Rhizobium nodulation protein NodA is a host-specific determinant of the transfer of fatty acids in Nod factor biosynthesis

In the biosynthesis of lipochitin oligosaccharides (LCOs) theRhizobium nodulation protein NodA plays an essential role in the transfer of an acyl chain to the chitin oligosaccharide acceptor molecule. The presence ofnodA in thenodABCIJ operon makes genetic studies difficult to interpret. In order to be able to investigate the biological and biochemical functions of NodA, we have constructed a test system in which thenodA, nodB andnodC genes are separately present on different plasmids. Efficient nodulation was only obtained ifnodC was present on a low-copy-number vector. Our results confirm the notion thatnodA ofRhizobium leguminosarum biovarviciae is essential for nodulation onVicia. Surprisingly, replacement ofR. l. bv.viciae nodA by that ofBradyrhizobium sp. ANU289 results in a nodulation-minus phenotype onVicia. Further analysis revealed that theBradyrhizobium sp. ANU289 NodA is active in the biosynthesis of LCOs, but is unable to direct the transfer of theR. l. bv.viciae nodF E-dependent multi-unsaturated fatty acid to the chitin oligosaccharide acceptor. These results lead to the conclusion that the original notion thatnodA is a commonnod gene should be revised.     

Structural identification of the iipo-chitin oligosaccharide nodulation signals of Rhizobium loti

Rhizobium loti is a fast-growing Rhizobium species that has been described as a microsymbiont of plants of the genus Lotus. Nodulation studies show that Lotus plants are nodulated by R loti, but not by most other Rhizobium strains, indicating that R. loti produces specific lipo-chitin oligosaccharides (LCOs) which are necessary for the nodulation of Lotus plants. The LCOs produced by five different Rhizobium ioti strains have been purified and were shown to be N-acetylglucosamine pentasaccharides of which the non-reducing residue is N-methylated and N-acylated with c/s-vaccenic acid (C18:1) or stearic acid (C18:O) and carries a carbamoyl group. In one R. loti strain, NZP2037, an additional carbamoyl group is present on the non-reducing terminal residue. The major class of LCO molecules is substituted on the reducing terminal residue with 4-O-acetylfucose. Addition of LCOs to the roots of Lotus plants results in abundant distortion, swelling and branching of the root hairs, whereas spot inoculation leads to the formation of nodule primordia.     

Rhizobium nodulation protein NodA is a host-specific determinant of the transfer of fatty acids in Nod factor biosynthesis

In the biosynthesis of lipochitin oligosaccharides (LCOs) theRhizobium nodulation protein NodA plays an essential role in the transfer of an acyl chain to the chitin oligosaccharide acceptor molecule. The presence ofnodA in thenodABCIJ operon makes genetic studies difficult to interpret. In order to be able to investigate the biological and biochemical functions of NodA, we have constructed a test system in which thenodA, nodB andnodC genes are separately present on different plasmids. Efficient nodulation was only obtained ifnodC was present on a low-copy-number vector. Our results confirm the notion thatnodA ofRhizobium leguminosarum biovarviciae is essential for nodulation onVicia. Surprisingly, replacement ofR. l. bv.viciae nodA by that ofBradyrhizobium sp. ANU289 results in a nodulation-minus phenotype onVicia. Further analysis revealed that theBradyrhizobium sp. ANU289 NodA is active in the biosynthesis of LCOs, but is unable to direct the transfer of theR. l. bv.viciae nodF E-dependent multi-unsaturated fatty acid to the chitin oligosaccharide acceptor. These results lead to the conclusion that the original notion thatnodA is a commonnod gene should be revised.     

Rhizobial Resource Associated with Epidemic Legumes in Tibet

A total of 128 bacterial test strains originated from Astragalus, Caragana, Gueldenstaedtia, Medicago, Melilotus, Oxytropis, Trifolium, and Vicia grown in Tibet were characterized phenotypically and genomically. Based upon the consensus of grouping results, they were identified as 16 putative species. Twenty-five test strains belonging to seven putative species of Agrobacterium, Bradyrhizobium, and Rhizobium might be nonsymbiotic bacteria and the remaining 103 test strains were symbiotic bacteria belonging to Mesorhizobium, Rhizobium, and Sinorhizobium meliloti. Although no novel taxon was detected in the symbiotic bacteria, several characters including the alkaliphilic psychrotolerance revealed that the Tibetan rhizobia could be ecotypes adapted to the local conditions. The results also demonstrated that frequent lateral transfer of symbiotic genes might have happened in the Tibetan rhizobia since nodC genes similar to that of S. meliloti were found in several Rhizobium test strains and all the Mesorhizobium species had very similar nodC genes despite their genomic background. All of these findings demonstrated that the Tibetan rhizobia were an important resource for further studies on rhizobial ecology and application. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Induction of nodule primordia on Phaseolus and Acacia by lipo-chitin oligosaccharide nodulation signals from broad-host-range Rhizobium strain GRH2

