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PubMed2005

Diverse bacteria isolated from root nodules of Phaseolus vulgaris and species within the genera Campylotropis and Cassia grown in China.

Eighty bacterial isolates from root nodules of the leguminous plants Phaseolus vulgaris, Campylotropis spp. and Cassia spp. grown in China were classified into five groups by phenotypic analyses, SDS-PAGE of whole-cell proteins, PCR-based 16S rRNA gene restriction-fragment-length-polymorphism and sequencing. Thirty-three isolates from the three plant genera were identified as Agrobacterium tumefaciens because they are closely related to the type strain of A. tumefaciens. Fourteen isolates from P. vulgaris grown in Yunnan and Inner Mongolia were classified as R. leguminosarum bv. phaseoli based on their close relationship with the type strain in numerical taxonomy and in 16S rDNA phylogeny. Twenty-seven isolates from Campylotropis delavayi, P. vulgaris and four species of Cassia grown in the central zones of China were classified into three groups within the genus Bradyrhizobium. One of these three groups could be defined as Bradyrhizobium japonicum. Our results demonstrated that P. vulgaris and the species of Campylotropis and Cassia could form nodules with diverse rhizobia in Chinese soils, including novel lineages associated with P. vulgaris. These results also offered information about the convergent evolution between rhizobia and legumes since the rhizobial populations associated with P. vulgaris in Chinese soils were completely different from those in Mexico, the original cite of this plant. Some rhizobial species could be found in all of the three leguminous genera.

Agrobacterium tumefaciens↗
PubMed2006

Micromonospora coriariae sp. nov., isolated from root nodules of Coriaria myrtifolia.

An actinomycete strain, NAR01(T), was isolated from root nodules of a Coriaria plant. The 16S rRNA gene sequence of strain NAR01(T) showed most similarity to the type strains of Micromonospora endolithica (98.94 %) and Micromonospora chersina (98.4 %). The chemotaxonomic results obtained confirmed the taxonomic position of the isolate within the genus Micromonospora, and revealed differences at the species level. Physiological and biochemical tests showed that strain NAR01(T) could be clearly distinguished from its closest phylogenetic neighbours, while DNA-DNA hybridization results indicated that the isolate represents a novel species. On the basis of these results, strain NAR01(T) (=DSM 44875(T)=LMG 23557(T)) is proposed as the type strain of the novel species Micromonospora coriariae sp. nov.

Bacterial Typing Techniques↗
PubMedDate not supplied

Mass spectrometric analysis of lipo-chitin oligosaccharides--signal molecules mediating the host-specific legume-rhizobium symbiosis.

Lipo-chitin oligosaccharides (LCOs) are novel bacterial glycolipid signal molecules that mediate the species--specific symbiosis between rhizobial bacteria and leguminous plants. Nodulation of the legume roots and nitrogen-fixation in the resulting nodules by Rhizobia is controlled by the bacterial nodulation genes that encode the LCO biosynthetic enzymes. The length of the LCO chitin backbone, the length and degree of unsaturation of the fatty acyl chain attached to it, and the combination of different chemical substituents on the reducing- and nonreducing-terminal residues all contribute to the species--specificity of the signal. LCOs are bioactive in the nanomolar and subnanomolar concentration range and are produced as heterogeneous mixtures, making determination of their structures a difficult task, most successfully approached by the application of modern mass spectrometric methods in combination with specific chemical treatments aimed at identifying specific chemical moieties. This review presents an overview of these methods as they are being used for the structural elucidation of LCOs, and discusses the role of structural diversity in mediating species-specificity.

Carbohydrate Sequence↗
PubMed2003

Herbaspirillum lusitanum sp. nov., a novel nitrogen-fixing bacterium associated with root nodules of Phaseolus vulgaris.

Several bacterial strains were isolated from root nodules of Phaseolus vulgaris plants grown in a soil from Portugal. The strains were Gram-negative, aerobic, curved rod-shaped and motile. The isolates were catalase- and oxidase-positive. The TP-RAPD (two-primer randomly amplified polymorphic DNA) patterns of all strains were identical, suggesting that they belong to the same species. The complete 16S rDNA sequence of a representative strain was obtained and phylogenetic analysis based on the neighbour-joining method indicated that this bacterium belongs to the beta-Proteobacteria and that the closest related genus is Herbaspirillum. The DNA G+C content ranged from 57.9 to 61.9 mol%. Growth was observed with many different carbohydrates and organic acids including caprate, malate, citrate and phenylacetate. No growth was observed with maltose, meso-inositol, meso-erythritol or adipate as sole carbon source. According to the phenotypic and genotypic data obtained in this work, the bacterium represents a novel species of the genus Herbaspirillum, and the name Herbaspirillum lusitanum sp. nov. is proposed. The type strain is P6-12(T) (=LMG 21710(T)=CECT 5661(T)).

