nonflooding conditions . Upon flooding, root hair growth was prevented, invasion on accessible root hairs was inhibited, and intercellular invasion was recruited . The plant hormone ethylene was involved in these processes . The occurrence of both invasion pathways on the same host plant enabled a comparison to be made of the structural requirements for the perception of nodulation factors, which were more stringent for the epidermal root hair invasion than for the cortical intercellular invasion at lateral root bases.

Plant J, 2004 Apr, 38(2), 203 - 14
Cytokinins play opposite roles in lateral root formation, and nematode and Rhizobial symbioses; Lohar DP et al.; We used the cytokinin-responsive Arabidopsis response regulator (ARR)5 gene promoter fused to a beta-glucuronidase (GUS) reporter gene, and cytokinin oxidase (CKX) genes from Arabidopsis thaliana (AtCKX3) and maize (ZmCKX1) to investigate the roles of cytokinins in lateral root formation and symbiosis in Lotus japonicus . ARR5 expression was undetectable in the dividing initial cells at early stages of lateral root formation, but later we observed high expression in the base of the lateral root primordium . The root tip continues to express ARR5 during subsequent development of the lateral root . These results suggest a dynamic role for cytokinin in lateral root development . We observed ARR5 expression in curled/deformed root hairs, and also in nodule primordia in response to Rhizobial inoculation . This expression declined once the nodule emerged from the parent root . Root penetration and migration of root-knot nematode (RKN) second-stage larvae (L2) did not elevate ARR5 expression, but a high level of expression was induced when L2 reached the differentiating vascular bundle and during early stages of the nematode-plant interaction . ARR5 expression was specifically absent in mature giant cells (GCs), although dividing cells around the GCs continued to express this reporter . The same pattern was observed using a green fluorescent protein (GFP) reporter driven by the ARR5 promoter in tomato . Overexpression of CKX genes rendered the transgenic hairy roots resistant to exogenous application of the cytokinin {N6-(Delta2 isopentenyl) adenine riboside} (iPR) . CKX roots have significantly more lateral roots, but fewer nodules and nematode-induced root galls per plant, than control hairy roots.

J Plant Physiol, 2004 Mar, 161(3), 329 - 38
Nitrogenase and antioxidant enzyme activities in Phaseolus vulgaris nodules formed by Rhizobium tropici isogenic strains with varying tolerance to salt stress; Tejera NA et al.; Common bean plants inoculated with salt-tolerant Rhizobium tropici wild-type strain CIAT899 formed a more active symbiosis than did its decreased salt-tolerance (DST) mutant derivatives (HB8, HB10, HB12 and HB13) . The mutants formed partially effective (HB10, HB12) or almost ineffective (HB8, HB13) nodules (Fix(d)) under non-saline conditions . The DST mutant formed nodules that accumulated more proline than did the wild-type nodules, while soluble sugars were accumulated mainly in ineffective nodules . Under salt stress, plant growth, nitrogen fixation, and the activities of the antioxidant defense enzymes of nodules were affected in all symbioses tested . Overall, mutant nodules showed lower antioxidant enzyme activities than wild-type nodules . Levels of nodule catalase appeared to correlate with symbiotic nitrogen-fixing efficiency . Superoxide dismutase and dehydroascorbate reductase seem to function in the molecular mechanisms underlying the tolerance of nodules to salinity.

Mikrobiologiia, 2004 Jan-Feb, 73(1), 62 - 7
{The influence of lipopolysaccharides and glucans from two Rhizobium leguminosarum bv . viciae strains on the formation and efficiency of their symbioses with pea plants}; Antipchuk AF et al.; The influence of lipopolysaccharides (LPS), glucans, and their unseparated complexes on nodulation activity of rhizobia and efficiency of their symbioses with pea plants was studied in vegetation experiments . Two Rhizobium leguminosarum bv . viciae strains which differed in their symbiotic properties were used: strain 31 (fix+, efficient, moderately virulent, moderately competitive), and strain 248b (fix-, inefficient, highly virulent, highly competitive) . Preparations of LPS-glucan complex and the respective LPS from the highly virulent strain 248b increased the nodulation activity of both strains by 10-26% . Analogous preparations from a less virulent strain 31 did not have this ability . Unseparated LPS-glucan complexes from these strains increased the productivity of plants infected with the efficient strain by 18-23% but did not change it in plants inoculated with the other, inefficient strain . No significant influence of LPS preparations on the symbiosis productivity was observed . Glucans from both strains enhanced the nodulation ability of the highly virulent strain by 36-56% . In addition, treatment of pea plants with glucan from strain 248b increased nitrogen fixation by root nodules by 27% in plants inoculated with strain 31 . In general, the formation and efficiency of the symbiosis of R . leguminosarum bv . viciae with pea plants was more influenced by preparations from strain 248b, highly virulent but deficient in nitrogen fixation, than by preparations from the nitrogen fixation-proficient but less virulent strain 31.

J Hered, 2004 Mar-Apr, 95(2), 154 - 7
Genetics of promiscuous nodulation in soybean: nodule dry weight and leaf color score; Gwata ET et al.; The symbiotic relationship between the soybean plant and rhizobium results in fixation of atmospheric nitrogen (N(2)) in the root nodules, with the result that nitrogenous fertilization of the soybean is unnecessary . The effectiveness of nodule formation and N(2) fixation with rhizobial strains is under genetic control with two general categories identified: (1) promiscuous, which produces functional nodules with cowpea-type rhizobial strains; and (2) nonpromiscuous, which forms no or nonfunctional nodules with these strains . The segregation pattern of this promiscuity trait was studied using nodule dry weight (NDW) and leaf color score (LCS) as indicators of N(2) fixation effectiveness . Individual plants in each of six populations {P(1) = nonpromiscuous, P(2) = promiscuous, F(1) = P(1) x P(2) (and the reciprocal cross), BC(1)(P(1)) = F(1) (female) x P(1), BC(1)(P(2)) = F(1) (female) x P(2), F(2)} were scored for these characters after inoculation with a rhizobial strain that would distinguish between both types . For NDW, nonpromiscuity was found to be partially dominant (h/d = 0.37), controlled by four loci . For LCS, nonpromiscuity was shown to be almost completely dominant (h/d = 0.74), controlled by two loci . LCS was a more meaningful estimate of N(2) fixation because it represented the total effectiveness of nodulation to provide nitrogen for the plant.

Proc Natl Acad Sci U S A, 2004 Mar 30, 101(13), 4701 - 5 Epub 2004 Mar 01.
A Ca2+/calmodulin-dependent protein kinase required for symbiotic nodule development: Gene identification by transcript-based cloning; Mitra RM et al.; In the establishment of the legume-rhizobial symbiosis, bacterial lipochitooligosaccharide signaling molecules termed Nod factors activate the formation of a novel root organ, the nodule . Nod factors elicit several responses in plant root hair cells, including oscillations in cytoplasmic calcium levels (termed calcium spiking) and alterations in root hair growth . A number of plant mutants with defects in the Nod factor signaling pathway have been identified . One such Medicago truncatula mutant, dmi3, exhibits calcium spiking and root hair swelling in response to Nod factor, but fails to initiate symbiotic gene expression or cell divisions for nodule formation . On the basis of these data, it is thought that the dmi3 mutant perceives Nod factor but fails to transduce the signal downstream of calcium spiking . Additionally, the dmi3 mutant is defective in the symbiosis with mycorrhizal fungi, indicating the importance of the encoded protein in multiple symbioses . We report the identification of the DMI3 gene, using a gene cloning method based on transcript abundance . We show that transcript-based cloning is a valid approach for cloning genes in barley, indicating the value of this technology in crop plants . DMI3 encodes a calcium/calmodulin-dependent protein kinase . Mutants in pea sym9 have phenotypes similar to dmi3 and have alterations in this gene . The DMI3 class of proteins is well conserved among plants that interact with mycorrhizal fungi, but it is less conserved in Arabidopsis thaliana, which does not participate in the mycorrhizal symbiosis.

Int Rev Cytol, 2004, 234, 201 - 62
Organogenesis of legume root nodules; Patriarca EJ et al.; The N(2)-fixing nodules elicited by rhizobia on legume roots represent a useful model for studying plant development . Nodule formation implies a complex progression of temporally and spatially regulated events of cell differentiation/dedifferentiation involving several root tissues . In this review we describe the morphogenetic events leading to the development of these histologically well-structured organs . These events include (1) root hair deformation, (2) development and growth of infection threads, (3) induction of the nodule primordium, and (4) induction, activity, and persistence of the nodular meristem and/or of foci of meristematic activities . Particular attention is given to specific aspects of the symbiosis, such as the early stages of intracellular invasion and to differentiation of the intracellular form of rhizobia, called symbiosomes . These developmental aspects were correlated with (1) the regulatory signals exchanged, (2) the plant genes expressed in specific cell types, and (3) the staining procedures that allow the recognition of some cell types . When strictly linked with morphogenesis, the nodulation phenotypes of plant and bacterial mutants such as the developmental consequence of the treatment with metabolic inhibitors, metabolic intermediates, or the variation of physical parameters are described . Finally, some aspects of nodule senescence and of regulation of nodulation are discussed.

Carbohydr Res, 2004 Apr 28, 339(6), 1061 - 7
Acacia senegal and Prosopis chilensis-nodulating rhizobia Sinorhizobium arboris HAMBI 2361 and S . kostiense HAMBI 2362 produce tetra- and pentameric LCOs that are N-methylated, O-6-carbamoylated and partially sulfated; Nowak P et al.; Sinorhizobium arboris and S . kostiense are rhizobia that nodulate the tropical leguminous trees Acacia senegal and Prosopis chilensis . The lipochito-oligosaccharidic signalling molecules (LCOs) of S . arboris HAMBI 2361 and S . kostiense HAMBI 2362 were analyzed by mass spectrometry . The major LCOs produced by the strains were shown to be pentameric, acylated with common fatty acids, N-methylated, O-6-carbamoylated and partially sulfated, as are the LCOs characterized to date for other Acacia-nodulating rhizobia . Besides the major LCOs the two strains produced (i) tetrameric LCOs, (ii) LCOs acylated with fatty acids other than those commonly found, (iii) LCOs with only an acyl substituent and (iv) noncarbamoylated LCOs . Production of LCOs (i) to (iii) are novel among Acacia-nodulating rhizobia . The roles of the different structural characteristics of LCOs in the rhizobium-A . senegal symbiosis are discussed . Specific structural features of the LCOs are proposed to be important in the selection of effective nitrogen-fixing rhizobia by A . senegal.

J Bacteriol, 2004 Apr, 186(8), 2439 - 48
Expression islands clustered on the symbiosis island of the Mesorhizobium loti genome; Uchiumi T et al.; Rhizobia are symbiotic nitrogen-fixing soil bacteria that are associated with host legumes . The establishment of rhizobial symbiosis requires signal exchanges between partners in microaerobic environments that result in mutualism for the two partners . We developed a macroarray for Mesorhizobium loti MAFF303099, a microsymbiont of the model legume Lotus japonicus, and monitored the transcriptional dynamics of the bacterium during symbiosis, microaerobiosis, and starvation . Global transcriptional profiling demonstrated that the clusters of genes within the symbiosis island (611 kb), a transmissible region distinct from other chromosomal regions, are collectively expressed during symbiosis, whereas genes outside the island are downregulated . This finding implies that the huge symbiosis island functions as clustered expression islands to support symbiotic nitrogen fixation . Interestingly, most transposase genes on the symbiosis island were highly upregulated in bacteroids, as were nif, fix, fdx, and rpoN . The genome region containing the fixNOPQ genes outside the symbiosis island was markedly upregulated as another expression island under both microaerobic and symbiotic conditions . The symbiosis profiling data suggested that there was activation of amino acid metabolism, as well as nif-fix gene expression . In contrast, genes for cell wall synthesis, cell division, DNA replication, and flagella were strongly repressed in differentiated bacteroids . A highly upregulated gene in bacteroids, mlr5932 (encoding 1-aminocyclopropane-1-carboxylate deaminase), was disrupted and was confirmed to be involved in nodulation enhancement, indicating that disruption of highly expressed genes is a useful strategy for exploring novel gene functions in symbiosis.

J Gen Appl Microbiol, 2004 Feb, 50(1), 17 - 27
A novel symbiotic nitrogen-fixing member of the Ochrobactrum clade isolated from root nodules of Acacia mangium; Ngom A et al.; Ten strains of root nodule bacteria were isolated from the nodules of Acacia mangium grown in the Philippines and Thailand . Partial sequences (approx . 300 bp) of the 16S rRNA gene of each isolate were analyzed . The nucleotide sequences of strain DASA 35030 indicated high homology (>99%) with members of the genus Ochrobactrum in Brucellaceae, although the sequences of other isolates were homologous to those of two distinct genera Bradyrhizobium and Rhizobium . The strain DASA 35030 was strongly suggested to be a strain of Ochrobactrum by full length sequences of the 16S rRNA gene, fatty acids composition, G+C contents of the DNA, and other physiological characteristics . Strain DASA 35030 induced root nodules on A . mangium, A . albida and Paraserianthes falcataria . The nodules formed by strain DASA 35030 fixed nitrogen and the morphology of the nodules is the same as those of nodules formed by the other isolates . This is the first report that the strain of Ochrobactrum possesses complete symbiotic ability with Acacia.

Appl Biochem Biotechnol, 2004 Spring, 113-116, 639 - 52
Effect of process parameters on production of a biopolymer by Rhizobium sp; Duta FP et al.; The production of biopolymers by a Rhizobium strain was studied under batch and bioreactor conditions . The best viscosity levels were obtained under low mannitol concentrations as well as low agitation and aeration conditions . Infrared spectra indicated the presence of chemical groups characteristic of microbially produced biopolymers, including C = O and O-acetyl groups . Thermogravimetric analysis showed the characteristic degradation profiles of the exopolysaccharide produced (T(onset) = 290degreesC) . The experimental design showed that a low substrate concentration (10.0 g/L), and low aeration (0.2 vvm) and agitation (200 rpm) levels should be used . The maximum yield of the process was a Yp/s (g/g) of 0.19 +/- 0.1, obtained under optimized conditions.

J Ind Microbiol Biotechnol, 2004 Mar, 31(3), 122 - 6 Epub 2004 Mar 30.
Cheese whey: an alternative growth and protective medium for Rhizobium loti cells; Estrella MJ et al.; Cheese whey (CW)-based growth medium efficiently protects Rhizobium loti cells during freezing and desiccation and can maintain their growth in a manner similar to that of traditional mannitol-based medium (YEM) . The cheese-whey-based medium (CW) improved viability when used to re-suspend cell pellets kept at -20 degrees C and -80 degrees C and resulted in the survival of over 90% of the cells . Moreover, bacterial pellets obtained from cells grown in CW withstand desiccation better than cells grown in YEM . Survival was over 60% after 30 days at 4 degrees C . No differences were observed in nodulation efficiency between YEM-grown and CW-grown cells . Fast protein liquid chromatography (FPLC) protocols are presented for total protein profile analyses of sweet and acid cheese whey.

Can J Microbiol, 2001 Jan, 47(1), 81 - 5
Genetic diversity of rhizobia nodulating Trifolium ambiguum in North America; Seguin P et al.; Kura clover (Trifolium ambiguum M.B.) is a persistent rhizomatous forage legume, whose use in the U.S.A . is limited by establishment difficulties in part attributable to nodulation problems . In this study, soil was collected from established stands of Kura clover growing in 9 diverse North American environments . Rhizobia were plant-trapped using Kura clover cv . Endura as host, then rhizobia from nodules fingerprinted using BOX-PCR . The diversity of isolates from North America was then contrasted to that of rhizobia from a single Caucasian environment (Russia), the center of origin for this species . Populations were characterized using clustering methods, and genetic diversity estimated using the Shannon-Weaver diversity index . The genetic diversity of the North American populations was extremely limited, all isolates being closely related to two of the strains found in a locally available commercial inoculant . In contrast, Russian isolates formed a distinct cluster with significant internal genetic diversity . Genetic diversity indices for the North American and Russian populations were 3.5 and 10.76, respectively . The implication of this and other studies is that Kura clover is highly specific in Rhizobium requirement . If the performance of this legume in the U.S.A . is to be improved, either by modifying current establishment practices or plant breeding, it is essential that these studies be paralleled by more collections and evaluation of rhizobia from its center of origin, given the extremely limited diversity of rhizobia found in North America.