Rhizobium wild-type strain GRH2 was originally isolated from the tree, Acacia cyanophylla, and has a broad host-range which includes herbaceous legumes, such as Phaseolus and Trifolium species. Here we show that strains of Rhizobium sp. GRH2, into which heterologous nodD alleles have been introduced, produce a large diversity of both sulphated and non-sulphated lipo-chitin oligosaccharides (LCOs). Most of the molecular species contain an N-methyl group on the reducing-terminal N-acetyl-glucosamine. The LCOs vary in the nature of the fatty acyl chain and in the length of the chitin backbone. The majority of the LCOs have an olgosaccharide chain length of five GlcNAc residues, but a few are oligomers having six GlcNAc units. LCOs purified from GRH2 are able to induce root hair formation and deformation on Acacia cyanophylla and A. melanoxylon plants. We show that an N-vaccenoyl-chitopentaose bearing an N-methyl group is able to induce nodule primordia on Phaseolus vulgaris, A. cyanophylla, and A. melanoxylon, indicating that for these plants an N-methyl modification is sufficient for nodule primordia induction.     

<Emphasis Type="Italic">Bradyrhizobium</Emphasis> spp. and <Emphasis Type="Italic">Sinorhizobium fredii</Emphasis> are predominant in root nodules of <Emphasis Type="Italic">Vigna angularis</Emphasis>, a native legume crop in the subtropical region of China

Adzuki bean (Vigna angularis) is an important legume crop native to China, but its rhizobia have not been well characterized. In the present study, a total of 60 rhizobial strains isolated from eight provinces of China were analyzed with amplified 16S rRNA gene RFLP, IGS-RFLP, and sequencing analyses of 16S rRNA, atpD, recA, and nodC genes. These strains were identified as genomic species within Rhizobium, Sinorhizobium, Mesorhizobium, Bradyrhizobium, and Ochrobactrum. The most abundant groups were Bradyrhizobium species and Sinorhizobium fredii. Diverse nodC genes were found in these strains, which were mainly co-evolved with the housekeeping genes, but a possible lateral transfer of nodC from Sinorhizobium to Rhizobium was found. Analyses of the genomic and symbiotic gene backgrounds showed that adzuki bean shared the same rhizobial gene pool with soybean (legume native to China) and the exotic Vigna species. All of these data demonstrated that nodule formation is the interaction of rhizobia, host plants, and environment characters. Electronic Supplementary Material  Supplementary material is available for this article at and is accessible for authorized users.     

Appearance and accumulation of nodulin mRNAs and their relationship to the effectiveness of root nodules

Summary Cloned cDNAs corresponding to mRNAs which accumulate in nitrogen-fixing root nodules of soybean (nodulin mRNAs) were used as probes to investigate the sizes, sequence relationships, tissue specificities and developmental accumulations of individual nodulin mRNA sequences. Northern blot analysis indicated that the NodB, NodC and NodD mRNA sequences are 1 150, 770, and 3 150 nucleotides long, respectively, which is consistent with the previously determined sizes of the hybrid-selected translation products (27 000, 24 000 and 100 000 MW, respectively). The NodA clones pNodA15 and pNodA25 hybridized to two mRNAs of lengths 1 600 and 1 100 nucleotides, indicating that they contain significant sequence homologies. However, increasing the hybridization stringency showed that the pNodA15 clone encodes the 1 600 nucleotide mRNA corresponding to the major NodA hybrid-selected translation product (44 000 MW) while pNodA25 encodes an mRNA of 1 100 nucleotides. The latter probably corresponds to one of two smaller (23 500 and 24 500 MW) in vitro translation products. RNA dot-blot hybridizations indicated that nodulin and leghemoglobin mRNAs began to appear and accumulate in Rhizobium infected root tissue very early (day 3 to 5) and reached fully induced levels by day 11. This accumulation was specific for nodule tissue (except for the NodD sequence) and preceded the accumulation of nitrogen fixation activity. Nodules produced by different effective Rhizobium strains accumulated similar levels of leghemoglobin and nodulin mRNAs while ineffective strains had a pleiotropic affect. While one ineffective strain (61A24) gave reduced levels of all these mRNAs, the other (SM5) gave levels which were nearly normal by the time nitrogen fixation activity should have reached its maximal level (day 17). Thus, leghemoglobin and nodulin genes are switched on soon after infection, prior to nodule morphogenesis, and the switch occurs prior to and is independent of nitrogen fixation activity.     