Geography↗
PubMed2002

Analysis of genomic diversity among photosynthetic stem-nodulating rhizobial strains from northeast Argentina.

The genomic diversity among photosynthetic rhizobia from northeast Argentina was assessed. Forty six isolates obtained from naturally occurring stem and root nodules of Aeschynomene rudis plants were analyzed by three molecular typing methods with different levels of taxonomic resolution: repetitive sequence-based PCR (rep-PCR) genomic fingerprinting with BOX and REP primers, amplified 16S rDNA restriction analysis (ARDRA), and 16S-23S rDNA intergenic spacer-restriction fragment length polymorphism (IGS-RFLP) analysis. The in vivo absorption spectra of membranes of strains were similar in the near infrared region with peaks at 870 and 800 nm revealing the presence of light harvesting complex I, bacteriochlorophyll-binding polypeptides (LHI-Bchl complex). After extraction with acetone-methanol the spectra differed in the visible part displaying peaks belonging to canthaxanthin or spirilloxanthin as the main carotenoid complement. The genotypic characterization by rep-PCR revealed a high level of genomic diversity among the isolates and almost all the photosynthetic ones have identical ARDRA patterns and fell into one cluster different from Bradyrhizobium japonicum and Bradyrhizobium elkanii. In the combined analysis of ARDRA and rep-PCR fingerprints, 7 clusters were found including most of the isolates. Five of those contained only photosynthetic isolates; all canthaxanthin-containing strains grouped in one cluster, most of the other photosynthetic isolates were grouped in a second large cluster, while the remaining three clusters contained a few strains. The other two clusters comprising reference strains of B. japonicum and B. elkanii, respectively. The IGS-RFLP analysis produced similar clustering for almost all the strains. The 16S rRNA gene sequence of one representative isolate was determined and the DNA sequence analysis confirmed the position of photosynthetic rhizobia in a distinct phylogenetic group within the Bradyrhizobium rDNA cluster.

Argentina↗
PubMed2003

Rhizobium-lnduced calcium spiking in Lotus japonicus.

Legumes and rhizobium bacteria form a symbiosis that results in the development of nitrogen-fixing nodules on the root of the host plant. The earliest plant developmental changes are triggered by bacterially produced nodulation (Nod) factors. Within minutes of exposure to Nod factors, sharp oscillations in cytoplasmic calcium levels (calcium spiking) occur in epidermal cells of several closely related legumes. We found that Lotus japonicus, a legume that follows an alternate developmental pathway, responds to both its bacterial partner and to the purified bacterial signal with calcium spiking. Thus, calcium spiking is not restricted to a particular pathway of nodule development and may be a general component of the response of host legumes to their bacterial partner. Using Nod factor-induced calcium spiking as a tool to identify mutants blocked early in the response to Nod factor, we show that the L. japonicus Ljsym22-1 mutant but not the Ljsym30 mutant fails to respond to Nod factor with calcium spiking.

Calcium Signaling↗
PubMed2003

Distinct patterns of symbiosis-related gene expression in actinorhizal nodules from different plant families.

Phylogenetic analyses suggest that, among the members of the Eurosid I clade, nitrogen-fixing root nodule symbioses developed multiple times independently, four times with rhizobia and four times with the genus Frankia. In order to understand the degree of similarity between symbiotic systems of different phylogenetic subgroups, gene expression patterns were analyzed in root nodules of Datisca glomerata and compared with those in nodules of another actinorhizal plant, Alnus glutinosa, and with the expression patterns of homologous genes in legumes. In parallel, the phylogeny of actinorhizal plants was examined more closely. The results suggest that, although relationships between major groups are difficult to resolve using molecular phylogenetic analysis, the comparison of gene expression patterns can be used to inform evolutionary relationships. In this case, stronger similarities were found between legumes and intracellularly infected actinorhizal plants (Alnus) than between actinorhizal plants of two different phylogenetic subgroups (Alnus/Datisca).

Amino Acid Sequence↗
PubMed1990

Symbiotic host-specificity of Rhizobium meliloti is determined by a sulphated and acylated glucosamine oligosaccharide signal.