Plant Cell Physiol, 2004 Mar, 45(3), 300 - 8
Proteomic analysis on symbiotic differentiation of mitochondria in soybean nodules; Hoa le TP et al.; Symbiotic interactions between legume plants and rhizobia induce specific metabolisms and intracellular organelles in nodules . For surveying symbiotic differentiation of a key organelle, mitochondria, protein constituents of soybean nodule and root mitochondria were compared after two-dimensional (2-D) electrophoresis, and the proteins were characterized in combination with matrix-assisted desorption/ionization time-of-flight mass spectrometry, electrospray ionization mass spectrometry and N-terminal amino acid sequencing . Of the proteins that were detected only in nodule mitochondria, phosphoserine aminotransferase, flavanone 3-hydroxylase, coproporphyrinogen III oxidase, one ribonucleoprotein and three unknown proteins were identified . Seven up-regulated, eight down-regulated and two strongly suppressed protein spots in nodule mitochondria were also assigned protein identities . The physiological roles of these differential expressions were discussed in relation to nodule-specific metabolisms in soybean nodules.

Plant Biol (Stuttg), 2004 Mar-Apr, 6(2), 105 - 8
Gene expression analysis in cucumber leaves primed by root colonization with Pseudomonas chlororaphis O6 upon challenge-inoculation with Corynespora cassiicola; Kim MS et al.; Root colonization by Pseudomonas chlororaphis O6, a non-pathogenic rhizobacterium, induced systemic resistance in cucumber against target leaf spot caused by Corynespora cassiicola . A cDNA library was constructed using mRNA extracted from cucumber leaves 12 h after inoculation with C . cassiicola, using plants colonized by O6 . To identify genes involved in O6-mediated induced systemic resistance (ISR), we employed a subtractive hybridization method using mRNAs extracted from pathogen-challenged cucumber leaves of plants lacking colonization . Differential screening of the cDNA library led to the isolation of six distinct genes encoding a GTP binding protein, a 60S ribosomal protein, a hypersensitive-induced reaction protein, a ubiquitin extension protein, a pyridine nucleotide-disulfide oxidoreductase, and a signal recognition particle receptor . Expression of these genes was not induced by O6 colonization alone . Rather, transcript accumulation of these genes increased significantly faster and stronger in the O6 colonized than in non-colonized plants after challenge infection . Therefore, O6-mediated ISR may be associated with an enhanced capacity for the rapid and effective activation of cellular defence responses after challenge inoculation.

Nat Rev Microbiol, 2004 Jan, 2(1), 43 - 56
Trichoderma species--opportunistic, avirulent plant symbionts; Harman GE et al.; Trichoderma spp . are free-living fungi that are common in soil and root ecosystems . Recent discoveries show that they are opportunistic, avirulent plant symbionts, as well as being parasites of other fungi . At least some strains establish robust and long-lasting colonizations of root surfaces and penetrate into the epidermis and a few cells below this level . They produce or release a variety of compounds that induce localized or systemic resistance responses, and this explains their lack of pathogenicity to plants . These root-microorganism associations cause substantial changes to the plant proteome and metabolism . Plants are protected from numerous classes of plant pathogen by responses that are similar to systemic acquired resistance and rhizobacteria-induced systemic resistance . Root colonization by Trichoderma spp . also frequently enhances root growth and development, crop productivity, resistance to abiotic stresses and the uptake and use of nutrients.

Physiol Plant, 2004 Jan, 120(1), 132 - 139
Phytohormonal responses in enod40-overexpressing plants of Medicago truncatula and rice; Dey M et al.; Phytohormones are well-known regulators of the symbiotic Rhizobium-legume association in the plant host . The enod40 nodulin gene is associated with the earliest phases of the nodule organogenesis programme in the legume host and modifying its expression resulted in perturbations of nodule development in Medicago truncatula . Therefore in our pursuit to mimic the initial signal transduction steps of legume nodulation in the alien physiological set-up of a rice plant, we have expressed the Mtenod40 gene in rice . Molecular data confirm the stable integration, inheritance and transcription of the foreign gene in this non-legume . We have compared the phytohormonal responses of Mtenod40-overexpressing and control plants in a homologous legume background (M . truncatula) and in the non-legume rice . An enod40-mediated root growth response, induced by inhibition of ethylene biosynthesis, was observed in both plants . On the other hand, a significant differential effect of cytokinins was observed only in rice plants . This suggests that ethylene inhibits enod40 action both in legumes and non-legumes and reinforces that some of the early signal transduction steps of the nodule developmental programme may function in rice.

Physiol Plant, 2004 Apr, 120(4), 546 - 555
Rhizobial nod gene-inducing activity in pea nodulation mutants: dissociation of nodulation and flavonoid response; Novak K et al.; The root exudates of 24 pea (Pisum sativum L.) non-nodulating mutants, originating from cv . 'Finale', were tested for Rhizobium nodulation (nod) gene-inducing activity to reveal potential differences from the wild type . The interaction with flavonoid receptor NodD of R . leguminosarum bv . viciae strain 248 was monitored as the activity of the regulated operon nodABC fused to the reporter gene lacZ . In the uninoculated plants, the only decreased exudate activity was found in the line Risnod17 and ascribed to the impaired root growth in this line . Consequently, constitutive deficiency in the flavonoid nod gene inducers is not a cause of the asymbiotic phenotypes in the studied set . On the other hand, line Risnod5 had constitutively higher nod gene-inducing activity which might be responsible for its conditional nodulation and Fix(-) phenotype when the nodules are present . Monitoring of the response of wild-type plants to inoculation revealed an increase in nod gene-inducing activity (Ini response) starting first day after inoculation . The chosen mutant lines Risnod22 (putative locus sym9) and Risnod25 (putatively sym8) showed Ini as well indicating that the flavonoid burst can be mutationally dissociated from nodule initiation . On the other hand, absence of Ini in Risnod20 (assumed sym19) suggests branching of the signal transduction pathway downstream of this mutation and places it upstream of the two former mutations in the transduction of the bacterial Nod factor signal . More intense Ini response observed in the supernodulating mutant RisfixC, included in the study for its contrasting symbiotic phenotype, suggests common regulation of nodulation and Ini response via systemic shoot factor . The time shift in the exudation of isoflavonoid phytoalexin pisatin compared to Ini, its increased level in Risnod25 and lowered level in RisfixC are consistent with the regulatory role of plant defence in nodule initiation, which might be mediated directly by isoflavonoids as negative regulators.

Antonie Van Leeuwenhoek, 2004 Feb, 85(2), 115 - 27
Phenotypic and genotypic analysis of rhizobia isolated from pasture legumes native of Sardinia and Asinara Island; Safronova VI et al.; Thirty-five rhizobial strains were isolated from nodules of Lotus edulis, L . ornithopodioides, L . cytisoides, Hedysarum coronarium, Ornithopus compressus and Scorpiurus muricatus growing in Sardinia and Asinara Island . Basic characteristics applied to identification of rhizobia such as symbiotic properties, antibiotic- and salt-resistance, temperate-sensitivities, utilization of different sources of carbon and nitrogen were studied . The results from the 74 metabolic tests were used for cluster analysis of the new rhizobial isolates and 28 reference strains, belonging to previously classified and unclassified fast-, intermediate- and slow-growing rhizobia . All strains examined were divided into two large groups at a linkage distance of 0.58 . None of the reference strains clustered with the new rhizobial isolates, which formed five subgroups almost respective of their plant origin . RFLP analysis of PCR-amplified 16S-23S rDNA IGS showed that the levels of similarity between rhizobial isolates from Ornithopus, Hedysarum and Scorpiurus, and the type strains of Rhizobium leguminosarum, Mesorhizobium loti, M . ciceri, M . mediterraneum, Sinorhizobium meliloti and Bradyrhizobium japonicum were not more than 30% . Thus, it can be assumed that these groups of new rhizobial isolates are not closely related to the validly described rhizobial species.

Antonie Van Leeuwenhoek, 2004 Apr, 85(3), 217 - 27
Regulation of pssA and pssB gene expression in Rhizobium leguminosarum bv . trifolii in response to environmental factors; Janczarek M et al.; Exopolysaccharide (EPS) produced by Rhizobium leguminosarum plays an important role in indeterminate-type nodulation . The pssA gene, encoding the first glycosyl transferase and pssB, encoding inositol monophosphatase, are involved in EPS synthesis and regulation in R . leguminosarum bv . trifolii . Using pssA-lacZ and pssB-lacZ plasmid-borne transcriptional fusions, a stimulating effect of phosphate on the transcription of both genes was observed . The increase in the transcription of pssA and pssB genes was correlated with a significant enhancement in EPS production in rhizobia grown in the high phosphate concentrations . In the presence of ammonium, transcription of the pss-lacZ fusions was repressed, especially the pssB-lacZ fusion, but EPS production still remained enhanced . Root exudate positively influenced the level of pss-lacZ transcription . These results indicate a complex regulation of pss gene transcription by several environmental factors . In addition, the expression of pssA and pssB genes in the symbiotic state was examined using pss-gusA plasmid-borne transcriptional fusions . It was found that the pssA-gusA fusion was not expressed in the nodules . The pssB-gusA fusion was expressed mainly in the infection zone and interzone of the nodule and the pattern of expression was dependent on the plant phosphate availability.

Arch Microbiol, 2004 May, 181(5), 337 - 44 Epub 2004 Mar 13.
Rhizobium etli maize populations and their competitiveness for root colonization; Rosenblueth M et al.; Rhizobium etli, which normally forms nitrogen-fixing nodules on Phaseolus vulgaris (common bean), is a natural maize endophyte . The genetic diversity of R . etli strains from bulk soil, bean nodules, the maize rhizosphere, the maize root, and inside stem tissue in traditional fields where maize is intercropped with P . vulgaris-beans was analyzed . Based on plasmid profiles and alloenzymes, it was determined that several R . etli types were preferentially encountered as putative maize endophytes . Some of these strains from maize were more competitive maize-root colonizers than other R . etli strains from the rhizosphere or from bean nodules . The dominant and highly competitive strain Ch24-10 was the most tolerant to 6-methoxy-2-benzoxazolinone (MBOA), a maize antimicrobial compound that is inhibitory to some bacteria and fungi . The R . tropici strain CIAT899, successfully used as inoculant of P . vulgaris, was also found to be a competitive maize endophyte in inoculation experiments.

Carbohydr Res, 2004 Feb 25, 339(3), 519 - 27
Synthesis and characterization of carboxymethylated cyclosophoraose, and its inclusion complexation behavior; Lee S et al.; Carboxymethylated cyclosophoraoses (CM-Cys) were synthesized by chemical modification of a family of neutral cyclosophoraoses isolated from Rhizobium leguminosarum biovar trifolii . Structural analyses of the CM-Cys were carried out using NMR and FTIR spectroscopies, and the molecular weight distributions were confirmed with MALDI-TOF mass spectrometry . Based on structural characterization, native cyclosophoraoses were successfully substituted with carboxymethyl groups at the OH-4 and OH-6 of the glucose residues with degrees of substitution (DS) ranging from 0.012 to 0.290 . CM-Cys was also used as a host for the inclusion complexation with hydrobenzoin (HB) and N-acetyltryptophan (N-AcTrp) as guest molecules . NMR spectroscopic analyses of the complexes showed that the CM-Cys induced chemical shifts of some protons of the guest molecules upon the complexation . Phase solubility studies of the guest molecules by CM-Cys were performed using HPLC, and the results were compared with those of native cyclosophoraoses . The solubility of HB and N-AcTrp was enhanced by the CM-Cys about 5.1- and 299-fold, respectively.

Carbohydr Res, 2004 Feb 25, 339(3), 461 - 8
Cyclosophoraose as a catalytic carbohydrate for methanolysis; Lee S et al.; A novel catalytic methanolysis can be induced by a natural cyclooligosaccharide, a cyclosophoraose (cyclic-(1-->2)-beta-D-glucan, Cys), which is a member of a family of unbranched cyclooligosaccharides produced as intra- or extraoligosaccharides by soil microorganisms of the genus, Rhizobium . Cys catalyzed the methanolysis for 5(4H)-oxazolones and various phospholipids . Cys enhanced the methanolysis reaction about 9200-fold for a benzylidene oxazolone or 250-fold for dipalmitoylphosphatidylcholine comparing with control . In this study, we describe that natural cyclosophoraoses isolated from the Rhizobium species function as catalytic carbohydrates for the methanolysis.

Mol Phylogenet Evol, 2004 Mar, 30(3), 720 - 32
Phylogenetic analyses of symbiotic nodulation genes support vertical and lateral gene co-transfer within the Bradyrhizobium genus; Moulin L et al.; Symbiotic nitrogen fixing bacteria-known as rhizobia-harbour a set of nodulation (nod) genes that control the synthesis of modified lipo-chitooligosaccharides, called Nod factors that are required for legume nodulation . The nodA gene, which is essential for symbiosis, is responsible for the attachment of the fatty acid group to the oligosaccharide backbone . The nodZ, nolL, and noeI genes are involved in specific modifications of Nod factors common to bradyrhizobia, i.e., the transfer of a fucosyl group on the Nod factor core, fucose acetylation and fucose methylation, respectively . PCR amplification, sequencing and phylogenetic analysis of nodA gene sequences from a collection of diverse Bradyrhizobium strains revealed the monophyletic character with the possible exception of photosynthetic Bradyrhizobium, despite high sequence diversity . The distribution of the nodZ, nolL, and noeI genes in the studied strains, as assessed by gene amplification, hybridization or sequencing, was found to correlate with the nodA tree topology . Moreover, the nodA, nodZ, and noeI phylogenies were largely congruent, but did not closely follow the taxonomy of the strains shown by the housekeeping 16S rRNA and dnaK genes . Additionally, the distribution of nodZ, noeI, and nolL genes suggested that their presence may be related to the requirements of their legume hosts . These data indicated that the spread and maintenance of nodulation genes within the Bradyrhizobium genus occurred through vertical transmission, although lateral gene transfer also played a significant role.

Annu Rev Phytopathol, 1998, 36, 453 - 83
Systemic resistance induced by rhizosphere bacteria; van Loon LC et al.; Nonpathogenic rhizobacteria can induce a systemic resistance in plants that is phenotypically similar to pathogen-induced systemic acquired resistance (SAR) . Rhizobacteria-mediated induced systemic resistance (ISR) has been demonstrated against fungi, bacteria, and viruses in Arabidopsis, bean, carnation, cucumber, radish, tobacco, and tomato under conditions in which the inducing bacteria and the challenging pathogen remained spatially separated . Bacterial strains differ in their ability to induce resistance in different plant species, and plants show variation in the expression of ISR upon induction by specific bacterial strains . Bacterial determinants of ISR include lipopolysaccharides, siderophores, and salicylic acid (SA) . Whereas some of the rhizobacteria induce resistance through the SA-dependent SAR pathway, others do not and require jasmonic acid and ethylene perception by the plant for ISR to develop . No consistent host plant alterations are associated with the induced state, but upon challenge inoculation, resistance responses are accelerated and enhanced . ISR is effective under field conditions and offers a natural mechanism for biological control of plant disease.

Annu Rev Phytopathol, 1998, 36, 165 - 205
Developing sustainable systems for nematode management; Barker KR et al.; Early researchers identified key concepts and developed tactics for multiple-option management of nematodes . Although the emphasis on integrated pest management over the past three decades has promoted strategies and tactics for nematode management, comprehensive studies on the related soil biology-ecology are relatively recent . Traditional management tactics include host resistance (where available), cultural tactics such as rotation with nonhosts, sanitation and avoidance, and destruction of residual crop roots, and the judicious use of nematicides . There have been advances in biological control of nematodes, but field-scale exploitation of this tactic remains to be realized . New technologies and resources are currently becoming central to the development of sustainable systems for nematode-pest-crop management: molecular diagnostics for nematode identification, genetic engineering for host resistance, and the elucidation and application of soil biology for general integrated cropping systems . The latter strategy includes the use of nematode-pest antagonistic cover crops, animal wastes, and limited tillage practices that favor growth-promoting rhizobacteria, earthworms, predatory mites, and other beneficial organisms while suppressing parasitic nematodes and other plant pathogens . Certain rhizobacteria may induce systemic host resistance to nematodes and, in some instances, to foliage pathogens . The systems focusing on soil biology hold great promise for sustainable crop-nematode management, but only a few research programs are currently involved in this labor-intensive endeavor.