Root nodules of Genista germanica harbor Bradyrhizobium and Rhizobium bacteria exchanging nodC and nodZ genes

A collection of 18 previously unstudied strains isolated from root nodules of Genista germanica (German greenweed) grown in southeast Poland was evaluated for the level of genetic diversity using the BOX-PCR technique and the phylogenetic relationship based on both core (16S rRNA, dnaK, ftsA, glnII, gyrB, recA, rpoB) and nodulation (nodC and nodZ) gene sequences. Each of the 18 G. germanica root nodule isolates displayed unique BOX-PCR patterns, indicating their high level of genomic heterogeneity. Based on the comparative 16S rDNA sequence analysis, 12 isolates were affiliated to the Bradyrhizobium genus and the other strains were most similar to Rhizobium species. Phylogenetic analysis of the core gene sequences indicated that the studied Bradyrhizobium bacteria were most closely related to Bradyrhizobium japonicum, whereas Rhizobium isolates were most closely related to Rhizobium lusitanum and R. leguminosarum. The phylogenies of nodC and nodZ for the Rhizobium strains were incongruent with each other and with the phylogenies inferred from the core gene sequences. All Rhizobium nodZ gene sequences acquired in this study were grouped with the sequences of Bradyrhizobium strains. Some of the studied Rhizobium isolates were placed in the nodC phylogenetic tree together with reference Rhizobium species, while the others were closely related to Bradyrhizobium bacteria. The results provided evidence for horizontal transfer of nodulation genes between Bradyrhizobium and Rhizobium. However, the horizontal transfer of nod genes was not sufficient for Rhizobium strains to form nodules on G. germanica roots, suggesting that symbiotic genes have to be adapted to the bacterial genome.     

Feedback regulation of the Bradyrhizobium japonicum nodulation genes

Lipochitin Nod signals are produced by rhizobia and are required for the establishment of a nitrogen-fixing symbiosis with a legume host. The nodulation genes encode products required for the synthesis of this signal and are induced in response to plant-produced flavonoid compounds. The addition of chitin and lipo-chitin oligomers to Bradyrhizobium japonicum cultures resulted in a significant reduction in the expression of a nod–lacZ fusion. Intracellular expression of NodC, encoding a chitin synthase, also reduced nod gene expression. In contrast, expression of the ChiB chitinase increased nod gene expression. The chain length of the oligosaccharide was important in feedback regulation, with chitotetraose molecules the best modulators of nod gene expression. Feedback regulation is mediated by the induction of nolA by chitin, resulting in elevated levels of the repressor protein, NodD2.     

Genetic Diversity and Host Range of Rhizobia Nodulating Lotus tenuis in Typical Soils of the Salado River Basin (Argentina)

A total of 103 root nodule isolates were used to estimate the diversity of bacteria nodulating Lotus tenuis in typical soils of the Salado River Basin. A high level of genetic diversity was revealed by repetitive extragenic palindromic PCR, and 77 isolates with unique genomic fingerprints were further differentiated into two clusters, clusters A and B, after 16S rRNA restriction fragment length polymorphism analysis. Cluster A strains appeared to be related to the genus Mesorhizobium, whereas cluster B was related to the genus Rhizobium. 16S rRNA sequence and phylogenetic analysis further supported the distribution of most of the symbiotic isolates in either Rhizobium or Mesorhizobium: the only exception was isolate BA135, whose 16S rRNA gene was closely related to the 16S rRNA gene of the genus Aminobacter. Most Mesorhizobium-like isolates were closely related to Mesorhizobium amorphae, Mesorhizobium mediterraneum, Mesorhizobium tianshanense, or the broad-host-range strain NZP2037, but surprisingly few isolates grouped with Mesorhizobium loti type strain NZP2213. Rhizobium-like strains were related to Rhizobium gallicum, Rhizobium etli, or Rhizobium tropici, for which Phaseolus vulgaris is a common host. However, no nodC or nifH genes could be amplified from the L. tenuis isolates, suggesting that they have rather divergent symbiosis genes. In contrast, nodC genes from the Mesorhizobium and Aminobacter strains were closely related to nodC genes from narrow-host-range M. loti strains. Likewise, nifH gene sequences were very highly conserved among the Argentinian isolates and reference Lotus rhizobia. The high levels of conservation of the nodC and nifH genes suggest that there was a common origin of the symbiosis genes in narrow-host-range Lotus symbionts, supporting the hypothesis that both intrageneric horizontal gene transfer and intergeneric horizontal gene transfer are important mechanisms for the spread of symbiotic capacity in the Salado River Basin.     

