Diversity of rhizobia associated with Amorpha fruticosa isolated from Chinese soils and description of Mesorhizobium amorphae sp. nov.
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Fifty-five Chinese isolates from nodules of Amorpha fruticosa were characterized and compared with the type strains of the species and genera of bacteria which form nitrogen-fixing symbioses with leguminous host plants. A polyphasic approach, which included RFLP of PCR-amplified 16S rRNA genes, multilocus enzyme electrophoresis (MLEE), DNA-DNA hybridization, 16S rRNA gene sequencing, electrophoretic plasmid profiles, cross-nodulation and a phenotypic study, was used in the comparative analysis. The isolates originated from several different sites in China and they varied in their phenotypic and genetic characteristics. The majority of the isolates had moderate to slow growth rates, produced acid on YMA and harboured a 930 kb symbiotic plasmid (pSym). Five different RFLP patterns were identified among the 16S rRNA genes of all the isolates. Isolates grouped by PCR-RFLP of the 16S rRNA genes were also separated into groups by variation in MLEE profiles and by DNA-DNA hybridization. A representative isolate from each of these DNA homology groups had a separate position in a phylogenetic tree as determined from sequencing analysis of the 16S rRNA genes. A new species, Mesorhizobium amorphae, is proposed for the majority of the isolates, which belonged to a moderately slow- to slow-growing, acid-producing group based upon their distinct phylogenetic position, their unique electrophoretic type, their low DNA homology with reference strains representing the species within the genus Mesorhizobium and their distinct phenotypic features. Strain ACCC 19665 was chosen as the type strain for M. amorphae sp. nov. (+info)
NADH-glutamate synthase in alfalfa root nodules. Genetic regulation and cellular expression.
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NADH-dependent glutamate synthase (NADH-GOGAT; EC 1.4.1.14) is a key enzyme in primary nitrogen assimilation in alfalfa (Medicago sativa L.) root nodules. Here we report that in alfalfa, a single gene, probably with multiple alleles, encodes for NADH-GOGAT. In situ hybridizations were performed to assess the location of NADH-GOGAT transcript in alfalfa root nodules. In wild-type cv Saranac nodules the NADH-GOGAT gene is predominantly expressed in infected cells. Nodules devoid of bacteroids (empty) induced by Sinorhizobium meliloti 7154 had no NADH-GOGAT transcript detectable by in situ hybridization, suggesting that the presence of the bacteroid may be important for NADH-GOGAT expression. The pattern of expression of NADH-GOGAT shifted during root nodule development. Until d 9 after planting, all infected cells appeared to express NADH-GOGAT. By d 19, a gradient of expression from high in the early symbiotic zone to low in the late symbiotic zone was observed. In 33-d-old nodules expression was seen in only a few cell layers in the early symbiotic zone. This pattern of expression was also observed for the nifH transcript but not for leghemoglobin. The promoter of NADH-GOGAT was evaluated in transgenic alfalfa plants carrying chimeric beta-glucuronidase promoter fusions. The results suggest that there are at least four regulatory elements. The region responsible for expression in the infected cell zone contains an 88-bp direct repeat. (+info)
Azorhizobium caulinodans PII and GlnK proteins control nitrogen fixation and ammonia assimilation.
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We herein report that Azorhizobium caulinodans PII and GlnK are not necessary for glutamine synthetase (GS) adenylylation whereas both proteins are required for complete GS deadenylylation. The disruption of both glnB and glnK resulted in a high level of GS adenylylation under the condition of nitrogen fixation, leading to ammonium excretion in the free-living state. PII and GlnK also controlled nif gene expression because NifA activated nifH transcription and nitrogenase activity was derepressed in glnB glnK double mutants, but not in wild-type bacteria, grown in the presence of ammonia. (+info)
Susceptibility to hydrogen peroxide and catalase activity of root nodule bacteria.
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The root nodule bacteria (free-living cells) tested had higher susceptibility to hydrogen peroxide (H2O2) than the other genera of aerobic or facultative anaerobic bacteria tested. The catalase activities tended to have a positive correlation with H2O2 resistance among all bacteria tested. Addition of a catalase inhibitor such as 3-amino-1, 2, 4-triazole increased the susceptibility to H2O2. These results suggest that the lower catalase activity brings about the higher susceptibility of root nodule bacteria to H2O2. Root nodule bacteria seemed to have two or three catalase isozymes during growth and their catalase activities were higher in log phase than in stationary phase, contrary to other genera of bacteria tested. (+info)
Nodule-inducing activity of synthetic Sinorhizobium meliloti nodulation factors and related lipo-chitooligosaccharides on alfalfa. Importance of the acyl chain structure.