Annu Rev Plant Physiol Plant Mol Biol, 1997 Jun, 48, 493 - 523
METABOLITE TRANSPORT ACROSS SYMBIOTIC MEMBRANES OF LEGUME NODULES; Udvardi MK et al.; Infection of legume roots or stems with soil bacteria of the Rhizobiaceae results in the formation of nodules that become symbiotic nitrogen-fixing organs . Within the infected cells of these nodules, bacteria are enveloped in a membrane of plant origin, called the peribacteroid membrane (PBM), and divide and differentiate to form nitrogen-fixing bacteroids . The organelle-like structure comprised of PBM and bacteroids is termed the symbiosome, and is the basic nitrogen-fixing unit of the nodule . The major exchange of nutrients between the symbiotic partners is reduced carbon from the plant, to fuel nitrogenase activity in the bacteroid, and fixed nitrogen from the bacteroid, which is assimilated in the plant cytoplasm . However, many other metabolites are also exchanged . The metabolic interaction between the plant and the bacteroids is regulated by a series of transporters and channels on the PBM and the bacteroid membrane, and these form the focus of this review.

Mol Genet Genomics, 2004 May, 271(4), 416 - 25 Epub 2004 Mar 06.
Sinorhizobium meliloti RpoH1 is required for effective nitrogen-fixing symbiosis with alfalfa; Mitsui H et al.; Sinorhizobium meliloti is a root-nodulating, nitrogen-fixing bacterium . An S . meliloti strain that is mutant for the rpoH(1) gene, which encodes a sigma(32)-like protein, elicits the formation of ineffective nodules on the host plant alfalfa . We characterized the rpoH(1) mutant for phenotypes related to symbiosis . Alfalfa nodules formed by the rpoH(1) mutant exhibited greatly reduced levels of acetylene reduction activity compared to the wild-type nodules . Whereas intracellular colonization by rhizobia was observed in a zone just below the apical meristem, we found ultrastructural abnormalities and signs of degeneration of bacteroids within many host cells in the proximally adjacent zone . In the proximal part of the nodule, only a few nodule cells contained bacteroids . In contrast, the rpoH(1) mutant showed normal induction of nitrogen fixation gene expression in microaerobic culture . These results suggest that the rpoH(1) mutation causes early senescence of bacteroids during the endosymbiotic process, but does not affect the invasion process or the synthesis of the nitrogenase machinery . The rpoH(1) mutant exhibited increased sensitivity to various agents and to acid pH, suggesting that RpoH(1) is required to protect the bacterial cell against environmental stresses encountered within the host . Since RpoH(1) was previously reported to be required for the synthesis of some heat shock proteins (Hsps), we examined the transcription of several genes for Hsp homologs . We found that transcription of groESL(5), lon, and clpB after heat shock was RpoH(1)-dependent, and conserved nucleotide sequences were found in the -35 and -10 regions upstream of the transcription start sites of these genes . Although groESL(5) expression is almost completely dependent on RpoH(1), we found that a groESL(5) mutant strain is still capable of normal symbiotic nitrogen fixation on alfalfa.

Appl Environ Microbiol, 2004 Mar, 70(3), 1537 - 44
2-O-methylation of fucosyl residues of a rhizobial lipopolysaccharide is increased in response to host exudate and is eliminated in a symbiotically defective mutant; Noel KD et al.; When Rhizobium etli CE3 was grown in the presence of Phaseolus vulgaris seed extracts containing anthocyanins, its lipopolysaccharide (LPS) sugar composition was changed in two ways: greatly decreased content of what is normally the terminal residue of the LPS, di-O-methylfucose, and a doubling of the 2-O-methylation of other fucose residues in the LPS O antigen . R . etli strain CE395 was isolated after Tn5 mutagenesis of strain CE3 by screening for mutant colonies that did not change antigenically in the presence of seed extract . The LPS of this strain completely lacked 2-O-methylfucose, regardless of whether anthocyanins were present during growth . The mutant gave only pseudonodules in association with P . vulgaris . Interpretation of this phenotype was complicated by a second LPS defect exhibited by the mutant: its LPS population had only about 50% of the normal amount of O-antigen-containing LPS (LPS I) . The latter defect could be suppressed genetically such that the resulting strain (CE395 alpha 395) synthesized the normal amount of an LPS I that still lacked 2-O-methylfucose residues . Strain CE395 alpha 395 did not elicit pseudonodules but resulted in significantly slower nodule development, fewer nodules, and less nitrogenase activity than lps(+) strains . The relative symbiotic deficiency was more severe when seeds were planted and inoculated with bacteria before they germinated . These results support previous conclusions that the relative amount of LPS I on the bacterial surface is crucial in symbiosis, but LPS structural features, such as 2-O-methylation of fucose, also may facilitate symbiotic interactions.

Appl Environ Microbiol, 2004 Mar, 70(3), 1287 - 96
Regulatory role of Rhizobium etli CNPAF512 fnrN during symbiosis; Moris M et al.; The Rhizobium etli CNPAF512 fnrN gene was identified in the fixABCX rpoN(2) region . The corresponding protein contains the hallmark residues characteristic of proteins belonging to the class IB group of Fnr-related proteins . The expression of R . etli fnrN is highly induced under free-living microaerobic conditions and during symbiosis . This microaerobic and symbiotic induction of fnrN is not controlled by the sigma factor RpoN and the symbiotic regulator nifA or fixLJ, but it is due to positive autoregulation . Inoculation of Phaseolus vulgaris with an R . etli fnrN mutant strain resulted in a severe reduction in the bacteroid nitrogen fixation capacity compared to the wild-type capacity, confirming the importance of FnrN during symbiosis . The expression of the R . etli fixN, fixG, and arcA genes is strictly controlled by fnrN under free-living microaerobic conditions and in bacteroids during symbiosis with the host . However, there is an additional level of regulation of fixN and fixG under symbiotic conditions . A phylogenetic analysis of the available rhizobial FnrN and FixK proteins grouped the proteins in three different clusters.

Mol Plant Microbe Interact, 2004 Mar, 17(3), 283 - 91
A Rhizobium leguminosarum lipopolysaccharide lipid-A mutant induces nitrogen-fixing nodules with delayed and defective bacteroid formation; Vedam V et al.; Lipopolysaccharides from pea-nodulating strain Rhizobium leguminosarum by . viciae 3841, as all other members of the family Rhizobiaceae with the possible exception of Azorhizobium caulinodans, contains a very long chain fatty acid; 27-hydroxyoctacosanoic acid (27OHC28:0) in its lipid A region . The exact function and importance of this residue, however, is not known . In this work, a previously constructed mutant, Rhizobium leguminosarum by . viciae 22, deficient in the fatty acid residue, was analyzed for its symbiotic phenotype . While the mutant was able to form nitrogen-fixing nodules, a detailed study of the timing and efficiency of nodulation using light and electron microscopy showed that there was a delay in the onset of nodulation and nodule tissue invasion . Further, microscopy showed that the mutant was unable to differentiate normally forming numerous irregularly shaped bacteroids, that the resultant mature bacteroids were unusually large, and that several bacteroids were frequently enclosed in a single symbiosome membrane, a feature not observed with parent bacteroids . In addition, the mutant nodules were delayed in the onset of nitrogenase production and showed reduced nitrogenase throughout the testing period . These results imply that the lack of 27OHC28:0 in the lipid A in mutant bacteroids results in altered membrane properties that are essential for the development of normal bacteroids.

Microbiology, 2004 Mar, 150(Pt 3), 665 - 75
Characterization of a new internal promoter (P3) for Rhizobium leguminosarum hydrogenase accessory genes hupGHIJ; Martinez M et al.; Synthesis of the Rhizobium leguminosarum {NiFe} hydrogenase requires the participation of 16 accessory genes (hupCDEFGHIJKhypABFCDEX) besides the genes encoding the structural proteins (hupSL) . Transcription of hupSL is controlled by a -24/-12-type promoter (P(1)), located upstream of hupS and regulated by NifA . In this work, a second -24/-12-type promoter (P(3)), located upstream of the hupG gene and transcribing hupGHIJ genes in R . leguminosarum pea (Pisum sativum L.) bacteroids, has been identified in the hup gene cluster . Promoter P(3) was also active in R . leguminosarum free-living cells, as evidenced by genetic complementation of hydrogenase mutants . Both NifA and NtrC activated P(3) expression in the heterologous host Klebsiella pneumoniae . Also, P(3) activity was highly stimulated by K . pneumoniae NifA in Escherichia coli . This NifA activation of P(3) expression only required the sigma(54)-binding site, and it was independent of any cis-acting element upstream of the sigma(54) box, which suggests a direct interaction of free NifA with the RNA polymerase holoenzyme . P(3)-dependent hupGHIJ expression in pea nodules started in interzone II/III, spanned through nitrogen-fixing zone III, and was coincident with the NifA-dependent nifH expression pattern . However, P(3) was dispensable for hupGHIJ transcription and hydrogenase activity in pea bacteroids due to transcription initiated at P(1) . This fact and the lack of an activator recruitment system suggest that P(3) plays a secondary role in symbiotic hupGHIJ expression.

Environ Int, 2004 May, 30(3), 293 - 300
Revegetating fly ash landfills with Prosopis juliflora L.: impact of different amendments and Rhizobium inoculation; Rai UN et al.; A revegetation trial was conducted to evaluate the feasibility of growing a legume species, Prosopis juliflora L., on fly ash ameliorated with combination of various organic amendments, blue-green algal biofertilizer and Rhizobium inoculation . Significant enhancements in plant biomass, photosynthetic pigments, protein content and in vivo nitrate reductase activity were found in the plants grown on ameliorated fly ash in comparison to the plants growing in unamended fly ash or garden soil . Higher growth was obtained in fly ash amended with blue-green algae (BGA) than farmyard manure or press mud (PM), a waste from sugar-processing industry, due to the greater contribution of plant nutrients, supply of fixed nitrogen and increased availability of phosphorus . Nodulation was suppressed in different amendments of fly ash with soil in a concentration-duration-dependent manner, but not with other amendments . Plants accumulated higher amounts of Fe, Mn, Cu, Zn and Cr in various fly ash amendments than in garden soil . Further, inoculation of the plant with a fly ash tolerant Rhizobium strain conferred tolerance for the plant to grow under fly ash stress conditions with more translocation of metals to the above ground parts . The results showed the potential of P . juliflora to grow in plantations on fly ash landfills and to reduce the metal contents of fly ash by bioaccumulation in its tissues.

FEMS Microbiol Lett, 2004 Feb 16, 231(2), 267 - 75
Disruption of dTDP-rhamnose biosynthesis modifies lipopolysaccharide core, exopolysaccharide production, and root colonization in Azospirillum brasilense; Jofre E et al.; The interaction between Azospirillum brasilense and plants is not fully understood, although several bacterial surface components like exopolysaccharides (EPS), flagella, and capsular polysaccharides are required for attachment and colonization . While in other plant-bacteria associations (Rhizobium-legume, Pseudomonas-potato), lipopolysaccharides (LPS) play a key role in the establishment of an effective association, their role in the root colonization by Azospirillum had not been determined . In this study, we isolated a Tn5 mutant of A . brasilense Cd (EJ1) with an apparently modified LPS core structure, non-mucoid colony morphology, increased EPS production, and affected in maize root colonization . A 3790-bp region revealed the presence of three complete open reading frames designated rmlC, rmlB and rmlD . The beginning of a fourth open reading frame was found and designated rmlA . These genes are organized in a cluster which shows homology to the cluster involved in the synthesis of dTDP-rhamnose in other bacteria . Additionally, the analysis of the monosaccharide composition of LPSs showed a diminution of rhamnose compared to the wild-type strain.

Rev Argent Microbiol, 2003 Oct-Dec, 35(4), 193 - 7
{Characterization of rhizobia causing nodules on leguminous trees native to Uruguay using the rep-PCR technique}; Rodriguez A et al.; Methods for identifying and following microorganisms in the environment such as soils, water and plant association have been highly developed in recent years . In this study, we used rep-PCR for the characterization of Rhizobium bacteria isolated from legume trees native of Uruguay which permitted to follow them in plant inoculation assays . Among the primers used, ERIC and BOX1AR, the latter allowed the differentiation of isolates from different legumes and the finding of a high level of homology among rhizobia that nodulate the same legume species . Besides, it demonstrated that the profiles of the isolates recovered from nodules of Acacia caven, were identical to the ones used as inoculants; this confirmed the efficiency of these methods to follow these diazotrophs in the environment.

Tree Physiol, 1991 Dec, 9(4), 501 - 6
A simple method for measuring acetylene reduction of intact, nodulated black locust seedlings; Johnsen KH et al.; A simple method is described for measuring acetylene (C(2)H(2)) reduction of nodulated black locust (Robinia pseudoacacia L.) seedlings . The C(2)H(2) reduction chamber consists of a standard plant pot sealed at one end with a stopper having holes for the seedling and a gas inlet port, and sealed at the other end with a stoppered collar with a gas port . The chamber temperature is regulated by circulating water at a controlled temperature through a copper coil surrounding the plant pot . A pump recirculates an approximately ten percent C(2)H(2) atmosphere through the system . Nine-week-old black locust seedlings, grown in sand culture and inoculated with Rhizobium, were used to obtain ethylene (C(2)H(4)) production curves . Ethylene production was linear for up to 60 minutes indicating that, for this particular symbiosis, (1) a simple closed system is adequate and (2) a short duration assay (10 minutes) will reliably estimate C(2)H(2) reduction over a longer period (up to at least 60 minutes) . The water content of the soil medium had no influence on the C(2)H(2)/(C(2)H(2) + C(2)H(4)) ratio, which supports the suitability of C(2)H(2) as an internal standard for calculating C(2)H(2) reduction . This system has several desirable features . First, plant disturbance is minimized . Second, plants can be grown in inexpensive pots and no transplanting is necessary . Third, gas exchange and mixing are facilitated by a recirculating pump . Finally, root and nodule temperature can be controlled.

Proc Natl Acad Sci U S A, 2004 Mar 2, 101(9), 2742 - 6 Epub 2004 Feb 17.
ADP reduces the oxygen-binding affinity of a sensory histidine kinase, FixL: the possibility of an enhanced reciprocating kinase reaction; Nakamura H et al.; The rhizobial FixL/FixJ system, a paradigm of heme-based oxygen sensors, belongs to the ubiquitous two-component signal transduction system . Oxygen-free (deoxy) FixL is autophosphorylated at an invariant histidine residue by using ATP and catalyzes the concomitant phosphoryl transfer to FixJ, but oxygen binding to the FixL heme moiety inactivates the kinase activity . Here we demonstrate that ADP acts as an allosteric effector, reducing the oxygen-binding affinity of the sensor domain in FixL when it is produced from ATP in the kinase reaction . The addition of ADP to a solution of purified wild-type FixL resulted in an approximately 4- to 5-fold decrease in oxygen-binding affinity in the presence of FixJ . In contrast, phosphorylation-deficient mutants, in which the well conserved ATP-binding catalytic site of the kinase domain is impaired, showed no such allosteric effect . This discovery casts light on the significance of homodimerization of two-component histidine kinases; ADP, generated in the phosphorylation reaction in one subunit of the homodimer, enhances the histidine kinase activity of the other, analogous to a two-cylinder reciprocating engine by reducing the ligand-binding affinity.

Plant Physiol, 2004 Feb, 134(2), 871 - 9
NopL, an effector protein of Rhizobium sp . NGR234, thwarts activation of plant defense reactions; Bartsev AV et al.; Bacterial effector proteins delivered into eukaryotic cells via bacterial type III secretion systems are important virulence factors in plant-pathogen interactions . Type III secretion systems have been found in Rhizobium species that form symbiotic, nitrogen-fixing associations with legumes . One such bacterium, Rhizobium sp . NGR234, secretes a number of type III effectors, including nodulation outer protein L (NopL, formerly y4xL) . Here, we show that expression of nopL in tobacco (Nicotiana tabacum) prevents full induction of pathogenesis-related (PR) defense proteins . Transgenic tobacco plants that express nopL and were infected with potato virus Y (necrotic strain 605) exhibited only very low levels of chitinase (class I) and beta-1,3-glucanase (classes I and III) proteins . Northern-blot analysis indicated that expression of nopL in plant cells suppresses transcription of PR genes . Treatment with ethylene counteracted the effect of NopL on chitinase (class I) . Transgenic Lotus japonicus plants that expressed nopL exhibited delayed development and low chitinase levels . In vitro experiments showed that NopL is a substrate for plant protein kinases . Together, these data suggest that NopL, when delivered into the plant cell, modulates the activity of signal transduction pathways that culminate in activation of PR proteins.

