Mtsym6, a gene conditioning Sinorhizobium strain-specific nitrogen fixation in Medicago truncatula.
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The availability of a wide range of independent lines for the annual medic Medicago truncatula led us to search for natural variants in the symbiotic association with Sinorhizobium meliloti. Two homozygous lines, Jemalong 6 and DZA315.16, originating from an Australian cultivar and a natural Algerian population, respectively, were inoculated with two wild-type strains of S. meliloti, RCR2011 and A145. Both plant lines formed nitrogen-fixing (effective) nodules with the RCR2011 strain. However, the A145 strain revealed a nitrogen fixation polymorphism, establishing an effective symbiosis (Nod(+)Fix(+)) with DZA315.16, whereas only small, white, non-nitrogen fixing nodules (Nod(+)Fix(-)) were elicited on Jemalong 6. Cytological studies demonstrated that these non-fixing nodules are encircled by an endodermis at late stages of development, with no visible meristem, and contain hypertrophied and autofluorescent infection threads, suggesting the induction of plant defense reactions. The non-fixing phenotype is independent of growth conditions and determined by a single recessive allele (Mtsym6), which is located on linkage group 8. (+info)
New substrates for the dicarboxylate transport system of Sinorhizobium meliloti.
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The dicarboxylate transport (Dct) system of Sinorhizobium meliloti, which is essential for a functional nitrogen-fixing symbiosis, has been thought to transport only dicarboxylic acids. We show here that the permease component of the Dct system, DctA, can transport orotate, a monocarboxylic acid, with an apparent K(m) of 1.7 mM and a V(max) of 163 nmol min(-1) per mg of protein in induced cells. DctA was not induced by the presence of orotate. The transport of orotate was inhibited by several compounds, including succinamic acid and succinamide, which are not dicarboxylic acids. The dicarboxylic acid maleate (cis-butenedioic acid) was not an inhibitor of orotate transport, which suggests that it was not recognized by DctA. However, maleate was an excellent inducer of DctA expression. Our evaluation of 17 compounds as inducers and inhibitors of transport suggests that substrates recognized by S. meliloti DctA must have appropriately spaced carbonyl groups and an extended conformation, while good inducers are more likely to have a curved conformation. (+info)
Alfalfa root nodule invasion efficiency is dependent on Sinorhizobium meliloti polysaccharides.
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The soil bacterium Sinorhizobium meliloti is capable of entering into a nitrogen-fixing symbiosis with Medicago sativa (alfalfa). Particular low-molecular-weight forms of certain polysaccharides produced by S. meliloti are crucial for establishing this symbiosis. Alfalfa nodule invasion by S. meliloti can be mediated by any one of three symbiotically important polysaccharides: succinoglycan, EPS II, or K antigen (also referred to as KPS). Using green fluorescent protein-labeled S. meliloti cells, we have shown that there are significant differences in the details and efficiencies of nodule invasion mediated by these polysaccharides. Succinoglycan is highly efficient in mediating both infection thread initiation and extension. However, EPS II is significantly less efficient than succinoglycan at mediating both invasion steps, and K antigen is significantly less efficient than succinoglycan at mediating infection thread extension. In the case of EPS II-mediated symbioses, the reduction in invasion efficiency results in stunted host plant growth relative to plants inoculated with succinoglycan or K-antigen-producing strains. Additionally, EPS II- and K-antigen-mediated infection threads are 8 to 10 times more likely to have aberrant morphologies than those mediated by succinoglycan. These data have important implications for understanding how S. meliloti polysaccharides are functioning in the plant-bacterium interaction, and models are discussed. (+info)
Identification of essential amino acid residues in the Sinorhizobium meliloti glucosyltransferase ExoM.
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ExoM is a beta(1-4)-glucosyltransferase involved in the assembly of the repeat unit of the exopolysaccharide succinoglycan from Sinorhizobium meliloti. By comparing the sequence of ExoM to those of other members of the Pfam Glyco Domain 2 family, most notably SpsA (Bacillus subtilis) for whom the three-dimensional structure has been resolved, three potentially important aspartic acid residues of ExoM were identified. Single substitutions of each of the Asp amino acids at positions 44, 96, and 187 with Ala resulted in the loss of mutant recombinant protein activity in vitro as well as the loss of succinoglycan production in an in vivo rescue assay. Mutants harboring Glu instead of Asp-44 or Asp-96 possessed no in vitro activity but could restore succinoglycan production in vivo. However, replacement of Asp-187 with Glu completely inactivated ExoM as judged by both the in vitro and in vivo assays. These results indicate that Asp-44, Asp-96, and Asp-187 are essential for the activity of ExoM. Furthermore, these data are consistent with the functions proposed for each of the analogous aspartic acids of SpsA based on the SpsA-UDP structure, namely, that Asp-44 and Asp-96 are involved in UDP substrate binding and that Asp-187 is the catalytic base in the glycosyltransferase reaction. (+info)
Effect of field inoculation with Sinorhizobium meliloti L33 on the composition of bacterial communities in rhizospheres of a target plant (Medicago sativa) and a non-target plant (Chenopodium album)-linking of 16S rRNA gene-based single-strand conformation polymorphism community profiles to the diversity of cultivated bacteria.