Rhizobia are symbiotic bacteria that elicit the formation on leguminous plants of specialized organs, root nodules, in which they fix nitrogen. In various Rhizobium species, such as R. leguminosarum and R. meliloti, common and host-specific nodulation (nod) genes have been identified which determine infection and nodulation of specific hosts. Common nodABC genes as well as host-specific nodH and nodQ genes were shown recently, using bioassays, to be involved in the production of extracellular Nod signals. Using R. meliloti strains overproducing symbiotic Nod factors, we have purified the major alfalfa-specific signal, NodRm-1, by gel permeation, ion exchange and C18 reverse-phase high performance liquid chromatography. From mass spectrometry, nuclear magnetic resonance, (35)S-labelling and chemical modification studies, NodRm-1 was shown to be a sulphated beta-1,4-tetrasaccharide of D-glucosamine (Mr 1,102) in which three amino groups were acetylated and one was acylated with a C16 bis-unsaturated fatty acid. This purified Nod signal specifically elicited root hair deformation on the homologous host when added in nanomolar concentration.

Acylation↗
PubMed1996

The C-terminal domain of the Rhizobium leguminosarum chitin synthase NodC is important for function and determines the orientation of the N-terminal region in the inner membrane.

The nodC genes from rhizobia encode an N-acetylglucosaminyl transferase (chitin synthase) involved in the formation of lipo-chito-oligosaccharide Nod factors that initiate root nodule morphogenesis in legume plants. NodC proteins have two hydrophobic domains, one of about 21 residues at the N-terminus and a longer one, which could consist of two or three transmembrane spans, near the C-terminus. These two hydrophobic domains flank a large hydrophilic region that shows extensive homology with other beta -glycosyl transferases. The topology NodC in the inner membrane of Rhizobium leguminosarum biovar viciae was analysed using a series of gene fusions encoding proteins in which NodC was fused to alkaline phosphatase (PhoA) lacking an N-terminal transit sequence or to beta-galactosidase (LacZ). Our data support a model in which the N-terminal hydrophobic domain spans the membrane in a Nout-Cin orientation, with the adjacent large hydrophilic domain being exposed to the cytoplasm. This orientation appears to depend upon the presence of the hydrophobic region near the C-terminus. We propose that this hydrophobic region contains three transmembrane spans, such that the C-terminus of NodC is located in the periplasm. A short region of about 40 amino acids, encompassing the last transmembrane span, is essential for the function of NodC. Our model for NodC topology suggests that most of NodC, including the region showing most similarity to other beta-glycosyl transferases, is exposed to the cytoplasm, where it is likely that polymerization of N-acetyl glucoasamine occurs. Such a model is incompatible with previous reports suggesting that NodC spans both inner and other membranes.

Alkaline Phosphatase↗
PubMed2001

Identification and structure of the Rhizobium galegae common nodulation genes: evidence for horizontal gene transfer.

Rhizobia are soil bacteria able to fix atmospheric nitrogen in symbiosis with leguminous plants. In response to a signal cascade coded by genes of both symbiotic partners, a specific plant organ, the nodule, is formed. Rhizobial nodulation (nod) genes trigger nodule formation through the synthesis of Nod factors, a family of chitolipooligosaccharides that are specifically recognized by the host plant at the first stages of the nodulation process. Here, we present the organization and sequence of the common nod genes from Rhizobium galegae, a symbiotic member of the RHIZOBIACEAE: This species has an intriguing phylogenetic position, being symbiotic among pathogenic agrobacteria, which induce tumors instead of nodules in plant shoots or roots. This apparent incongruence raises special interest in the origin of the symbiotic apparatus of R. galegae. Our analysis of DNA sequence data indicated that the organization of the common nod gene region of R. galegae was similar to that of Sinorhizobium meliloti and Rhizobium leguminosarum, with nodIJ downstream of nodABC and the regulatory nodD gene closely linked to the common nod operon. Moreover, phylogenetic analyses of the nod gene sequences showed a close relationship especially between the common nodA sequences of R. galegae, S. meliloti, and R. leguminosarum biovars viciae and trifolii. This relationship in structure and sequence contrasts with the phylogeny based on 16S rRNA, which groups R. galegae close to agrobacteria and separate from most other rhizobia. The topology of the nodA tree was similar to that of the corresponding host plant tree. Taken together, these observations indicate that lateral nod gene transfer occurred from fast-growing rhizobia toward agrobacteria, after which the symbiotic apparatus evolved under host plant constraint.

Bacterial Proteins↗
PubMed2006

The Sinorhizobium meliloti chromosomal origin of replication.

The predicted chromosomal origin of replication (oriC) from the alfalfa symbiont Sinorhizobium meliloti is shown to allow autonomous replication of a normally non-replicating plasmid within S. meliloti cells. This is the first chromosomal replication origin to be experimentally localized in the Rhizobiaceae and its location, adjacent to hemE, is the same as for oriC in Caulobacter crescentus, the only experimentally characterized alphaproteobacterial oriC. Using an electrophoretic mobility shift assay and purified S. meliloti DnaA replication initiation protein, binding sites for DnaA were mapped in the S. meliloti oriC region. Mutations in these sites eliminated autonomous replication. S. meliloti that expressed DnaA from a plasmid lac promoter was observed to form pleomorphic filamentous cells, suggesting that cell division was perturbed. Interestingly, this cell phenotype is reminiscent of differentiated bacteroids found inside plant cells in alfalfa root nodules.