Genetika, 2003 Dec, 39(12), 1594 - 605
{Microevolution of nodule bacteria in emergence of mutants with changed viability in the "plant-soil" system}; Provorov NA et al.; Simulation of cyclic processes in the plant-soil system was used to analyze the effects of factors responsible for the population dynamics of rhizobia on generation of mutants with changed ex planta viability . Rhizobial evolution in a system of ecological niches (soil, rhizosphere, nodules) was described with recurrent equations . Computer experiments were carried out with parameters determining the mutation pressure, selection, and amplitude of the population wave arising in soil on the release of bacteria from nodules and the rhizosphere . Analysis of the model showed that (1) mutants with enhanced ex planta viability do not completely replace the parental strain and (2) mutants with impaired ex planta viability may be fixed in the population . The maintenance of genotypes subject to elimination from the soil and rhizosphere by Darwinian selection was associated with frequency-dependent selection (FDS), which is effective in competition for nodulation . The FDS index was proposed to characterize FDS pressure and was shown to determine the population polymorphism for adaptive traits . An increase in population wave amplitude proved to increase the fixation level (the proportion in the limiting state of the system) of mutants with enhanced viability and to decrease it in mutants with low viability . The results obtained with the model agreed with the data that, in edaphic stress, rhizobial populations remain highly polymorphic, which is associated with the maintenance of sensitive strains . The simulation procedure may be employed in estimating the genetic consequences of introduction of modified rhizobial strains in the environment.

Mol Plant Microbe Interact, 2004 Feb, 17(2), 216 - 23
Cell surface interactions of Rhizobium bacteroids and other bacterial strains with symbiosomal and peribacteroid membrane components from pea nodules; Bolanos L et al.; Samples of Rhizobium bacteroids isolated from pea nodule symbiosomes reacted positively with a monoclonal antibody recognizing N-linked glycan epitopes on plant glycoproteins associated with the peribacteroid membrane and peribacteroid fluid . An antiserum recognizing the symbiosomal lectin-like glycoprotein PsNLEC-1 also reacted positively . Samples of isolated bacteroids also reacted with an antibody recognizing a glycolipid component of the peribacteroid membrane and plasma membrane . Bacterial cells derived from free-living cultures then were immobilized on nitrocellulose sheets and tested for their ability to associate with components of plant extracts derived from nodule fractionation . A positive antibody-staining reaction indicated that both PsNLEC-1 and membrane glycolipid had become associated with the bacterial surface . A range of rhizobial strains with mutants affecting cell surface polysaccharides all showed similar interactions with PsNLEC-1 and associated plant membranes, with the exception of strain B659 (a deep-rough lipopolysaccharide mutant of Rhizobium leguminosarum) . However, the presence of a capsule of extracellular polysaccharide apparently prevented interactions between rhizobial cells and these plant components . The importance of a close association between peribacteroid membranes, PsNLEC-1, and the bacterial surface is discussed in the context of symbiosome development.

Science, 2004 Feb 27, 303(5662), 1361 - 4 Epub 2004 Feb 12.
A putative Ca2+ and calmodulin-dependent protein kinase required for bacterial and fungal symbioses; Levy J et al.; Legumes can enter into symbiotic relationships with both nitrogen-fixing bacteria (rhizobia) and mycorrhizal fungi . Nodulation by rhizobia results from a signal transduction pathway induced in legume roots by rhizobial Nod factors . DMI3, a Medicago truncatula gene that acts immediately downstream of calcium spiking in this signaling pathway and is required for both nodulation and mycorrhizal infection, has high sequence similarity to genes encoding calcium and calmodulin-dependent protein kinases (CCaMKs) . This indicates that calcium spiking is likely an essential component of the signaling cascade leading to nodule development and mycorrhizal infection, and sheds light on the biological role of plant CCaMKs.

Science, 2004 Feb 27, 303(5662), 1364 - 7 Epub 2004 Feb 12.
Medicago truncatula DMI1 required for bacterial and fungal symbioses in legumes; Ane JM et al.; Legumes form symbiotic associations with both mycorrhizal fungi and nitrogen-fixing soil bacteria called rhizobia . Several of the plant genes required for transduction of rhizobial signals, the Nod factors, are also necessary for mycorrhizal symbiosis . Here, we describe the cloning and characterization of one such gene from the legume Medicago truncatula . The DMI1 (does not make infections) gene encodes a novel protein with low global similarity to a ligand-gated cation channel domain of archaea . The protein is highly conserved in angiosperms and ancestral to land plants . We suggest that DMI1 represents an ancient plant-specific innovation, potentially enabling mycorrhizal associations.

J Appl Microbiol, 2004, 96(3), 473 - 80
Screening plant growth-promoting rhizobacteria for improving growth and yield of wheat; Khalid A et al.; AIMS: Plant growth promoting rhizobacteria (PGPR) are commonly used as inoculants for improving the growth and yield of agricultural crops, however screening for the selection of effective PGPR strains is very critical . This study focuses on the screening of effective PGPR strains on the basis of their potential for in vitro auxin production and plant growth promoting activity under gnotobiotic conditions . METHODS AND RESULTS: A large number of bacteria were isolated from the rhizosphere soil of wheat plants grown at different sites . Thirty isolates showing prolific growth on agar medium were selected and evaluated for their potential to produce auxins in vitro . Colorimetric analysis showed variable amount of auxins (ranging from 1.1 to 12.1 mg l-1) produced by the rhizobacteria in vitro and amendment of the culture media with l-tryptophan (l-TRP), further stimulated auxin biosynthesis (ranging from 1.8 to 24.8 mg l-1) . HPLC analysis confirmed the presence of indole acetic acid (IAA) and indole acetamide (IAM) as the major auxins in the culture filtrates of these rhizobacteria . A series of laboratory experiments conducted on two cv . of wheat under gnotobiotic (axenic) conditions demonstrated increases in root elongation (up to 17.3%), root dry weight (up to 13.5%), shoot elongation (up to 37.7%) and shoot dry weight (up to 36.3%) of inoculated wheat seedlings . Linear positive correlation (r = 0.99) between in vitro auxin production and increase in growth parameters of inoculated seeds was found . Based upon auxin biosynthesis and growth-promoting activity, four isolates were selected and designated as plant growth-promoting rhizobacteria (PGPR) . Auxin biosynthesis in sterilized vs nonsterilized soil inoculated with selected PGPR was also monitored that revealed superiority of the selected PGPR over indigenous microflora . Peat-based seed inoculation with selected PGPR isolates exhibited stimulatory effects on grain yields of tested wheat cv . in pot (up to 14.7% increase over control) and field experiments (up to 27.5% increase over control); however, the response varied with cv . and PGPR strains . CONCLUSIONS: It was concluded that the strain, which produced the highest amount of auxins in nonsterilized soil, also caused maximum increase in growth and yield of both the wheat cv . SIGNIFICANCE AND IMPACT OF STUDY: This study suggested that potential for auxin biosynthesis by rhizobacteria could be used as a tool for the screening of effective PGPR strains.

Chirality, 2004 Mar, 16(3), 204 - 10
Chiral recognition based on enantioselective interactions of propranolol enantiomers with cyclosophoraoses isolated from Rhizobium meliloti; Lee S et al.; Cyclosophoraoses isolated from Rhizobium meliloti, as an NMR chiral shift agent, were used to discriminate propranolol enantiomers . Continuous variation plot made from the complex of cyclosophoraoses with propranolol showed that the diastereomeric complex had predominantly 1:1 stoichiometry through UV spectroscopic analysis . The chiral recognition of propranolol enantiomers by cyclosophoraoses was investigated through the determination of binding constant based on the (13)C NMR chemical shift changes . The averaged K(obs) values from the plots were 55.7 M(-1) for (R)-(+)-propranolol and 36.6 M(-1) for (S)-(-)-propranolol, respectively . Enantioselectivity (alpha = K(R+)/K(S(-)) of 1.52 was then obtained . Computational calculation also revealed that (R)-(+) propranolol was more tightly bound with cyclosophoraose than (S)-(-)-propranolol due to the enhanced van der Waals interaction .

Microbiology, 2004 Feb, 150(Pt 2), 399 - 406
Biochemical characterization of a Rhizobium etli monovalent cation-stimulated acyl-coenzyme A carboxylase with a high substrate specificity constant for propionyl-coenzyme A; Dunn MF et al.; Biotin has a profound effect on the metabolism of rhizobia . It is reported here that the activities of the biotin-dependent enzymes acetyl-coenzyme A carboxylase (ACC; EC 6.4.1.2) and propionyl-coenzyme A carboxylase (PCC; EC 6.4.1.3) are present in all species of the five genera comprising the Rhizobiaceae which were examined . Evidence is presented that the ACC and PCC activities detectable in Rhizobium etli extracts are catalysed by a single acyl-coenzyme A carboxylase . The enzyme from R . etli strain 12-53 was purified 478-fold and displayed its highest activity with propionyl-CoA as substrate, with apparent K(m) and V(max) values of 0.064 mM and 2885 nmol min(-1) (mg protein)(-1), respectively . The enzyme carboxylated acetyl-CoA and butyryl-CoA with apparent K(m) values of 0.392 and 0.144 mM, respectively, and V(max) values of 423 and 268 nmol min(-1) (mg protein)(-1), respectively . K(+), or Cs(+) markedly activated the enzyme, which was essentially inactive in their absence . Electrophoretic analysis indicated that the acyl-CoA carboxylase was composed of a 74 kDa biotin-containing alpha subunit and a 45 kDa biotin-free beta subunit, and gel chromatography indicated a total molecular mass of 620 000 Da . The strong kinetic preference of the enzyme for propionyl-CoA is consistent with its participation in an anaplerotic pathway utilizing this substrate.

Plant Physiol, 2004 Feb, 134(2), 664 - 75 Epub 2004 Feb 05.
PvUPS1, an allantoin transporter in nodulated roots of French bean; Pelissier HC et al.; Nodulated legumes receive their nitrogen via nitrogen-fixing rhizobia, which exist in a symbiotic relationship with the root system . In tropical legumes like French bean (Phaseolus vulgaris) or soybean (Glycine max), most of the fixed nitrogen is used for synthesis of the ureides allantoin and allantoic acid, the major long-distance transport forms of organic nitrogen in these species . The purpose of this investigation was to identify a ureide transporter that would allow us to further characterize the mechanisms regulating ureide partitioning in legume roots . A putative allantoin transporter (PvUPS1) was isolated from nodulated roots of French bean and was functionally characterized in an allantoin transport-deficient yeast mutant showing that PvUPS1 transports allantoin but also binds its precursors xanthine and uric acid . In beans, PvUPS1 was expressed throughout the plant body, with strongest expression in nodulated roots, source leaves, pods, and seed coats . In roots, PvUPS1 expression was dependent on the status of nodulation, with highest expression in nodules and roots of nodulated plants compared with non-nodulated roots supplied with ammonium nitrate or allantoin . In situ RNA hybridization localized PvUPS1 to the nodule endodermis and the endodermis and phloem of the nodule vasculature . These results strengthen our prediction that in bean nodules, PvUPS1 is involved in delivery of allantoin to the vascular bundle and loading into the nodule phloem.

Mol Microbiol, 2004 Jan, 51(2), 335 - 47
Flavonoids induce temporal shifts in gene-expression of nod-box controlled loci in Rhizobium sp . NGR234; Kobayashi H et al.; Rhizobia, soil bacteria of the Rhizobiales, enter the roots of homologous legumes, where they induce the formation of nitrogen-fixing nodules . Signals emanating from both symbiotic partners control nodule development . Efficient nodulation requires precise, temporal regulation of symbiotic genes . Roots continuously release flavonoids that interact with transcriptional activators of the LysR family . NodD proteins, which are members of this family, act both as sensors of the environment and modulate the expression of genes preceded by conserved promoter sequences called nod-boxes . The symbiotic plasmid of the broad host-range Rhizobium sp . NGR234 caries 19 nod-boxes (NB1 to NB19), all of which were cloned upstream of a lacZ-reporter gene . A flavonoid, daidzein was able to induce 18 of the 19 nod-boxes in a NodD1-dependent manner . Interestingly, induction of four nod-boxes (NB6, NB15, NB16 and NB17) is highly dependent on NodD2 and was delayed in comparison with the others . In turn, NodD2 is involved in the repression of the NB8 nodABCIJnolOnoeI operon . Activation of transcription of nodD2 is also dependent on flavonoids despite the absence of a nod-box like sequence in the upstream promoter region . Mutational analysis showed that syrM 2 (another member of the LysR family), which is controlled by NB19, is also necessary for expression of nodD 2 . Thus, NodD1, NodD2 and SyrM2 co-modulate a flavonoid-inducible regulatory cascade that coordinates the expression of symbiotic genes with nodule development.

Res Microbiol, 2004 Jan-Feb, 155(1), 47 - 51
A novel alpha-Proteobacterium, Nordella oligomobilis gen . nov., sp . nov., isolated by using amoebal co-cultures; La Scola B et al.; Using an amoebal co-culture procedure, a novel alpha-Proteobacterium phylogenetically close to two uncultured aquatic bacteria was isolated . On the basis of phenotypic characterization and 16S rRNA gene sequence analysis, we propose the new genus and species Nordella oligomobilis gen . nov., sp . nov . The genus Nordella forms a well separated taxon in the order Rhizobiales within the alpha-2 subgroup of Proteobacteria . Its close relatives are environmental uncultured bacteria.

Ecotoxicol Environ Saf, 2004 Feb, 57(2), 190 - 201
Effects of pendimethalin at lower trophic levels--a review; Strandberg M et al.; Pendimethalin's herbicidal action lies in its inhibition of the steps in plant cell division responsible for chromosome separation and cell wall formation . Terrestrial studies show that 10-20% of the herbicide evaporates during the first weeks after application . The remainder may dissipate biologically or chemically . Half-maximal dissipation time, or half-life (DT(50)) values vary from a few days to >200 days . Field and laboratory studies showed that reduced temperatures and drought prolong dissipation time to as long as 72-2094 days . In freshwater, pendimethalin concentrations reach 6 microg/L after runoff . In water the 10% lethal concentration (LC(10)) for Daphnia was 6 microg/L when exposed via suspended food . The LC(50) was 78 microg/L . An invertebrate field study showed that soil nematodes were reduced by 35-60% at pendimethalin application rates of 0.75 and 1.0 kg/ha, respectively . After application, soil microbiota is affected by pendimethalin for approximately 4 weeks . Plant-Rhizobium symbiosis is affected at application rates of 0.5-1.0 kg/ha.

Environ Manage, 2003 Aug, 32(2), 246 - 51
Comparison of four Sesbania species to remediate Pb/Zn and Cu mine tailings; Chan GY et al.; A 6-month greenhouse pot trial was performed, aimed at screening appropriate Sesbania species for remediation of Pb/Zn and Cu mine tailings . Performances of young seedlings of four Sesbania species (S . cannabina, S . grandiflora, S . rostrata, and S . sesban) were compared with and without inoculation of rhizobia . Seedlings were planted in two types of tailings amended with garden soil or garden soil mixed with river sediment . The results indicated that inoculated plants generally produced a higher biomass than samples without inoculation . Pb/Zn mine tailings containing rather high concentrations of total and water-soluble Cu, Pb, and Zn were toxic to plant growth compared with Cu mine tailings, according to the growth performance of the four species . Sesbania sesban and S . rostrata showed superior growth performance, compared to the other two species . Thus, they can serve as pioneer species to modify the barren environment, by providing organic matter and essential nutrients such as nitrogen, upon decomposition, in a relatively short period of time . This is especially true for S . rostrata, which is an annual plant that forms both stem and root nodules . However, a longer-term field trial should be conducted to investigate if superior species can beneficially modify the habitat for the growth of subsequent plant communities.

Environ Pollut, 2004 May, 129(1), 5 - 12
Evaluation of factors influencing root-induced changes of copper fractionation in rhizosphere of a calcareous soil; Tao S et al.; Major factors influencing the root-induced copper fractionation changes within the rhizosphere of maize, wheat, pea, and soybean seedlings were evaluated using a contaminated calcareous soil . The effects of acidification, alkalization, and introduction of root exudates were investigated by addition of acid, alkaline and root exudates from solution cultures, prior to incubation and copper fractionation . Raw and sterilized soils were compared for changes of copper fractionation in the rhizosphere using rhizoboxes with maize, wheat, pea and soybean seedlings . The results indicated that the general trend in considerable changes was similar among the plant species studied . The rhizosphere experienced a depletion of carbonate associated and organic bound copper along with an accumulation of exchangeable and Fe-Mn oxide bound copper . The resulting significant influence of root exudates on copper fractionation appears to have been produced through complexation rather than acidification or alkalization . The increase in exchangeable copper in rhizosphere was strengthened by microorganisms.