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Sinorhizobium meliloti nodulation factors (NFs) elicit a number of symbiotic responses in alfalfa (Medicago sativa) roots. Using a semiquantitative nodulation assay, we have shown that chemically synthesized NFs trigger nodule formation in the same range of concentrations (down to 10(-10) M) as natural NFs. The absence of O-sulfate or O-acetate substitutions resulted in a decrease in morphogenic activity of more than 100-fold and approximately 10-fold, respectively. To address the question of the influence of the structure of the N-acyl chain, we synthesized a series of sulfated tetrameric lipo-chitooligosaccharides (LCOs) having fatty acids of different lengths and with unsaturations either conjugated to the carbonyl group (2E) or located in the middle of the chain (9Z). A nonacylated, sulfated chitin tetramer was unable to elicit nodule formation. Acylation with short (C8) chains rendered the LCO active at 10(-7) M. The optimal chain length was C16, with the C16-LCO being more than 10-fold more active than the C12- and C18-LCOs. Unsaturations were important, and the diunsaturated 2E,9Z LCO was more active than the monounsaturated LCOs. We discuss different hypotheses for the role of the acyl chain in NF perception. (+info)
Comparison of the evolutionary dynamics of symbiotic and housekeeping loci: a case for the genetic coherence of rhizobial lineages.
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In prokaryotes, lateral gene transfer across chromosomal lineages may be mediated by plasmids, phages, transposable elements, and other accessory DNA elements. However, the importance of such transfer and the evolutionary forces that may restrict gene exchange remain largely unexplored in native settings. In this study, tests of phylogenetic congruence are employed to explore the range of horizontal transfer of symbiotic (sym) loci among distinct chromosomal lineages of native rhizobia, the nitrogen-fixing symbiont of legumes. Rhizobial strains isolated from nodules of several host plant genera were sequenced at three loci: symbiotic nodulation genes (nodB and nodC), the chromosomal housekeeping locus glutamine synthetase II (GSII), and a portion of the 16S rRNA gene. Molecular phylogenetic analysis shows that each locus generally subdivides strains into the same major groups, which correspond to the genera Rhizobium, Sinorhizobium, and Mesorhizobium. This broad phylogenetic congruence indicates a lack of lateral transfer across major chromosomal subdivisions, and it contrasts with previous studies of agricultural populations showing broad transfer of sym loci across divergent chromosomal lineages. A general correspondence of the three rhizobial genera with major legume groups suggests that host plant associations may be important in the differentiation of rhizobial nod and chromosomal loci and may restrict lateral transfer among strains. The second major result is a significant incongruence of nod and GSII phylogenies within rhizobial subdivisions, which strongly suggests horizontal transfer of nod genes among congenerics. This combined evidence for lateral gene transfer within, but not between, genetic subdivisions supports the view that rhizobial genera are "reproductively isolated" and diverge independently. Differences across rhizobial genera in the specificity of host associations imply that the evolutionary dynamics of the symbiosis vary considerably across lineages in native settings. (+info)
The early nodulin gene MtN6 is a novel marker for events preceding infection of Medicago truncatula roots by Sinorhizobium meliloti.
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MtN6 belongs to a series of cDNA clones representing Medicago truncatula genes transcriptionally activated during nodulation by Sinorhizobium meliloti (P. Gamas, F. de Carvalho Niebel, N. Lescure, and J. V. Cullimore, Mol. Plant-Microbe Interact. 9:233-242, 1996). We show here by in situ hybridization that MtN6 transcripts specifically accumulate first at very localized regions in the outer root cell layers, corresponding to outer cortical cells containing preinfection threads. At later stages, MtN6 expression is observed ahead of growing infection threads, including in the infection zone of mature root nodules. Interestingly, regulation of MtN6 is clearly distinct from that of other early nodulins expressed in the same region of the nodule, in terms of response to bacterial symbiotic mutants and to purified Nod factors. We thus suggest that MtN6 represents the first specific marker of a pathway involved in preparation to infection, which is at least partly controlled by Nod factors. Finally, we discuss the intriguing sequence homology shown by MtN6 to a protein from Emericella (Aspergillus) nidulans, FluG, that plays a key role in controlling the organogenesis of conidiophores (B. N. Lee and T. H. Adams, Genes Dev. 8:641-651, 1994). (+info)
Brucella outer membrane lipoproteins share antigenic determinants with bacteria of the family Rhizobiaceae.
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Brucellae have been reported to be phylogenetically related to bacteria of the family Rhizobiaceae. In the present study, we used a panel of monoclonal antibodies (MAbs) to Brucella outer membrane proteins (OMPs) to determine the presence of common OMP epitopes in some representative bacteria of this family, i.e., Ochrobactrum anthropi, Phyllobacterium rubiacearum, Rhizobium leguminosarum, and Agrobacterium tumefaciens, and also in bacteria reported to serologically cross-react with brucella, i.e., Yersinia enterocolitica O:9, Escherichia coli O:157, and Salmonella urbana. In particular, most MAbs to the Brucella outer membrane lipoproteins Omp10, Omp16, and Omp19 cross-reacted with O. anthropi and P. rubiacearum, which are actually the closest relatives of brucellae. Some of them also cross-reacted, but to a lower extent, with R. leguminosarum and A. tumefaciens. The putative Omp16 and Omp19 homologs in these bacteria showed the same apparent molecular masses as their Brucella counterparts. None of the antilipoprotein MAbs cross-reacted with Y. enterocolitica O:9, E. coli O:157, or S. urbana. (+info)