Bacterial Proteins↗
PubMed1981

Carbon Dioxide Fixation in Soybean Roots and Nodules: I. CHARACTERIZATION AND COMPARISON WITH N(2) FIXATION AND COMPOSITION OF XYLEM EXUDATE DURING EARLY NODULE DEVELOPMENT.

These studies demonstrate that soybean (Merr) roots and nodules possess an active system for fixing CO(2). The maximum rates of CO(2) fixation observed for roots and nodules of intact plants were 120 and 110 nanomoles CO(2) fixed per milligram dry weight per hour, respectively. Results of labeling studies suggest a primary role for phosphoenolpyruvate carboxylase in CO(2) assimilation in these tissues. After pulse-labeling with (14)CO(2) for 2 minutes, 70% of the total radioactivity was lost within 18 minutes via respiration and/or translocation out of nodules. During the vegetative stages of growth of soybeans grown symbiotically, CO(2) fixation in nodules increased at the onset of N(2) fixation but declined to a lower level prior to the decrease in N(2) fixation. This decrease coincided with a decrease in the transport of amino acids, especially asparagine, and an increase in the export of ureides. These findings are consistent with a dual role for CO(2) fixation, providing substrates for energy-yielding metabolism and supplying carbon skeletons for NH(4) (+) assimilation and amino acid biosynthesis.

Journal Article↗
PubMed1981

Dinitrogen Fixation by Cultures of Frankia sp CpIl Demonstrated by N(2) Incorporation.

The filamentous bacterium Frankia of the Actinomycetales, isolated from the nitrogen-fixing root nodules of certain woody plants, has shown nitrogenase activity in culture, using the acetylene reduction method. In the present work, nitrogenase activity in pure cultures of Frankia sp. CpIl is confirmed using mass spectrometric measurements of (15)N(2) incorporation. After addition of carrier NH(4) (+) to digested cultures, those exposed to (15)N(2) (25 atom%) had a (15)N content of 3.16 atom% compared to 0.354 atom% (15)N in the controls.

Journal Article↗
PubMed1990

Nitrogenase Activity and Nodule Gas Permeability Response to Rhizospheric NH(3) in Soybean.

This study was conducted on soybean (Glycine max L. Merr.) nodules to determine if exogenous NH(3) exerts a controlling influence over nitrogenase activity through changes in nodule gas permeability (P), and if decreasing carbohydrate availability, as a result of low-light treatment, increases the sensitivity of root nodules to NH(3). Nodulated root systems of intact plants were exposed to one of several NH(3) concentrations ranging from 0 to 821 microliters per liter for an 8-hour period. Treatments were conducted under high-light (2300 micromoles per square meter per second) or low-light (800 micromoles per square meter per second) conditions. Increasing the NH(3) concentration and length of exposure of NH(3) caused a progressive decline in acetylene reduction activity (ARA). There was generally a greater reduction in ARA under the low-light treatment compared to the high-light treatment at a particular NH(3) concentration. The NH(3) concentration necessary to decrease P was greater than that needed to decrease ARA, and there was no evidence of a causal relationship between P and ARA in response to NH(3).

Journal Article↗
PubMed1992

Genetics of competition for nodulation of legumes.

An economically important problem in microbial ecology concerns the efficacy of rhizobial inoculants for the formation of nitrogen-fixing root nodules on legume crop plants such as soybean, alfalfa, and clover. Some strains of rhizobia can increase symbiotic nitrogen fixation under controlled conditions. However, attempts to improve nitrogen fixation under agricultural conditions with such strains often fail, usually as a result of the presence of indigenous rhizobia limiting nodulation by the inoculum strains. This problem is referred to as the Rhizobium competition problem, and molecular genetics is being used to address the problem from two perspectives. First, the host specificity of rhizobia is being characterized with the long term goal of developing strains that can nodulate a very strain-specific host-legume genotype. Second, the genetic basis of competitiveness in several strains is being examined. Genetic determinants of nodulation competitiveness have been isolated and mechanisms for their stable integration into the genome of superior nitrogen-fixing strains have been developed. Several phenotypes have been identified as playing an important role in nodulation competitiveness including antibiosis, motility, speed of nodulation, cell-surface characteristics, and nodulation efficiency. Several solutions to this problem are likely to result from these strategies and will be useful for certain legumes in specific locations.

Fabaceae↗