Nodulation protein NodL of Rhizobium leguminosarum O-acetylates lipo-oligosaccharides, chitin fragments and N-acetylglucosamine in vitro

Upon induction of their nodulation genes, the root nodule-inducing Rhizobium bacteria produce lipo-oligosaccharide signal molecules. All lipo-oligosaccharides identified from Rhizobium leguminosarum bv. viciae carry an O-acetyl group at the C-6 position of the non-reducing terminal sugar, the presence of which is important for biological activity and host specificity. Previously we showed that a functional nodL gene product is required for the presence of this O-acetyl moiety. The production of polyclonal antibodies against isolated NodL protein, using a NodL- overproducing Escherichia coli strain is described. These antibodies were used (i) to elucidate the subcellular localization of the NodL protein, which appeared to be present in the cytosol, and (ii) for the purification of native NodL protein from E. coli. Here we provide biochemical proof that purified NodL protein has transacetylating activity in vitro with acetyl-CoA as the acetyl donor. NodL protein appeared to be able to acetylate various substrates, such as lipo-oligosaccharides, chitin fragments and N-acetylglucosamine. For chitinpentaose as the substrate we have shown, using mass spectrometry and NMR spectroscopy, that NodL protein substitutes one O-acetyl group at the C-6 position of the non-reducing terminal sugar.     

Evolutionary genomics of LysM genes in land plants

Background  

The ubiquitous LysM motif recognizes peptidoglycan, chitooligosaccharides (chitin) and, presumably, other structurally-related oligosaccharides. LysM-containing proteins were first shown to be involved in bacterial cell wall degradation and, more recently, were implicated in perceiving chitin (one of the established pathogen-associated molecular patterns) and lipo-chitin (nodulation factors) in flowering plants. However, the majority of LysM genes in plants remain functionally uncharacterized and the evolutionary history of complex LysM genes remains elusive.     

Factors effecting chitinase activity of <Emphasis Type="Italic">Rhizobium</Emphasis> sp. from <Emphasis Type="Italic">Sesbania sesban</Emphasis>

Twenty six Rhizobium strains isolated from root nodules of Sesbania sesban were studied for chitinase activity on chitin agar plates. Among them, only 12 strains showed chitinase activity. The strain showing the highest chitinase activity was selected based on maximum clear zone/colony size ratio on chitin agar plates and chitinase activity in culture filtrate. The strain was identified as Rhizobium sp. which showed a high degree of similarity with Rhizobium radiobacter (= Agrobacterium radiobacter). The cultural and nutritional conditions were optimized for maximum chitinase activity. The Rhizobium sp. exhibited maximum chitinase activity after 36 h of incubation, at neutral pH. Among the different nutritional sources, arabinose and yeast extract were found to be good inducers for chitinase activity. Rhizobium sp. could degrade and utilize dead mycelia of Aspergillus flavus, Aspergillus niger, Curvularia lunata, Fusarium oxysporum and Fusarium udum.     

The analysis of core and symbiotic genes of rhizobia nodulating Vicia from different continents reveals their common phylogenetic origin and suggests the distribution of Rhizobium leguminosarum strains together with Vicia seeds

In this work, we analysed the core and symbiotic genes of rhizobial strains isolated from Vicia sativa in three soils from the Northwest of Spain, and compared them with other Vicia endosymbionts isolated in other geographical locations. The analysis of rrs, recA and atpD genes and 16S–23S rRNA intergenic spacer showed that the Spanish strains nodulating V. sativa are phylogenetically close to those isolated from V. sativa and V. faba in different European, American and Asian countries forming a group related to Rhizobium leguminosarum. The analysis of the nodC gene of strains nodulating V. sativa and V. faba in different continents showed they belong to a phylogenetically compact group indicating that these legumes are restrictive hosts. The results of the nodC gene analysis allow the delineation of the biovar viciae showing a common phylogenetic origin of V. sativa and V. faba endosymbionts in several continents. Since these two legume species are indigenous from Europe, our results suggest a world distribution of strains from R. leguminosarum together with the V. sativa and V. faba seeds and a close coevolution among chromosome, symbiotic genes and legume host in this Rhizobium–Vicia symbiosis.     