C R Biol, 2003 Dec, 326(12), 1163 - 73
{Bioprotection mechanisms of the lentil plant by Rhizobium leguminosarum against Fusarium oxysporum f . sp . lentis}; Essalmani H et al.; Living and heat-killed bacterial cells of Rhizobium leguminosarum protected totally lentil plants against infection by the pathogen Fusarium oxysporum MR 84 . Culture filtrate of this rhizobacterium was also able to protect the plants to a high degree . However, when they were inoculated separately of the pathogen, living bacterial cells did not protect the plants whereas culture filtrate and killed bacterial cells protected them . These results suggest that Rhizobium cannot protect lentil plants without interaction with the pathogen, but the culture filtrate and the killed bacterial cells can protect them even in the absence of this interaction . It seems that the culture filtrate and the killed bacterial cells contain signals able to induce plant resistance . Those signals would be suppressed once Rhizobium is in contact with the plant.

Plant Cell Rep, 2004 Apr, 22(9), 705 - 10 Epub 2004 Jan 27.
Interspecific hybridization of Trifolium alexandrinum with T . constantinopolitanum using embryo rescue; Roy AK et al.; The embryo rescue technique was successfully used to raise hybrids between Trifolium alexandrinum and T . constantinopolitanum . As a result of its narrow genetic base, genetic improvement in Egyptian clover (syn . Berseem; T . alexandrinum), an important fodder crop in tropical and subtropical countries, is hampered, thereby making it imperative to introduce alien genes from related species . In a conventional interspecific hybridization program, hybrids could not be raised due to post-fertilization barriers . Of the several combinations tried, pollination 2 days after emasculation was found to be the best . Globular embryos were observed 5-7 days after pollination (DAP), followed by heart-shaped embryos 10-12 DAP . Embryos excised at the heart-shaped stage responded well to EC3 culture medium . Of 612 crosses, 33 healthy embryos could be excised and cultured on EC3 medium . The plumule emerged 8-12 days following inoculation . The embryo-rescued plants were hardened, inoculated with Rhizobium and transferred to the field . The hybrids showed intermediate morphological features with reduced pollen fertility (55-65%) and a chromosomal complement of 2n = 16 . Biochemical characterization using isozymes confirmed hybridity.

Ying Yong Sheng Tai Xue Bao, 2003 Sep, 14(9), 1521 - 4
{Identification on an antagonistic rhizobacterium X3 from rhizosphere of cucumber}; Shen A et al.; Classical physiological and biochemical methods, Biolog, and 16S rDNA analysis were used to identify the strain X3 which could control cucumber seedling damping-off . Physiological and biochemical identification showed that it belonged to Pseudomanas aeruginosa, but Biolog identification showed it was P . spinosa . 16S rDNA determination and analysis was used for further identification, which showed that the 16S rDNA sequence of X3 shared 93.7% homologies with published sequence of P . aeruginosa from GenBank, and both sequences constituted a branch in Phylogenetic tree . Based on these results, it is considered that the strain X3 belongs to one strain of P . aeruginosa.

Mol Microbiol, 2004 Feb, 51(3), 861 - 71
A HU-like gene mutation in Rhizobium leguminosarum bv . viciae affects the expression of nodulation genes; Li Q et al.; NodD is the major regulator of nod genes expression in rhizobia . Previously, a HU-like protein in Rhizobium leguminosarum bv . viciae has been identified to bind specifically with nod promoters and be involved in in vitro nodD transcription, but its in vivo function remained unknown . In this work we have cloned and sequenced the R . leguminosarum bv . viciae gene, named hurL, for this HU-like protein . Using the E . coli-expressed HurL proteins, we proved that HurL had high affinity to several nod promoters and showed a stimulation effect on in vitro nodD transcription at appropriate concentration . The R . leguminosarum bv . viciae hurL gene was mutated by insertion of a kanamycin resistance cassette . The obtained hurL mutant strain M704 exhibited poor growth under free-living conditions and failed to induce nodules on Pisum sativum cv . Frisson and Vicia hirsuta . Further studies of NodD production and nod genes-lacZ fusions expression in the hurL mutant revealed that inactivation of hurL led to severe impairment in the nodD expression, repression in the inducible expression of nodA and nodF, and slight enhancement in the expression of px2, a gene identified earlier in this lab . These results suggested that hurL might be required for maintaining the normal expression of nod genes in R . leguminosarum bv . viciae.

J Bacteriol, 2004 Feb, 186(3), 842 - 9
Regulation of L-alanine dehydrogenase in Rhizobium leguminosarum bv . viciae and its role in pea nodules; Lodwig E et al.; Alanine dehydrogenase (AldA) is the principal enzyme with which pea bacteroids synthesize alanine de novo . In free-living culture, AldA activity is induced by carboxylic acids (succinate, malate, and pyruvate), although the best inducer is alanine . Measurement of the intracellular concentration of alanine showed that AldA contributes to net alanine synthesis in laboratory cultures . Divergently transcribed from aldA is an AsnC type regulator, aldR . Mutation of aldR prevents induction of AldA activity . Plasmid-borne gusA fusions showed that aldR is required for transcription of both aldA and aldR; hence, AldR is autoregulatory . However, plasmid fusions containing the aldA-aldR intergenic region could apparently titrate out AldR, sometimes resulting in a complete loss of AldA enzyme activity . Therefore, integrated aldR::gusA and aldA::gusA fusions, as well as Northern blotting, were used to confirm the induction of aldA activity . Both aldA and aldR were expressed in the II/III interzone and zone III of pea nodules . Overexpression of aldA in bacteroids did not alter the ability of pea plants to fix nitrogen, as measured by acetylene reduction, but caused a large reduction in the size and dry weight of plants . This suggests that overexpression of aldA impairs the ability of bacteroids to donate fixed nitrogen that the plant can productively assimilate . We propose that the role of AldA may be to balance the alanine level for optimal functioning of bacteroid metabolism rather than to synthesize alanine as the sole product of N(2) reduction.

J Plant Physiol, 2003 Dec, 160(12), 1493 - 7
Recovery of development and functionality of nodules and plant growth in salt-stressed Pisum sativum--Rhizobium leguminosarum symbiosis by boron and calcium; Bolanos L et al.; Nodules developed in Pisum sativum L . cv . Argona inoculated with Rhizobium leguminosarum bv . viciae 3841 and growing under saline conditions (75 mmol/L NaCl) are non functional and had abnormal structure . The infected cells contained a low amount of endophytic bacteria, compared to treatments without salt . Addition of B (up to 55.8 micromol/L) and Ca2+ (up to 2.72 mmol/L) increased bacterial population of host plant cells in salt-stressed nodules . Furthermore, symbiosomes developed inside the nodules from salt treated plants presented a degraded peribacteroid membrane . This effect was also prevented by combined addition of B and Ca2+ . Given the importance of both nutrients in cell wall structure, the pectin fraction was studied by electron microscopy and immunological methods . Salt stress produced cells with walls dramatically altered or even degraded in several zones . Pectin polysaccharides, detected by JIM 5 monoclonal antibody, increased in cells under salinity . These effects resembled typical effects of B-deficiency reactions in cell walls, and the increase of both Ca2+ and especially B also prevented these alterations.

J Appl Physiol, 2004 Feb, 96(2), 774 - 83
Signal transduction by heme-containing PAS-domain proteins; Gilles-Gonzalez MA et al.; The most common physiological strategy for detecting the gases oxygen, carbon monoxide, and nitric oxide is signal transduction by heme-based sensors, a broad class of modular proteins in which a heme-binding domain governs the activity of a neighboring transmitter domain . Different structures are possible for the heme-binding domains in these sensors, but, so far, the Per-ARNT-Sim motif, or PAS domain, is the one most commonly encountered . Heme-binding PAS (heme-PAS) domains can accomplish ligand-dependent switching of a variety of partner domains, including histidine kinase, phosphodiesterase, and basic helix-loop-helix (bHLH) DNA-binding modules . Proteins with heme-PAS domains occur in all kingdoms of life and are quite diverse in their physiological roles . Examples include the neuronal bHLH-PAS carbon monoxide sensor NPAS2 that is implicated in the mammalian circadian clock, the acetobacterial oxygen sensor AxPDEA1 that directs cellulose production, and the rhizobial oxygen sensor FixL, which governs nitrogen fixation . What factors determine the range of detection of these sensors? How do they transduce their signal? This review examines the recent advances in answering these questions.

Mol Plant Microbe Interact, 2004 Jan, 17(1), 16 - 26
Expression of MsLEC1 transgenes in alfalfa plants causes symbiotic abnormalities; Brill LM et al.; Legume lectins have been proposed to have important symbiotic roles during Rhizobium-legume symbioses . To test this hypothesis, the symbiotic responses of transgenic alfalfa plants that express a portion of the putative alfalfa lectin gene MsLEC1 or MsLEC2 in either the antisense or sense orientation were analyzed following inoculation with wild-type Sinorhizobium meliloti 1021 . MsLEC1-antisense (LEC1AS) plants were stunted, exhibited hypernodulation, and developed not only abnormally large nodules but also numerous small nodules, both of which senesced prematurely . MsLEC2-antisense plants were intermediate in growth and nodule number compared with LEC1AS and vector control plants . The symbiotic abnormalities of MsLEC1-sense transgene plants were similar to but milder than the responses shown by the LEC1AS plants, whereas MsLEC2-sense transgene plants exhibited symbiotic responses that were identical to those of vector and nontransgenic control plants . MsLEC1 mRNA accumulation was not detected in nodule RNA by Northern blot analysis but was localized to alfalfa nodule meristems and the adjacent cells of the invasion zone by in situ hybridization; transcripts were also detected in root meristems . A similar spatial pattern of MsLEC2 expression was found by using a whole-mount in situ hybridization procedure . Moreover, mRNAs for an orthologous lectin gene (MaLEC) were detected in white sweetclover (Melilotus alba) nodules and root tips.

Appl Environ Microbiol, 2004 Jan, 70(1), 535 - 41
Bradyrhizobium elkanii rtxC gene is required for expression of symbiotic phenotypes in the final step of rhizobitoxine biosynthesis; Okazaki S et al.; We disrupted the rtxC gene on the chromosome of Bradyrhizobium elkanii USDA94 by insertion of a nonpolar aph cartridge . The rtxC mutant, designated DeltartxC, produced serinol and dihydrorhizobitoxine but no rhizobitoxine, both in culture and in planta . The introduction of cosmids harboring the rtxC gene into the DeltartxC mutant complemented rhizobitoxine production, suggesting that rtxC is involved in the final step of rhizobitoxine biosynthesis in B . elkanii USDA94 . Glycine max cv . Lee inoculated with DeltartxC or with a null mutant, Deltartx::Omega1, showed no foliar chlorosis, whereas the wild-type strain USDA94 caused severe foliar chlorosis . The two mutants showed significantly less nodulation competitiveness than the wild-type strain on Macroptilium atropurpureum . These results indicate that dihydrorhizobitoxine, the immediate precursor of the oxidative form of rhizobitoxine, has no distinct effect on nodulation phenotype in these legumes . Thus, desaturation of dihydrorhizobitoxine by rtxC-encoded protein is essential for the bacterium to show rhizobitoxine phenotypes in planta . In addition, complementation analysis of rtxC by cosmids differing in rtxC transcription levels suggested that rhizobitoxine production correlates with the amount of rtxC transcript.

Arch Microbiol, 2004 Feb, 181(2), 129 - 36 Epub 2003 Dec 18.
Ethiopian soils harbor natural populations of rhizobia that form symbioses with common bean ( Phaseolus vulgaris L.); Beyene D et al.; The diversity and taxonomic relationships of 83 bean-nodulating rhizobia indigenous to Ethiopian soils were characterized by PCR-RFLP of the internally transcribed spacer (ITS) region between the 16S and 23S rRNA genes, 16S rRNA gene sequence analysis, multilocus enzyme electrophoresis (MLEE), and amplified fragment-length polymorphism . The isolates fell into 13 distinct genotypes according to PCR-RFLP analysis of the ITS region . Based on MLEE, the majority of these genotypes (70%) was genetically related to the type strain of Rhizobium leguminosarum . However, from analysis of their 16S rRNA genes, the majority was placed with Rhizobium etli . Transfer and recombination of the 16S rRNA gene from presumptively introduced R . etli to local R . leguminosarum is a possible theory to explain these contrasting results . However, it seems unlikely that bean rhizobia originating from the Americas (or Europe) extensively colonized soils of Ethiopia because Rhizobium tropici, Rhizobium gallicum, and Rhizobium giardinii were not detected and only a single ineffective isolate of R . etli that originated from a remote location was identified . Therefore, Ethiopian R . leguminosarum may have acquired the determinants for nodulation of bean from a low number of introduced bean-nodulating rhizobia that either are poor competitors for nodulation of bean or that failed to survive in the Ethiopian environment . Furthermore, it may be concluded from the genetic data presented here that the evidence for separating R . leguminosarum and R . etli into two separate species is inconclusive.

FEMS Microbiol Lett, 2003 Dec 12, 229(2), 153 - 8
Identification of fast and slow growing rhizobia nodulating soybean (Glycine max {L.} Merr) by a multiplex PCR reaction; Pastorino GN et al.; Two DNA fragments, a 730-bp and a 900-bp fragment, one homologous to host cultivar specificity genes nolBT of Sinorhizobium fredii and the other one homologous to RSalpha, an insertion-like sequence present in Bradyrhizobium japonicum, were generated by polymerase chain reaction (PCR) with two pairs of primers . The amount of each fragment generated by the multiplex PCR was proportional to the amount of template DNA present . The amplification of the 900-bp RSalpha fragment was more sensitive, since it was amplified from a smaller amount of template DNA than the 730-bp nolBT fragment . By running the multiplex reaction in the presence of template DNA isolated from different sources, we confirmed that the reaction can discriminate between S . fredii, Bradyrhizobium japonicum and Sinorhizobium xinjiangensis.

Mikrobiologiia, 2003 Sep-Oct, 72(5), 633 - 8
{The biological activity of the Sinorhizobium meliloti glucan}; Kosenko LV et al.; The study of the effect of the periplasmic glucan isolated from the root-nodule bacterium S . meliloti CXM1-188 on the symbiosis of another strain (441) of the same root-nodule bacterium with alfalfa plants showed that this effect depends on the treatment procedure . The pretreatment of alfalfa seedlings with the glucan followed by their bacterization with S . meliloti 441 insignificantly influenced the nodulation parameters of symbiosis (the number of root nodules and their nitrogen-fixing activity) but induced a statistically significant increase in the efficiency of symbiosis (expressed as the masses of the alfalfa overground parts and roots) . At the same time, the pretreatment of S . meliloti 441 cells with the glucan brought about a considerable decrease in the nodulation parameters of symbiosis (the number of the root nodules and their nitrogen-fixing activity decreased by 2.5-11 and 7 times, respectively) . These data suggest that the stimulating effect of rhizobia on host plants may be due not only to symbiotrophic nitrogen fixation but also to other factors . Depending on the experimental conditions, the treatment of alfalfa plants with the glucan and their bacterization with rhizobial cells enhanced the activity of peroxidase in the alfalfa roots and leaves by 10-39 and 12-27%, respectively.

Clin Infect Dis, 2004 Jan 1, 38(1), 149 - 53 Epub 2003 Dec 04.
Clinical and microbiological characteristics of Rhizobium radiobacter infections; Lai CC et al.; Data obtained from 1996 to 2002 on 13 patients with Rhizobium radiobacter infections were analyzed . Ten patients (76%) had underlying hematological malignancy or solid-organ cancer . Six patients (46%) had febrile neutropenia during the course of R . radiobacter infection . The majority (54%) of infections were catheter-related bacteremia, and 92% of infections were hospital acquired . All the patients survived . Eighteen isolates were recovered from the 13 patients, and each isolate was susceptible to cefepime, piperacillin-tazobactam, carbapenems, and ciprofloxacin . The pulsed-field gel electrophoresis profiles differed among the isolates recovered from different patients, indicating the absence of nosocomial spread of the organism.