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Fourteen weeks after field release of luciferase gene-tagged Sinorhizobium meliloti L33 in field plots seeded with Medicago sativa, we found that the inoculant also occurred in bulk soil from noninoculated control plots. In rhizospheres of M. sativa plants, S. meliloti L33 could be detected in noninoculated plots 12 weeks after inoculation, indicating that growth in the rhizosphere preceded spread into bulk soil. To determine whether inoculation affected bacterial diversity, 1,119 bacteria were isolated from the rhizospheres of M. sativa and Chenopodium album, which was the dominant weed in the field plots. Amplified ribosomal DNA restriction analysis (ARDRA) revealed plant-specific fragment size frequencies. Dominant ARDRA groups were identified by 16S rRNA gene nucleotide sequencing. Database comparisons indicated that the rhizospheres contained members of the Proteobacteria (alpha, beta, and gamma subgroups), members of the Cytophaga-Flavobacterium group, and gram-positive bacteria with high G+C DNA contents. The levels of many groups were affected by the plant species and, in the case of M. sativa, by inoculation. The most abundant isolates were related to Variovorax sp., Arthrobacter ramosus, and Acinetobacter calcoaceticus. In the rhizosphere of M. sativa, inoculation reduced the numbers of cells of A. calcoaceticus and members of the genus Pseudomonas and increased the number of rhizobia. Cultivation-independent PCR-single-strand conformation polymorphism (SSCP) profiles of a 16S rRNA gene region confirmed the existence of plant-specific rhizosphere communities and the effect of the inoculant. All dominant ARDRA groups except Variovorax species could be detected. On the other hand, the SSCP profiles revealed products which could not be assigned to the dominant cultured isolates, indicating that the bacterial diversity was greater than the diversity suggested by cultivation. (+info)
PCR use of highly conserved DNA regions for identification of Sinorhizobium meliloti.
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A PCR identification method in which four primers that recognize homologous conserved regions in the Sinorhizobium meliloti genome are used was developed and tested. The regions used for identification were the nodbox 4 locus, which is located in one of the symbiotic megaplasmids, and the mucR gene, which is located in the chromosome. The new method was used to establish a collection of S. meliloti strains from polluted soils. (+info)
The Sinorhizobium meliloti ExpE1 protein secreted by a type I secretion system involving ExpD1 and ExpD2 is required for biosynthesis or secretion of the exopolysaccharide galactoglucan.
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In Sinorhizobium meliloti the biosynthesis of the exopolysaccharide galactoglucan (EPS II) is directed by the exp genes. The expD1 and expD2 gene products are homologous to components of type I secretion systems. ExpE1, the gene of which is located adjacent to expD1 and expD2, was detected in S. meliloti cells and culture supernatants. ExpD1 and ExpD2 were required for the secretion of ExpE1, indicating that ExpE1 is secreted by a type I secretion system involving ExpD1 and ExpD2. ExpE1 contains 15 aspartate- and glycine-rich nonapeptide repeats that were suggested to bind Ca(2+). The ability to bind Ca(2+) was demonstrated for a recombinant ExpE1 protein. Extracellular EPS II was not detected in cultures of non-polar expD1, expD2 and expE1 deletion mutants implying that these three genes are required for biosynthesis or secretion of galactoglucan. (+info)
Proteome analysis of differentially displayed proteins as a tool for the investigation of symbiosis.
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Two-dimensional gel electrophoresis was used to identify differentially displayed proteins expressed during the symbiotic interaction between the bacterium Sinorhizobium meliloti strain 1021 and the legume Melilotus alba (white sweetclover). Our aim was to characterize novel symbiosis proteins and to determine how the two symbiotic partners alter their respective metabolisms as part of the interaction, by identifying gene products that are differentially present between the symbiotic and non-symbiotic states. Proteome maps from control M. alba roots, wild-type nodules, cultured S. meliloti, and S. meliloti bacteroids were generated and compared. Over 250 proteins were induced or up-regulated in the nodule, compared with the root, and over 350 proteins were down-regulated in the bacteroid form of the rhizobia, compared with cultured cells. N-terminal amino acid sequencing and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry peptide mass fingerprint analysis, in conjunction with data base searching, were used to assign putative identity to nearly 100 nodule, bacterial, and bacteroid proteins. These included the previously identified nodule proteins leghemoglobin and NifH as well as proteins involved in carbon and nitrogen metabolism in S. meliloti. Bacteroid cells showed down-regulation of several proteins involved in nitrogen acquisition, including glutamine synthetase, urease, a urea-amide binding protein, and a PII isoform, indicating that the bacteroids were nitrogen proficient. The down-regulation of several enzymes involved in polyhydroxybutyrate synthesis and a cell division protein was also observed. This work shows that proteome analysis will be a useful strategy to link sequence information and functional genomics. (+info)