Visualization of chitin-wall formation in hyphal tips and anastomoses of Diplodia natalensis by fluorescein-conjugated wheat germ agglutinin and [3H] N-acetyl-D-glucosamine

Colonies of the fungus Diplodia natalensis produce ample anastomoses which are visible 0.5–1.0 mm inwards of the colony's periphery. Anastomose formation as well as other morphogenetic features, were followed by autoradiography, lectin binding and application of the chitin synthase inhibitor polyoxin D.Hyphal tips and septae were strongly labelled by short pulses of [³H] N-acetyl-D-glucosamine ([³H]GlcNAc) and were showing marked fluorescence after exposure to fluorescein isothiocyanate (FITC) conjugated wheat germ agglutinin (WGA).The dynamics of wall formation was followed by pulse and chase as well as by pulse and wash treatments in which the colony was shortly exposed to [³H]GlcNAc and then freed from the radioactive chitin precursor.Application of the chitin synthase inhibitor polyoxin D caused hyphal tip swellings as well as inflations and balloons along the hyphae at sites of initial new outgrowths and anastomoses. These structures were strongly fluorescenting after FITC-WGA application, indicating imbalance of wall formation and wall lysis.FITC-WGA binding, [³H]GlcNAc labelling and/or exposure to polyoxin D, indicated a process of anastomose formation which starts with short outgrowths of two juxtapositioned hyphae and ends with a complete bridge formation.Abbreviations FITC fluorescein isothiocyanate - WGA wheat germ agglutinin - GlcNAc N-acetyl-D-glucosamine     

Transmembrane orientation and receptor-like structure of the Rhizobium meliloti common nodulation protein NodC

The 46.8-kd NodC protein of Rhizobium meliloti is a membrane protein, essential for nodule formation. Gene fusions of nodC to a portion of the λ cI repressor gene were used to define the membrane-anchor domain which is necessary for membrane insertion of the NodC protein into the membrane. The transmembrane orientation of NodC was confirmed by surface-specific radiolabeling and proteolysis experiments. A highly hydrophobic transmembrane-anchor domain was found near the carboxyl terminus, separating a large extracellular domain which contains an unusual cysteine-rich cluster from a short putative intracellular domain. Cross-linking studies showed that the NodC protein exists in the membrane probably as a dimer. The domain structure of the NodC protein shows striking similiarities with cell surface receptors. In nodules of various legumes a truncated form of the NodC protein was detected. The processed NodC was associated with the bacteroids and the amount of this protein increased during nodule development.     

<Emphasis Type="Italic">Rhizobium helanshanense</Emphasis> sp. nov., a bacterium that nodulates <Emphasis Type="Italic">Sphaerophysa salsula</Emphasis> (Pall.) DC. in China

Studying rhizobia in the root nodules of Sphaerophysa salsula (Pall.) DC in the northwest of China, we obtained five strains classified as genus Rhizobium on the basis of their 16S rRNA gene sequences. The sequence similarity of strain CCNWQTX14^T with the most related species was 99.0%. Further phylogenetic analysis of housekeeping genes (recA and atpD) suggested the five strains comprised a novel lineage within Rhizobium. The nifH and nodD gene sequences of CCNWQTX14^T were phylogenetically closely related with those of Sinorhizobium kummerowiae and R. sphaerophysae, respectively. The five strains isolated from different places were also distinct from related Rhizobium species using ERIC fingerprint profiles. The DNA–DNA hybridization value was 41.8% between CCNWQTX14^T and Rhizobium sphaerophysae CCNWGS0238^T. Our novel strains were only able to form effective nodules on its original host Sphaerophysa salsula. Our data showed that the five Rhizobium strains formed a unique genomic species, for which a novel species Rhizobium helanshanense sp. nov. is proposed. The type strain is CCNWQTX14^T (=ACCC 16237^T =HAMBI 3083^T).     