J Exp Bot, 2004 Jan, 55(394), 89 - 97
Nodule-enhanced protease inhibitor gene: emerging patterns of gene expression in nodule development on Sesbania rostrata; Lievens S et al.; A novel marker for the early stages of nodulation of Sesbania rostrata was found to encode a putative member of the Kunitz family of protease inhibitors (SrPI1) . Its expression was enhanced during nodulation, and was not up-regulated by wounding or upon infection with wide host-range pathogens . In situ expression patterns resembled those previously described for functions that may be implicated in delimiting infected nodule tissues from the rest of the plant . Thus, SrPI1 may be a component of a multi-layered barrier that restrains the invading rhizobia.

Syst Appl Microbiol, 2003 Nov, 26(4), 611 - 23
Genetic diversity of bradyrhizobial populations from diverse geographic origins that nodulate Lupinus spp . and Ornithopus spp; Jarabo-Lorenzo A et al.; The genetic diversity of 45 bradyrhizobial isolates that nodulate several Lupinus and Ornithopus species in different geographic locations was investigated by 16S rDNA PCR-RFLP and sequence analysis, 16S-23S rDNA intergenic spacer (IGS) PCR-RFLP analysis, and ERIC-PCR genomic fingerprinting . Reference strains of Bradyrhizobium japonicum, B . liaoningense and B . elkanii and some Canarian isolates from endemic woody legumes in the tribe Genisteae were also included . The 16S rDNA-RFLP analysis resolved 9 genotypes of lupin isolates, a group of fourteen isolates presented restriction-genotypes identical or very similar to B . japonicum, while another two main groups of isolates (69%) presented genotypes that clearly separated them from the reference species of soybean . 16S rDNA sequencing of representative strains largely agreed with restriction analysis, except for a group of six isolates, and showed that all the lupin isolates are relatives of B . japonicum, but different lineages were observed . The 16S-23S IGS-RFLP analysis showed a high resolution level, resolving 19 distinct genotypes among 30 strains analysed, and so demonstrating the heterogeneity of the 16S-RFLP groups . ERIC-PCR fingerprint analysis showed an enormous genetic diversity producing a different pattern for each but two of the isolates . Phylogeny of nodC gene was independent from the 16S rRNA phylogeny, and showed a tight relationship in the symbiotic region of the lupin isolates with isolates from Canarian genistoid woody legumes, and in concordance, cross-nodulation was found . We conclude that Lupinus is a promiscuous host legume that is nodulated by rhizobia with very different chromosomal genotypes, which could even belong to several species of Bradyrhizobium . No correlation among genomic background, original host plant and geographic location was found, so, different chromosomal genotypes could be detected at a single site and in a same plant species, on the contrary, an identical genotype was detected in very different geographical locations and plants.

Microbiol Mol Biol Rev, 2003 Dec, 67(4), 574 - 92
Quorum sensing in nitrogen-fixing rhizobia; Gonzalez JE et al.; Members of the rhizobia are distinguished for their ability to establish a nitrogen-fixing symbiosis with leguminous plants . While many details of this relationship remain a mystery, much effort has gone into elucidating the mechanisms governing bacterium-host recognition and the events leading to symbiosis . Several signal molecules, including plant-produced flavonoids and bacterially produced nodulation factors and exopolysaccharides, are known to function in the molecular conversation between the host and the symbiont . Work by several laboratories has shown that an additional mode of regulation, quorum sensing, intercedes in the signal exchange process and perhaps plays a major role in preparing and coordinating the nitrogen-fixing rhizobia during the establishment of the symbiosis . Rhizobium leguminosarum, for example, carries a multitiered quorum-sensing system that represents one of the most complex regulatory networks identified for this form of gene regulation . This review focuses on the recent stream of information regarding quorum sensing in the nitrogen-fixing rhizobia . Seminal work on the quorum-sensing systems of R . leguminosarum bv . viciae, R . etli, Rhizobium sp . strain NGR234, Sinorhizobium meliloti, and Bradyrhizobium japonicum is presented and discussed . The latest work shows that quorum sensing can be linked to various symbiotic phenomena including nodulation efficiency, symbiosome development, exopolysaccharide production, and nitrogen fixation, all of which are important for the establishment of a successful symbiosis . Many questions remain to be answered, but the knowledge obtained so far provides a firm foundation for future studies on the role of quorum-sensing mediated gene regulation in host-bacterium interactions.

Appl Environ Microbiol, 2003 Dec, 69(12), 7343 - 53
Concomitant induction of systemic resistance to Pseudomonas syringae pv . lachrymans in cucumber by Trichoderma asperellum (T-203) and accumulation of phytoalexins; Yedidia I et al.; Most studies on the reduction of disease incidence in soil treated with Trichoderma asperellum have focused on microbial interactions rather than on plant responses . This study presents conclusive evidence for the induction of a systemic response against angular leaf spot of cucumber (Pseudomonas syringae pv . lachrymans) following application of T . asperellum to the root system . To ascertain that T . asperellum was the only microorganism present in the root milieu, plants were grown in an aseptic hydroponic growth system . Disease symptoms were reduced by as much as 80%, corresponding to a reduction of 2 orders of magnitude in bacterial cell densities in leaves of plants pretreated with T . asperellum . As revealed by electron microscopy, bacterial cell proliferation in these plants was halted . The protection afforded by the biocontrol agent was associated with the accumulation of mRNA of two defense genes: the phenylpropanoid pathway gene encoding phenylalanine ammonia lyase (PAL) and the lipoxygenase pathway gene encoding hydroxyperoxide lyase (HPL) . This was further supported by the accumulation of secondary metabolites of a phenolic nature that showed an increase of up to sixfold in inhibition capacity of bacterial growth in vitro . The bulk of the antimicrobial activity was found in the acid-hydrolyzed extract containing the phenolics in their aglycone form . High-performance liquid chromatography analysis of phenolic compounds showed a marked change in their profile in the challenged, preelicited plants relative to that in challenged controls . The results suggest that similar to beneficial rhizobacteria, T . asperellum may activate separate metabolic pathways in cucumber that are involved in plant signaling and biosynthesis, eventually leading to the systemic accumulation of phytoalexins.

Plant Physiol, 2004 Jan, 134(1), 182 - 93 Epub 2003 Dec 04.
Molecular and cell biology of a family of voltage-dependent anion channel porins in Lotus japonicus; Wandrey M et al.; Voltage-dependent anion channels (VDACs) are generally considered as the main pathway for metabolite transport across the mitochondrial outer membrane . Recent proteomic studies on isolated symbiosome membranes from legume nodules indicated that VDACs might also be involved in transport of nutrients between plants and rhizobia . In an attempt to substantiate this, we carried out a detailed molecular and cellular characterization of VDACs in Lotus japonicus and soybean (Glycine max) . Database searches revealed at least five genes encoding putative VDACs in each of the legumes L . japonicus, Medicago truncatula, and soybean . We obtained and sequenced cDNA clones from L . japonicus encoding five full-length VDAC proteins (LjVDAC1.1-1.3, LjVDAC2.1, and LjVDAC3.1) . Complementation of a yeast (Saccharomyces cerevisiae) mutant impaired in VDAC1, a porin of the mitochondrial outer membrane, showed that LjVDAC1.1, LjVDAC1.2, LjVDAC2.1, and LjVDAC3.1, but not LjVDAC1.3, are functional and targeted to the mitochondrial outer membrane in yeast . Studies of the expression pattern of the five L . japonicus VDAC genes revealed largely constitutive expression of each throughout the plant, including nodules . Antibodies to LjVDAC1.1 of L . japonicus and the related POM36 protein of potato (Solanum tuberosum) recognized several proteins between 30 and 36 kD on western blots, including LjVDAC1.1, LjVDAC1.2, LjVDAC1.3, and LjVDAC2.1 . Immunolocalization of VDACs in L . japonicus and soybean root nodules demonstrated their presence on not only mitochondria but also on numerous, small vesicles at the cell periphery . No evidence was found for the presence of VDACs on the symbiosome membrane . Nonetheless, the data indicate that VDACs may play more diverse roles in plants than suspected previously.

Int J Syst Evol Microbiol, 2003 Nov, 53(Pt 6), 1853 - 9
Fulvimarina pelagi gen . nov., sp . nov., a marine bacterium that forms a deep evolutionary lineage of descent in the order "Rhizobiales"; Cho JC et al.; Three brownish-yellow bacterial strains were isolated from the western Sargasso Sea by high-throughput culturing methods and characterized by polyphasic approaches . All isolates were Gram-negative, strictly aerobic, chemoheterotrophic, non-motile short rods that contained carotenoid pigments . Phylogenetic analyses based on 16S rRNA gene sequences, DNA-DNA hybridization and DNA G+C content, along with phenotypic characteristics, revealed that they belonged to the same species . The strains utilized a wide range of substrates, including pentoses, hexoses, oligosaccharides, sugar alcohols, organic acids and amino acids, as sole carbon sources . The DNA G+C content of the isolates ranged from 57.6 to 59.9 mol% . The predominant cellular fatty acid constituent was C(18 : 1)omega7c, whilst C(16 : 0), C(18 : 0) and C(19 : 0)omega8c cyclo were also abundant . The organism related most closely to these strains, as determined by 16S rDNA sequence comparison, was the recently described species Aurantimonas coralicida (93.3-93.8 % similarity) . Phylogenetic analyses indicated that the strains formed a distinct and deep evolutionary lineage of descent, together with A . coralicida, within the order "Rhizobiales" of the alpha-Proteobacteria . This lineage could not be associated with any of the ten known families in the order "Rhizobiales" . From polyphasic evidence, it is proposed that the strains be placed into a novel genus and species, Fulvimarina pelagi gen . nov., sp . nov . (type strain, HTCC2506(T)=ATCC BAA-666(T)=KCTC 12091(T)=DSM 15513(T)).

Ying Yong Sheng Tai Xue Bao, 2003 Aug, 14(8), 1283 - 6
{Root colonization and nodulation of Sinorhizobium fredii HN01DL in Glycine max rhizosphere}; Li Y et al.; Rhizobox-soil microcosms studies on the colonization, dispersal and nodulation of Sinorhizobium fredii HN01DL marked with luxAB gene in Glycine max rhizosphere showed that the colonization dynamics and the density of HN01DL in non-sterilized rhizobox-soil microcosms were different from those in sterilized rhizobox-soil microcosms . The colonization density of the former reached the maximum (8.65 log cfu.g-1 root) 12 days after the coated seeds planted, and that of the latter decreased rapidly at the early stage and achieved the maximum (6.88 log cfu.g-1 root) 15 days afterwards . Furthermore, the colonization density of HN01DL reached the maximum (7.05 log cfu.g-1 root) in section A (0-4 cm) of root system 5 days after seeds planted, decreased slowly and kept a relative stable level until 19 days, and began to rise up again 33 days afterwards . The strain could also disperse to the place of 16 cm from seed to root tip by 46 days after seed planted . HN01DL maintained a constantly higher colonization density level in section A of root system, formed the largest number of luminescent nodules (total 16.3, dominantly located in main root of section A), and had the highest luminescent percentage (68.8%) . The luminescent nodule percentage decreased gradually along section A to E of root system, and no luminescent nodule was detected in section E of root system.

Ying Yong Sheng Tai Xue Bao, 2003 Aug, 14(8), 1278 - 82
{Effects of rhizobia on morphological distribution of Cu, Zn and Cd in soil}; Chen W et al.; Red soil from Chenzhou of Hunan and brown soil from Gongyi of Henan were collected and treated with Cu (NO3)2, Zn(NO3)2 or Cd(NO3)2, respectively for 2 weeks, and Rhizobium fredii strain HN01 was inoculated . Sequential extraction method was employed to investigate the forms of Cu, Zn and Cd in the examined soils with the absence and presence of rhizobia . The results showed that after inoculation, the total amount of solid-bound Zn decreased 10%, and the amount of Zn associated with carbonate, Mn oxides, and organic matter fraction decreased 9-26% . No significant change was observed for the total amount of Zn combined with the solid phase of red soil in the presence of rhizobia . However, the amount of specifically adsorbed and Mn oxides bound Zn decreased, while the amount of exchangeable Zn increased . Inoculation of rhizobia depressed the release of Cu to the soil solution, and increased the total amount of Cu associated with the solid phase of brown soil . The increase of the amount of exchangeable Cu and of the Cu in fractions of carbonate, Mn oxides and organic matter ranged from 20% to 54% . There was no significant change for the level of Cd in the solution in both soils after rhizobia inoculation . The amount of exchangeable and organic Cd increased 22% and 11%, while the specifically adsorbed, and Mn oxides bound Mn decreased 14% and 29%, respectively . The different influence of rhizobia on the morphological distribution of test heavy metals in two soils was mainly ascribed to the soil pH changes.

Mol Plant Microbe Interact, 2003 Dec, 16(12), 1085 - 93
Three genes encoding for putative methyl- and acetyltransferases map adjacent to the wzm and wzt genes and are essential for O-antigen biosynthesis in Rhizobium etli CE3; Lerouge I et al.; The elucidation of the structure of the O-antigen of Rhizobium etli CE3 predicts that the R . etli CE3 genome must contain genes encoding acetyl- and methyltransferases to confer the corresponding modifications to the O-antigen . We identified three open reading frames (ORFs) upstream of wzm, encoding the membrane component of the O-antigen transporter and located in the lps alpha-region of R . etli CE3 . The ORFs encode two putative acetyltransferases with similarity to the CysE-LacA-LpxA-NodL family of acetyltransferases and one putative methyltransferase with sequence motifs common to a wide range of S-adenosyl-L-methionine-dependent methyltransferases . Mutational analysis of the ORFs encoding the putative acetyltransferases and methyltransferase revealed that the acetyl and methyl decorations mediated by these specific enzymes are essential for O-antigen synthesis . Composition analysis and high performance anion exchange chromatography analysis of the lipopolysaccharides (LPSs) of the mutants show that all of these LPSs contain an intact core region and lack the O-antigen polysaccharide . The possible role of these transferases in the decoration of the O-antigen of R . etli is discussed.

Plant Cell Physiol, 2003 Nov, 44(11), 1208 - 14
A member of the germin-like protein family is a highly conserved mycorrhiza-specific induced gene; Doll J et al.; A Medicago truncatula cDNA encoding a germin-like protein (GLP) was isolated from a suppression subtractive hybridization cDNA library enriched for arbuscular mycorrhiza (AM)-induced genes . The MtGLP1 amino acid sequence shows some striking differences to previously described plant GLP sequences and might therefore represent a new subgroup of this multigene family . The MtGlp1 mRNA was strongly induced in roots and root cultures colonized by the AM fungus Glomus intraradices . Whereas MtGlp1 is strongly induced in AM, no transcripts of the gene were detected in non-infected roots or in roots after infection with the oomycete root pathogen Aphanomyces euteiches or with Rhizobia . Increased phosphate levels during fertilization also could not stimulate MtGlp1 transcription . Hence, MtGlp1 induction seems to be an AM-specific phenomenon . In situ hybridization showed that MtGlp1 is localized in arbuscule containing cells . A putative orthologue of this AM-specific GLP gene could be localized in a second legume Lotus japonicus, indicating that the regulation of a member of the GLP family belongs to a conserved mechanism in AM regulation in different plant species.

Plant Cell Physiol, 2003 Nov, 44(11), 1176 - 84
Lotus japonicus: a new model to study root-parasitic nematodes; Lohar DP et al.; Sedentary plant-parasitic nematodes engage in complex interactions, and induce specialized feeding structures by redirecting plant developmental pathways, and parallels have been observed with rhizobial nodule development on legumes . A model legume would greatly facilitate a better understanding of the differences between parasitic (nematode) and mutualistic (rhizobia and mycorrhizae) symbioses, and we have developed Lotus japonicus as such a model . Conditions for efficient parasitism by root-knot nematode (Meloidogyne spp.) of the widely used Lotus "Gifu" ecotype were established . Features of Lotus biology, such as thin and translucent roots, proved ideal for monitoring the progress of nematode infection both on live specimens and post-staining . We examined L . japonicus mutants with nodulation phenotypes . One, har1, which is a hypernodulated mutant defective in a CLAVATA1-like receptor kinase gene, was found to be hyperinfected by M . incognita . However, another hypernodulated Lotus mutant exhibited the same level of M . incognita infection as wild-type plants . We also established conditions for infection of Lotus by soybean cyst nematode (Heterodera glycines) . In contrast to the response to root-knot nematode, the Gifu ecotype is resistant to H . glycines, and elicits a hypersensitive response . This pattern of resistance recapitulates that seen on nematode-resistant soybean plants . We conclude that L . japonicus is a powerful model legume for studying compatible and incompatible plant-nematode interactions.

J Appl Microbiol, 2003, 95(6), 1367 - 74
Analysis of the legume-rhizobia symbiosis in shrubs from central western Spain; Rodriguez-Echeverria S et al.; AIMS: This work analyses the diversity of rhizobia associated with some of the predominant shrubby legumes in central-western Spain . Symbiotic promiscuity and effectiveness were studied using cross-inoculation experiments with shrubby species . MATERIAL AND RESULTS: Six new bradyrhizobia strains were isolated from nodules collected from wild plants of six leguminous species, Cytisus balansae, C . multiflorus, C . scoparius, C . striatus, Genista hystrix and Retama sphaerocarpa . These isolates were genetically characterized by 16S rDNA partial sequencing and random amplification of polymorphic DNA-PCR fingerprinting . The phylogenetic analysis revealed that these isolates could represent three new Bradyrhizobium species . Shrubby legumes and bradyrhizobia displayed a high symbiotic promiscuity both for infectivity and effectiveness . Symbioses were effective in more than 70% of the associations established by four of the six plant species . CONCLUSIONS: Native woody legumes in western Spain are nodulated by Bradyrhizobium strains . The high degree of symbiotic promiscuity and effectiveness highlights the complex dynamics of these communities in wild ecosystems under a Mediterranean-type climate . Furthermore, the results from this study suggest a potential importance of inoculation for these legume species in soil-restoration projects . SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first study, to our knowledge, that combines both molecular analysis and pot trials to study the rhizobia-legume symbiosis for wild legumes.

Plant Physiol, 2003 Dec, 133(4), 1893 - 910 Epub 2003 Nov 20.
Characterization of four lectin-like receptor kinases expressed in roots of Medicago truncatula . Structure, location, regulation of expression, and potential role in the symbiosis with Sinorhizobium meliloti; Navarro-Gochicoa MT et al.; To study the role of LecRK (lectin-like receptor kinase) genes in the legumerhizobia symbiosis, we have characterized the four Medicago truncatula Gaernt . LecRK genes that are most highly expressed in roots . Three of these genes, MtLecRK7;1, MtLecRK7;2, and MtLecRK7;3, encode proteins most closely related to the Class A LecRKs of Arabidopsis, whereas the protein encoded by the fourth gene, MtLecRK1;1, is most similar to a Class B Arabidopsis LecRK . All four genes show a strongly enhanced root expression, and detailed studies on MtLecRK1;1 and MtLecRK7;2 revealed that the levels of their mRNAs are increased by nitrogen starvation and transiently repressed after either rhizobial inoculation or addition of lipochitooligosaccharidic Nod factors . Studies of the MtLecRK1;1 and MtLecRK7;2 proteins, using green fluorescent protein fusions in transgenic M . truncatula roots, revealed that they are located in the plasma membrane and that their central transmembrane-spanning helix is required for correct sorting . Moreover, their lectin-like domains appear to be highly glycosylated . Of the four proteins, only MtLecRK1;1 shows a high conservation of key residues implicated in monosaccharide binding, and molecular modeling revealed that this protein may be capable of interacting with Nod factors . However, no increase in Nod factor binding was found in roots overexpressing a fusion in which the kinase domain of this protein had been replaced with green fluorescent protein . Roots expressing this fusion protein however showed an increase in nodule number, suggesting that expression of MtLecRK1;1 influences nodulation . The potential role of LecRKs in the legume-rhizobia symbiosis is discussed.

Mycorrhiza, 2004 Feb, 14(1), 1 - 10 Epub 2003 Nov 19.
Proteomics as a tool to monitor plant-microbe endosymbioses in the rhizosphere; Bestel-Corre G et al.; In recent years, outstanding molecular approaches have been used to investigate genes and functions involved in plant-microbe endosymbioses . In this review, we outline the use of proteomic analysis, based on two-dimensional electrophoresis and mass spectrometry, to characterize symbiosis-related proteins . During the last decade, proteomics succeeded in identifying about 400 proteins associated with the development and functioning of both mycorrhizal and rhizobial symbioses . Further progress in prefractionation procedures is expected to allow the detection of symbiotic proteins showing low abundance or being present in certain cell compartments.

J Exp Bot, 2004 Jan, 55(394), 27 - 34 Epub 2003 Nov 17.
Plant growth-promoting bacteria and nitrate availability: impacts on root development and nitrate uptake; Mantelin S et al.; Plant growth-promoting bacteria (PGPB) and NO-3 availability both affect NO-3 uptake and root architecture . The presence of external NO-3 induces the expression of NO-3 transporter genes and elicits lateral root elongation in the part of the root system exposed to the NO-3 supply . By contrast, an increase in NO-3 supply leads to a higher plant N status (low N demand), which represses both the NO-3 transporters and lateral root development . The effects of PGPB on NO-3 uptake and root development are similar to those of low NO-3 availability (concomitant stimulation of NO-3 uptake rate and lateral root development) . The mechanisms responsible for the localized and long-distance regulation of NO-3 uptake and root development by NO-3 availability are beginning to be elucidated . By contrast, the signalling and transduction pathways elicited by the rhizobacteria remain totally unknown . This review will compare the effects of NO-3 availability and PGPB on root morphogenesis and NO-3 uptake, in order to determine whether interactions exist between the NO-3-dependent and the PGPB-dependent regulatory pathways.

FEBS Lett, 2003 Nov 20, 554(3), 271 - 4
Purification and phosphorylation of the effector protein NopL from Rhizobium sp . NGR234; Bartsev AV et al.; Bacterial pathogens use type III secretion systems (TTSSs) to deliver virulence factors into eukaryotic cells . These effectors perturb host-defence responses, especially signal transduction pathways . A functional TTSS was identified in the symbiotic, nitrogen-fixing bacterium Rhizobium sp . NGR234 . NopL (formerly y4xL) of NGR234 is a putative symbiotic effector that modulates nodulation in legumes . To test whether NopL could interact with plant proteins, in vitro phosphorylation experiments were performed using recombinant nopL protein purified from Escherichia coli as well as protein extracts from Lotus japonicus and tobacco plants . NopL serves as a substrate for plant protein kinases as well as purified protein kinase A . Phosphorylation of NopL was inhibited by the Ser/Thr kinase inhibitor K252a as well as by PD98059, a mitogen-activated protein (MAP) kinase kinase inhibitor . It thus seems likely that, after delivery into the plant cell, NopL modulates MAP kinase pathways.

Environ Sci Technol, 2003 Nov 1, 37(21), 5008 - 14
Rhizosphere characteristics of the arsenic hyperaccumulator Pteris vittata L . and monitoring of phytoremoval efficiency; Fitz WJ et al.; Recently discovered As-hyperaccumulator ferns hold promise for phytoremediation of As-polluted soils . We investigated changes in the rhizosphere characteristics of Pteris vittata (Chinese Brake fern) relevant for its use in phytoextraction . Plants were grown in rhizoboxes filled with soil containing 2270 mg kg(-1) As . Dissolved organic carbon (DOC) concentrations in rhizosphere soil solution were increased by 86% and appeared to enhance total Fe solubility due to complexation reactions . Despite substantial removal of As by the fern, As was not significantly decreased in the rhizsophere soil solution after one cropping, apparently due to the large buffer capacity of the soil and possibly because of ion competition with DOC . However, the difference between 0.05 M (NH4)2SO4-extractable labile As in bulk and rhizosphere soil accounted for 8.9% of total As accumulated in the fern, indicating that As was mainly acquired from less available pools . Moreover, As depletion in the rhizosphere and limited resupply from less available pools were indicated by a 19.3% decreased As flux, measured using the technique of diffusive gradients in thin films (DGT) . Modeling of the DGT-soil system was able to show that the rate of release from solid phase to solution in the rhizosphere was one-third of that in the bulk soil . Applying the remedial strategy of bioavailable contaminant stripping, which aims at diminishing the phytoavailable pollutant fraction, DGT can be used as a monitoring tool to evaluate the efficiency of phytoextraction and to study the potential resupply of bioavailable pools after phytoextraction has ceased.

Mol Plant Microbe Interact, 2003 Nov, 16(11), 1039 - 46
Molecular analysis of the pathway for the synthesis of thiol tripeptides in the model legume Lotus japonicus; Matamoros MA et al.; The thiol tripeptides, glutathione (GSH) and homoglutathione (hGSH), perform multiple roles in legumes, including protection against toxicity of free radicals and heavy metals . The three genes involved in the synthesis of GSH and hGSH in the model legume, Lotus japonicus, have been fully characterized and appear to be present as single copies in the genome . The gamma-glutamylcysteine synthetase (gamma(ecs)) gene was mapped on the long arm of chromosome 4 (70.0 centimorgans {cM}) and consists of 15 exons, whereas the glutathione synthetase (gshs) and homoglutathione synthetase (hgshs) genes were mapped on the long arm of chromosome 1 (81.3 cM) and found to be arranged in tandem with a separation of approximately 8 kb . Both genes consist of 12 exons of exactly the same size (except exon 1, which is similar) . Two types of transcripts were detected for the gshs gene, which putatively encode proteins localized in the plastids and cytosol . Promoter regions contain cis-acting regulatory elements that may be involved in the plant's response to light, hormones, and stress . Determination of transcript levels, enzyme activities, and thiol contents in nodules, roots, and leaves revealed that gamma(ecs) and hgshs are expressed in all three plant organs, whereas gshs is significantly functional only in nodules . This strongly suggests an important role of GSH in the rhizobia-legume symbiosis.

J Biol Chem, 2004 Jan 23, 279(4), 2520 - 7 Epub 2003 Oct 31.
Expression of two Escherichia coli acetyl-CoA carboxylase subunits is autoregulated; James ES et al.; Acetyl-CoA carboxylase (ACC) catalyzes the first step of fatty acid biosynthesis, the synthesis of malonyl-CoA from acetyl-CoA using ATP and bicarbonate . In Escherichia coli and most other bacteria, ACC is composed of four subunits encoded by accA, accB, accC, and accD . Prior work from this laboratory showed that the in vivo expression of the accBC operon had a strikingly nonlinear response to gene copy number (Li, S.-J, and Cronan, J . E., Jr . (1993) J . Bacteriol . 175, 332-340) in that the presence of 50 or more copies of the accBC operon resulted in only a 2-3-fold increase in AccB and AccC . We now report that AccB functions to negatively regulate transcription of the accBC operon . Expression of a chimeric protein consisting of the N terminus of E . coli AccB and the C-terminal bioinylation domain of Bacillus subtilis AccB down-regulated transcription of the E . coli accBC operon . A truncated form of AccB consisting of the N-terminal 68 amino acids of E . coli AccB was sufficient to negatively regulate the accBC operon . In vivo bypass of acetyl-CoA carboxylase activity by expression of a malonyl-CoA synthase from Rhizobium trifolii allowed construction of strain deleted for the accA and accB genes . Unexpectedly, the deltaaccB mutation could not be resolved from the deltaaccA mutation . Transcription of the accBC operon in the deltaaccB deltaaccA strain continued well into stationary phase under growth conditions that normally result in greatly decreased transcription . These data support a model in which AccB acts as an autoregulator of accBC operon transcription.

Acta Microbiol Pol, 2003, 52(2), 195 - 9
Increased synthesis of dihydroxybenzoic acid in the presence of aluminum by Rhizobium MO1; De M et al.; Rhizobizum sp . MO1, a mung bean (Vigna radiata) symbiont, produces a catecholate type of siderophore, 2,3-dihydroxy benzoic acid (DHBA), in iron depleted medium . Addition of aluminum to the medium decreased the growth but increased the production of the siderophore.

Arch Microbiol, 2003 Dec, 180(6), 444 - 54 Epub 2003 Oct 24.
Diverse Mesorhizobium plurifarium populations native to Mexican soils; Wang ET et al.; Forty-six Mesorhizobium strains associated with the leguminous plants Leucaena leucocephala and Sesbania herbacea in an uncultivated Mexican field were characterized using a polyphasic approach . The strains were identified as Mesorhizobium plurifarium based upon the close relationships with the reference strains for this species in PCR-based restriction fragment length polymorphism analyses, sequencing of 16S rRNA genes, multilocus enzyme electrophoresis, and DNA-DNA hybridization . Although the strains isolated from both plants formed the same group in multilocus enzyme electrophoresis and cross-nodulations were observed in the laboratory, different electrophoretic types were obtained from the two plants grown in natural soils, indicating the existence of a preferable association between the plants and the rhizobia . The M . plurifarium strains from Mexico and the reference strains from Africa and Brazil formed different phenotypic clusters in a numerical taxonomy . The Mexican strains did not grow at 37 degrees C and were sensitive to salty-alkaline conditions, while the reference strains from Africa and Brazil grew at 42 degrees C and were more resistant to salty-alkaline conditions . These results demonstrate that both the plants and environmental factors affected the evolution of rhizobia and that the Mexican strains had adapted to the neutral soils and the cool climate where they were isolated.

Shi Yan Sheng Wu Xue Bao, 2003 Aug, 36(4), 301 - 6
{Changes and distribution of ATPase activity in the paranodules of Nicotiana tabacum}; Yang YH et al.; Lead phosphate deposition technique was used to investigate the changes and characteristic distribution of ATPase activity in the paranodules of tabacoo . We found that ATPase activity was closely associated with cell type and cell developmental state, thus it was different in various cells and rhizobia . No lead phosphate granules were located in the menstematic cells, although there were a small number of lead phosphate granules in the cytoplasm and organelles of the cells without rhizobia, they did not exist in the young and mature rhizobia . On the contrary, upon the senscence of the cells and rhizobia, a large number of lead phosphate granules appeared on the plasmalemmas and in the cell walls and the inside surfaces of the rhizobia . When the cells and rhizobia progressively senesced, lead phosphate granules increased in number, and they were widely distributed on the tonoplasts, plasmalemmas, cell walls, intercellular layers and in the intercellular spaces . At the same time, they also appeared on the surfaces and in the cytoplasm and nucleoids of the rhizobia . Due to cell disintegration, lead phosphategranules obviously decreased in number, they were only located on the plasmalemmas and membrane-vesicular structures which came from disintegrated organelles.

Antonie Van Leeuwenhoek, 2003, 84(3), 201 - 7
Solubility enhancement of a hydrophobic flavonoid, luteolin by the complexation with cyclosophoraoses isolated from Rhizobium meliloti; Lee S et al.; A plant flavone, luteolin is a well-known inducer of nod genes in the Rhizobium meliloti . Its poor aqueous solubility was greatly enhanced by the complexation with a family of cyclosophoraoses synthesized in R.meliloti . Nuclear magnetic resonance (NMR) spectroscopic analysis showed that the chemical shifts of the aromatic ring moieties of the luteolin were changed greatly by the complexation with cyclosophoraoses . Fourier transform infrared (FTIR) spectroscopic analysis also showed a restricted vibrational pattern in carbonyl stretching region of the luteolin due to the complexation . This effective complex formation of cyclosophoraoses with a plant flavone, luteolin, suggests that rhizobial cyclosophoraoses play an important role as a solubility enhancer of the hydrophobic legume-derived flavonoids.

Can J Microbiol, 2003 Jun, 49(6), 399 - 405
A calcium-dependent bacterial surface protein is involved in the attachment of rhizobia to peanut roots; Dardanelli M et al.; As part of a project to characterize molecules involved in the crack-entry infection process leading to nodule development, a microscopic assay was used to visualize the attachment of cells of Bradyrhizobium sp . strains SEMIA 6144 and TAL 1000 (labelled by introducing a plasmid expressing constitutively the green fluorescent protein GFP-S65T) to Arachis hypogaea L . (peanut) . Qualitative and quantitative results revealed that attachment was strongly dependent on the growth phase of the bacteria . Optimal attachment occurred when bacteria were at the late log or early stationary phase . Cell surface proteins from the Bradyrhizobium sp . strains inhibited the attachment when supplied prior to the attachment assay . Root incubation with a 14-kDa protein (eluted from sodium dodecyl sulphate - gel electrophoresis of the cell surface fraction) prior to the attachment assay resulted in a strong decrease of attachment . The adhesin appeared to be a calcium-binding protein, since cells treated with EDTA were found to be able to bind to adhesin-treated peanut roots . Since this protein has properties identical to those reported for rhicadhesin, we propose that this adhesin is also involved in the attachment process of rhizobia to root legumes that are infected by the crack-entry process.