Symbiosis of Astragalus cicer with its microsymbionts: partial nodC gene sequence, host plant specificity, and root nodule structure

Astragalus cicer (cicer milkvetch) nodule bacteria were investigated for host plant specificity and partial nodC gene sequences, whilst their native host was studied for the microscopic structure of root nodules. The strains under investigation formed nodules not only on the original host but also on Astragalus glycyphyllos, Astragalus sinicus, Lotus corniculatus, and Phaseolus vulgaris. The nodules induced on the cicer milkvetch were classified as indeterminate and characterized by apical, persistent meristem, a large bacteroid region with infected and uninfected cells, and elongated bacteroids singly located inside peribacteroid membranes. By comparison of the partial nodC gene sequences of a representative strain of astragali rhizobia to those contained in the GenBank database, a close symbiotic relationship of A. cicer microsymbionts to Rhizobium sp. (Oxytropis) was found.     

Rhizobium nodulation protein NodC is an important determinant of chitin oligosaccharide chain length in Nod factor biosynthesis.

Synthesis of chitin oligosaccharides by NodC is the first committed step in the biosynthesis of rhizobial lipochitin oligosaccharides (LCOs). The distribution of oligosaccharide chain lengths in LCOs differs between various Rhizobium species. We expressed the cloned nodC genes of Rhizobium meliloti, R. leguminosarum bv. viciae, and R. loti in Escherichia coli. The in vivo activities of the various NodC proteins differed with respect to the length of the major chitin oligosaccharide produced. The clearest difference was observed between strains with R. meliloti and R. loti NodC, producing chitintetraose and chitinpentaose, respectively. In vitro experiments, using UDP-[14C]GlcNAc as a precursor, show that this difference reflects intrinsic properties of these NodC proteins and that it is not influenced by the UDP-GlcNAc concentration. Analysis of oligosaccharide chain lengths in LCOs produced by a R. leguminosarum bv. viciae nodC mutant, expressing the three cloned nodC genes mentioned above, shows that the difference in oligosaccharide chain length in LCOs of R. meliloti and R. leguminosarum bv. viciae is due only to nodC. The exclusive production of LCOs which contain a chitinpentaose backbone by R. loti strains is not due to NodC but to end product selection by Nod proteins involved in further modification of the chitin oligosaccharide. These results indicate that nodC contributes to the host specificity of R. meliloti, a conclusion consistent with the results of several studies which have shown that the lengths of the oligosaccharide backbones of LCOs can strongly influence their activities on host plants.     

Discrimination of Rhizobium japonicum,Rhizobium lupini,Rhizobium trifolii,Rhizobium leguminosarum and of bacteriods by uptake of 2-ketoglutaric acid,glutamic acid and phosphate

Rhizobium strains (one each of Rh.japonicum, Rh. lupini, Rh. leguminosarum) take up 2-ketoglutaric acid in general much faster and from lower concentrations in the medium than strains of Escherichia coli, Bacillus subtilis and Chromobacterium violaceum. A strain of Enterobacter aerogenes, however, is more similar to some Rhizobium strains. The same strains of Rhizobium take up also phosphate much faster and from lower concentrations than the other bacteria tested. 4 strains of Rh. lupini proved to be significantly different from 4 strains of Rh. trifolii in taking up l-glutamic acid from three to ten times lower concentration within 5 h. A similar difference was noticed between 5 strains of Rh. leguminosarum and 2 strains of Rh. japonicum for the uptake of 2-ketoglutaric acid and of l-glutamic acid. Isolated bacteriods from nodules of Glycine max var. Chippeway have a reduced uptake capacity for glutamic acid and for 2-ketoglutaric acid during the first 10–12 h, but reach the same value after 24 h as free living Rh. japonicum cells. The differences in the uptake kinetics are independent of cell concentration. The group II Rhizobium strains (Rh. japonicum and Rh. lupini, slow growing Rhizobium) are characterized by a rapid uptake of glutamic acid to a lowremaining concentration of 1–3×10^-7 M and an uptake of 2-ketoglutaric acid to a remaining concentration of 2–5×10^-7 M. The group I Rhizobium strains (Rh. trifolii and Rh. leguminosarum, fast growing Rhizobium), can be characterized by a much slower uptake of both substances with a more than ten times higher concentration of both metabolites remaining in the medium after the same time.