Mol Plant Microbe Interact, 2003 Oct, 16(10), 903 - 15
The Medicago truncatula sucrose synthase gene MtSucS1 is activated both in the infected region of root nodules and in the cortex of roots colonized by arbuscular mycorrhizal fungi; Hohnjec N et al.; The MtSucS1 gene encodes a sucrose synthase (EC 2.4.1.13) in the model legume Medicago truncatula . To determine the expression pattern of this gene in different organs and in particular during root endosymbioses, we transformed M . truncatula with specific regions of MtSucS1 fused to the gusAint reporter gene . These fusions directed an induction to the vasculature of leaves, stems, and roots as well as to flowers, developing seeds, young pods, and germinating seedlings . In root nodules, strong promoter activity occurred in the infected cells of the nitrogen-fixing zone but was additionally observed in the meristematic region, the prefixing zone, and the inner cortex, including the vasculature . Concerning endomycorrhizal roots, the MtSucS1 promoter mediated strongest expression in cortical cells harboring arbuscules . Specifically in highly colonized root sections, GUS-staining was furthermore detected in the surrounding cortical cells, irrespective of a direct contact with fungal structures . In accordance with the presence of an orthologous PsSus1 gene, we observed a comparable regulation of MtSucS1 expression in the grain legume Pisum sativum in response to microbial symbionts . Unlike other members of the MtSucS gene family, the presence of rhizobial or Glomus microsymbionts significantly altered and enhanced MtSucS1 gene expression, leading us to propose that MtSucS1 is involved in generating sink-strength, not only in root nodules but also in mycorrhizal roots.

Mol Plant Microbe Interact, 2003 Oct, 16(10), 884 - 92
Identical accumulation and immobilization of sulfated and nonsulfated Nod factors in host and nonhost root hair cell walls; Goedhart J et al.; Nod factors are signaling molecules secreted by Rhizobium bacteria . These lipo-chitooligosaccharides (LCOs) are required for symbiosis with legumes and can elicit specific responses at subnanomolar concentrations on a compatible host . How plants perceive LCOs is unclear . In this study, using fluorescent Nod factor analogs, we investigated whether sulfated and nonsulfated Nod factors were bound and perceived differently by Medicago truncatula and Vicia sativa root hairs . The bioactivity of three novel sulfated fluorescent LCOs was tested in a root hair deformation assay on M . truncatula, showing bioactivity down to 0.1 to 1 nM . Fluorescence microscopy of plasmolyzed M . truncatula root hairs shows that sulfated fluorescent Nod factors accumulate in the cell wall of root hairs, whereas they are absent from the plasma membrane when applied at 10 nM . When the fluorescent Nod factor distribution in medium surrounding a root was studied, a sharp decrease in fluorescence close to the root hairs was observed, visualizing the remarkable capacity of root hairs to absorb Nod factors from the medium . Fluorescence correlation microscopy was used to study in detail the mobilities of sulfated and nonsulfated fluorescent Nod factors which are biologically active on M . truncatula and V . sativa, respectively . Remarkably, no difference between sulfated and nonsulfated Nod factors was observed: both hardly diffuse and strongly accumulate in root hair cell walls of both M . truncatula and V . sativa . The implications for the mode of Nod factor perception are discussed.

J Biol Chem, 2003 Dec 19, 278(51), 51347 - 59 Epub 2003 Oct 08.
Genetic locus and structural characterization of the biochemical defect in the O-antigenic polysaccharide of the symbiotically deficient Rhizobium etli mutant, CE166 . Replacement of N-acetylquinovosamine with its hexosyl-4-ulose precursor; Forsberg LS et al.; The O-antigen polysaccharide (OPS) of Rhizobium etli CE3 lipopolysaccharide (LPS) is linked to the core oligosaccharide via an N-acetylquinovosaminosyl (QuiNAc) residue . A mutant of CE3, CE166, produces LPS with reduced amounts of OPS, and a suppressed mutant, CE166 alpha, produces LPS with nearly normal OPS levels . Both mutants are deficient in QuiNAc production . Characterization of OPS from CE166 and CE166 alpha showed that QuiNAc was replaced by its 4-keto derivative, 2-acetamido-2,6-dideoxyhexosyl-4-ulose . The identity of this residue was determined by NMR and mass spectrometry, and by gas chromatography-mass spectrometry analysis of its 2-acetamido-4-deutero-2,6-dideoxyhexosyl derivatives produced by reduction of the 4-keto group using borodeuteride . Mass spectrometric and methylation analyses showed that the 2-acetamido-2,6-dideoxyhexosyl-4-ulosyl residue was 3-linked and attached to the core-region external Kdo III residue of the LPS, the same position as that of QuiNAc in the CE3 LPS . DNA sequencing revealed that the transposon insertion in strain CE166 was located in an open reading frame whose predicted translation product, LpsQ, falls within a large family of predicted open reading frames, which includes biochemically characterized members that are sugar epimerases and/or reductases . A hypothesis to be tested in future work is that lpsQ encodes UDP-2-acetamido-2,6-dideoxyhexosyl-4-ulose reductase, the second step in the synthesis of UDP-QuiNAc from UDP-GlcNAc.

Biotechnol Adv, 1991, 9(2), 173 - 84
Improvement of Rhizobium inoculants by mutation, genetic engineering and formulation; Paau AS; The use of mutation and genetic engineering techniques have resulted in Rhizobium strains with improved characteristics . The latter approach can provide improvement and new traits not achievable previously . Enhanced commercial Rhizobium inoculants, however, still rely solely on traditional approaches including formulation improvement, mutation, and strain selection . The lack of contribution to the commercial product lines by genetic engineering is mostly due to the time delay and financial burdens cast by the regulatory policies rather than a lack of progress in the application of modern genetic technologies . Such constraints may lessen in time as the public becomes more educated about the technologies and the subsequent political pressure decreases.

Biotechnol Adv, 1985, 3(2), 155 - 70
Ecology and genetics of tropical rhizobia species; Ahmad MH et al.; Biological nitrogen fixation (BNF) technology with special reference to Rhizobium-legume symbiosis is growing very rapidly with the hope of combatting world hunger by producing cheaper protein for animal and human consumption in the Third World . One can see rapid progress made in the biochemistry and molecular biology of symbiotic nitrogen fixation in general; however, less progress has been made on the ecological aspects despite the fact that an enormous amount of literature is available on inoculation problems and on agronomic aspects of symbiotic nitrogen fixation . So far most information on Rhizobium concerns fast-growing rhizobia and their host legume . Although it is essential that food production using BNF technology should be maximized in the Third World, the least work has been done on slow-growing rhizobia, which are generally found in tropical and sub-tropical soils . The majority of the developing countries are in tropical and sub-tropical regions . Except for R . japonicum, a microsymbiont partner of soybean (Glycine max), the majority of the slow-growing rhizobia belong to the cowpea group, and we refer to cowpea rhizobia as tropical rhizobia species . In this review we have tried to consolidate the recent progress made on ecology and genetics of tropical rhizobia . By using recombinant DNA technology techniques it is expected that super strains of rhizobia with desirable characteristics can be produced . One must evaluate the efficiency and effectiveness of these genetically manipulated laboratory strains under field conditions . In conclusion, if one aims at combatting hunger in the Third World using BNF technology, an intensive research programme on fundamental and applied aspects of tropical rhizobia species is suggested . This involves close cooperation between molecular biologists and microbial ecologists.

Biotechnol Adv, 1989, 7(1), 31 - 45
Molecular genetics of Rhizobium Meliloti symbiotic nitrogen fixation; Watson RJ; The application of recombinant DNA techniques to the study of symbiotic nitrogen fixation has yielded a growing list of Rhizobium meliloti genes involved in the processes of nodulation, infection thread formation and nitrogenase activity in nodules on the roots of the host plant, Medicago sativa (alfalfa) . Interaction with the plant is initiated by genes encoding sensing and motility systems by which the bacteria recognizes and approaches the root . Signal molecules, such as flavonoids, mediate a complex interplay of bacterial and plant nodulation genes leading to entry of the bacteria through a root hair . As the nodule develops, the bacteria proceed inward towards the cortex within infection threads, the formation of which depends on bacterial genes involved in polysaccharide synthesis . Within the cortex, the bacteria enter host cells and differentiate into forms known as bacteroids . Genes which encode and regulate nitrogenase enzyme are expressed in the mature nodule, together with other genes required for import and metabolism of carbon and energy sources offered by the plant.

Biotechnol Adv, 1999 Oct, 17(4-5), 319 - 39
Phosphate solubilizing bacteria and their role in plant growth promotion; Rodriguez H et al.; The use of phosphate solubilizing bacteria as inoculants simultaneously increases P uptake by the plant and crop yield . Strains from the genera Pseudomonas, Bacillus and Rhizobium are among the most powerful phosphate solubilizers . The principal mechanism for mineral phosphate solubilization is the production of organic acids, and acid phosphatases play a major role in the mineralization of organic phosphorous in soil . Several phosphatase-encoding genes have been cloned and characterized and a few genes involved in mineral phosphate solubilization have been isolated . Therefore, genetic manipulation of phosphate-solubilizing bacteria to improve their ability to improve plant growth may include cloning genes involved in both mineral and organic phosphate solubilization, followed by their expression in selected rhizobacterial strains . Chromosomal insertion of these genes under appropriate promoters is an interesting approach.

Biotechnol Adv, 1993, 11(4), 741 - 79
Molecular mechanism of host specificity in legume-rhizobium symbiosis; Sharma PK et al.; Rhizobium - legume symbiosis is a highly specific interaction between the two partners . Host specificity is evident at early stages of infection and results from multiple interactions involving signalling among bacteria and host plants . Host specific plant signals (flavanoids) convert the NodD protein to an active form and its binding with nod box initiates the transcription of inducible nod operons . Common nod genes (nodABC) code for an extracellular mitogenic Nod factor which is required for nodule organogenesis . Host specific genes (hsn) modify the Nod factor to induce root hair deformation on specific hosts . The structure of Nod factor controls host range distinction between species and biovars of rhizobia . Interactions of lectins and Exopolysaccharide/Lipopolysaccharide result in host specific attachment of Rhizobium and its subsequent invasion . Change in Expopolysaccharide structure by the transfer of hsn genes enables the Rhizobium to bind with heterologous host lectins . Conversely, changes in root lectins via gene manipulation enables the heterologous rhizobia to bind and initiate nodulation on heterologous hosts . Finally, host specific signals are required to initiate nitrogen fixation in nodules that are formed.

Biotechnol Adv, 1995, 13(3), 415 - 24
The development of Azospirillum as a commercial inoculant for improving crop yields; Okon Y et al.; Bacteria of the genus Azospirillum are nitrogen-fixing organisms that live in close association with plants in the rhizosphere . The Azospirillum-plant association leads to the enhanced development and yield of different host plants under appropriate growth conditions . This increase in yield is attributed mainly to an improvement in root development, an increase in the rate of water and mineral uptake by roots, and to a lesser extent, biological N(2) fixation . Worldwide data accumulated in the field over the past 20 years indicates that Azospirillum is capable of promoting the yield of agriculturally important crops in different soils and climatic regions . A.brasilense shows both chemotaxis and chemokinesis in response to temporal gradients of different chemoeffectors, thereby increasing the chance of root-bacterial interactions . Phytohormones synthesized by Azospirillum influence the host root respiration rate, metabolism and root proliferation and hence better the mineral and water uptake in inoculated plants . Positive effects of combined inoculation with Rhizobium have been reported for different legumes and were related to the favorable influence of Azospirillum on the nodule number, plant development, dry weight, and N(2) fixation . Additionally, A . brasilense produces the reserve material polyhydroxybutyrate comprising up to 70% of the cell dry weight This substance has received much attention recently as it can be extracted and formed into a biodegradable thermoplastic.

Plant J, 2003 Oct, 36(2), 177 - 88
Transcriptome analysis of Arabidopsis colonized by a plant-growth promoting rhizobacterium reveals a general effect on disease resistance; Cartieaux F et al.; RNA transcript levels of Arabidopsis plants, infected by the rhizobacterium Pseudomonas thivervalensis (strain MLG45), and axenic control plants were compared using cDNA microarrays representing approximately 14 300 genes . The analysis revealed an increase of defence-related transcripts in the shoots of bacterized plants relative to control (axenic) plants . These modifications of transcript levels were confirmed by physiological experiments . Plants infected with P . thivervalensis were more resistant to subsequent infections by the virulent pathogen P . syringae pv . tomato (strain DC3000) than control plants . In addition, photosynthesis rates were repressed consistently with the reduced growth of plants colonized by P . thivervalensis . These results highlight the value of molecular phenotyping to predict physiological changes.

Nature, 2003 Oct 9, 425(6958), 637 - 40
A receptor kinase gene of the LysM type is involved in legume perception of rhizobial signals; Madsen EB et al.; Plants belonging to the legume family develop nitrogen-fixing root nodules in symbiosis with bacteria commonly known as rhizobia . The legume host encodes all of the functions necessary to build the specialized symbiotic organ, the nodule, but the process is elicited by the bacteria . Molecular communication initiates the interaction, and signals, usually flavones, secreted by the legume root induce the bacteria to produce a lipochitin-oligosaccharide signal molecule (Nod-factor), which in turn triggers the plant organogenic process . An important determinant of bacterial host specificity is the structure of the Nod-factor, suggesting that a plant receptor is involved in signal perception and signal transduction initiating the plant developmental response . Here we describe the cloning of a putative Nod-factor receptor kinase gene (NFR5) from Lotus japonicus . NFR5 is essential for Nod-factor perception and encodes an unusual transmembrane serine/threonine receptor-like kinase required for the earliest detectable plant responses to bacteria and Nod-factor . The extracellular domain of the putative receptor has three modules with similarity to LysM domains known from peptidoglycan-binding proteins and chitinases . Together with an atypical kinase domain structure this characterizes an unusual receptor-like kinase.

Nature, 2003 Oct 9, 425(6958), 585 - 92
Plant recognition of symbiotic bacteria requires two LysM receptor-like kinases; Radutoiu S et al.; Although most higher plants establish a symbiosis with arbuscular mycorrhizal fungi, symbiotic nitrogen fixation with rhizobia is a salient feature of legumes . Despite this host range difference, mycorrhizal and rhizobial invasion shares a common plant-specified genetic programme controlling the early host interaction . One feature distinguishing legumes is their ability to perceive rhizobial-specific signal molecules . We describe here two LysM-type serine/threonine receptor kinase genes, NFR1 and NFR5, enabling the model legume Lotus japonicus to recognize its bacterial microsymbiont Mesorhizobium loti . The extracellular domains of the two transmembrane kinases resemble LysM domains of peptidoglycan- and chitin-binding proteins, suggesting that they may be involved directly in perception of the rhizobial lipochitin-oligosaccharide signal . We show that NFR1 and NFR5 are required for the earliest physiological and cellular responses to this lipochitin-oligosaccharide signal, and demonstrate their role in the mechanism establishing susceptibility of the legume root for bacterial infection.

Antonie Van Leeuwenhoek, 2003, 84(2), 115 - 24
Low sequence similarity and gene content of symbiotic clusters of Bradyrhizobium sp . WM9 (Lupinus) indicate early divergence of "lupin" lineage in the genus Bradyrhizobium; Stepkowski T et al.; Two sequenced nodulation regions of lupin Bradyrhizobium sp . WM9 carried the majority of genes involved in the Nod factor production . The nod region I harbored: nolA, nodD, nodA, nodB, nodC, nodS, nodI, nodJ, nolO, nodZ, fixR, nifA, fixA, nodM, nolK and noeL . This gene arrangement resembled that found in the nodulation region of Bradyrhizobium japonicum USDA110, however strain WM9 harbored only one nodD gene copy, while the nodM, nolK and noeL genes had no counterparts in the 410 kb symbiotic region of strain USDA110 . Region II harbored nolL and nodW, but lacked an nodV gene . Both regions carried ORFs that lacked similarity to the published USDA110 sequences, though they had homologues in symbiotic regions of Rhizobium etli, Sinorhizobium sp . NGR234 and Mesorhizobium loti . These differences in gene content, as well as a low average sequence identity (70%) of symbiotic genes with respect to B . japonicum USDA110 were in contrast with the phylogenetic relationship of USDA110 and WM9 revealed by the analysis of 16S rDNA and dnaK sequences . This most likely reflected an early divergence of symbiotic loci, and possible co-speciation with distinct legumes . During this process the loss of a noel gene and the acquisition of a nolL gene could be regarded as an adaptation towards these legumes that responded to Nod factors carrying 4-O-acetylfucose rather than 2-O-methylfucose . This explained various responses of lupins and serradella plants to infection by mutants in nodZ and nolL genes, knowing that serradella is a stringent legume while lupins are more promiscuous legumes.

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