Rhizobium tropiciteu genes involved in specific uptake of Phaseolus vulgaris bean-exudate compounds

Rhizobium tropici nodulates and fixes nitrogen in bean. In the R. tropici strain CFN299 we identified and characterized teu genes (tropiciexudate uptake) induced by bean root exudates, localized by insertion of a promoter-less Tn5-gusA1 transposon. teu genes are present on a plasmid of around 185 kb that is conserved in all R. tropici strains. Proteins encoded by teu genes show similarity to ABC transporters, specifically to ribose transport proteins. No induction of the teu genes was obtained by treatment with root exudates from any of several other plants tested, with the exception of Macroptilium atropurpureum, which is also a host plant for R. tropici. It appears that the inducing compound is characteristic of bean and closely related legumes. It is present in root exudates, but not in seeds. This compound is removed, presumably by metabolism, from the exudates by the majority of bean-nodulating rhizobia (such as R. etli, R. leguminosarum bv. phaseoli and R.␣giardinii). The principal inducing compound has not been identified, but some induction was obtained using trigonelline. The CFN299 strain seems to have an additional uptake system, as no phenotype is observed in two different mutants. R. tropici strain CIAT899, on the other hand, must have only one uptake system, since a mutant bearing an insertion in the teu genes could not remove the compound from the exudates as efficiently as the wild type, and it showed diminished nodulation competitiveness. Received: 21 November 1997 / Accepted: 18 March 1998     

Genes Essential for Nod Factor Production and Nodulation Are Located on a Symbiotic Amplicon (AMPRtrCFN299pc60) in Rhizobium tropici

Amplifiable DNA regions (amplicons) have been identified in the genome of Rhizobium etli. Here we report the isolation and molecular characterization of a symbiotic amplicon of Rhizobium tropici. To search for symbiotic amplicons, a cartridge containing a kanamycin resistance marker that responds to gene dosage and conditional origins of replication and transfer was inserted in the nodulation region of the symbiotic plasmid (pSym) of R. tropici CFN299. Derivatives harboring amplifications were selected by increasing the concentration of kanamycin in the cell culture. The amplified DNA region was mobilized into Escherichia coli and then into Agrobacterium tumefaciens. The 60-kb symbiotic amplicon, which we termed AMPRtrCFN299pc60, contains several nodulation and nitrogen fixation genes and is flanked by a novel insertion sequence ISRtr1. Amplification of AMPRtrCFN299pc60 through homologous recombination between ISRtr1 repeats increased the amount of Nod factors. Strikingly, the conjugal transfer of the amplicon into a plasmidless A. tumefaciens strain confers on the transconjugant the ability to produce R. tropici Nod factors and to nodulate Phaseolus vulgaris, indicating that R. tropici genes essential for the nodulation process are confined to an ampliable DNA region of the pSym.     

Phaseolus vulgaris seed-borne endophytic community with novel bacterial species such as Rhizobium endophyticum sp. nov.

The bacterial endophytic community present in different Phaseolus vulgaris (bean) cultivars was analyzed by 16S ribosomal RNA gene sequences of cultured isolates derived from surface disinfected roots and immature seeds. Isolated endophytes from tissue-macerates belonged to over 50 species in 24 different genera and some isolates from Acinetobacter, Bacillus, Enterococcus, Nocardioides, Paracoccus, Phyllobacterium, and Sphingomonas seem to correspond to new lineages. Phytate solubilizing bacteria were identified among Acinetobacter, Bacillus and Streptomyces bean isolates, phytate is the most abundant reserve of phosphorus in bean and in other seeds. Endophytic rhizobia were not capable of forming nodules. A novel rhizobial species Rhizobium endophyticum was recognized on the basis of DNA–DNA hybridization, sequence of 16S rRNA, recA, rpoB, atpD, dnaK genes, plasmid profiles, and phenotypic characteristics. R. endophyticum is capable of solubilizing phytate, the type strain is CCGE2052 (ATCC BAA-2116; HAMBI 3153) that became fully symbiotic by acquiring the R. tropici CFN299 symbiotic plasmid.     

Nitrogen-Fixing Nodules with Ensifer adhaerens Harboring Rhizobium tropici Symbiotic Plasmids

Ensifer adhaerens is a soil bacterium that attaches to other bacteria and may cause lysis of these other bacteria. Based on the sequence of its small-subunit rRNA gene, E. adhaerens is related to Sinorhizobium spp. E. adhaerens ATCC 33499 did not nodulate Phaseolus vulgaris (bean) or Leucaena leucocephala, but with symbiotic plasmids from Rhizobium tropici CFN299 it formed nitrogen-fixing nodules on both hosts. The nodule isolates were identified as E. adhaerens isolates by growth on selective media.     

Increased Bean (Phaseolus vulgaris L.) Nodulation Competitiveness of Genetically Modified Rhizobium Strains

Rhizobium leguminosarum bv. phaseoli strain collections harbor heterogeneous groups of bacteria in which two main types of strains may be distinguished, differing both in the symbiotic plasmid and in the chromosome. We have analyzed under laboratory conditions the competitive abilities of the different types of Rhizobium strains capable of nodulating Phaseolus vulgaris L. bean. R. leguminosarum bv. phaseoli type I strains (characterized by nif gene reiterations and a narrow host range) are more competitive than type II strains (that have a broad host range), and both types are more competitive than the promiscuous rhizobia isolated from other tropical legumes able to nodulate beans. Type I strains become even more competitive by the transfer of a non-Sym, 225-kilobase plasmid from type II strain CFN299. This plasmid has been previously shown to enhance the nodulation and nitrogen fixation capabilities of Agrobacterium tumefaciens transconjugants carrying the Sym plasmid of strain CFN299. Other type I R. leguminosarum bv. phaseoli transconjugants carrying two symbiotic plasmids (type I and type II) have been constructed. These strains have a diminished competitive ability. The increase of competitiveness obtained in some transconjugants seems to be a transient property.     

Phaseolus vulgaris recognizes Azorhizobium caulinodans Nod factors with a variety of chemical substituents.

Phaseolus vulgaris is a promiscuous host plant that can be nodulated by many different rhizobia representing a wide spectrum of Nod factors. In this study, we introduced the Rhizobium tropici CFN299 Nod factor sulfation genes nodHPQ into Azorhizobium caulinodans. The A. caulinodans transconjugants produce Nod factors that are mostly if not all sulfated and often with an arabinosyl residue as the reducing end glycosylation. Using A. caulinodans mutant strains, affected in reducing end decorations, and their respective transconjugants in a bean nodulation assay, we demonstrated that bean nodule induction efficiency, in decreasing order, is modulated by the Nod factor reducing end decorations fucose, arabinose or sulfate, and hydrogen.     

Sucrose transport and hydrolysis inRhizobium tropici

TheRhizobium tropici strain CFN 299 was maintained on PY medium and was grown in minimal medium (MM) with sucrose, glucose, fructose and glutamate (or their combination) as carbon sources. Bacteria were able to simultaneously use different carbon sources and, with a combination sucrose and glutamate, the growth rate was faster than with either carbon source alone. Sucrose transport was induced by sucrose and partially repressed by glucose and glutamate if they were included in MM as additional carbon sources. The transport of sucrose was active because both an uncoupler (dinitrophenol, DNP) and inhibitors of terminal oxidation (KCN, NaN₃) severely reduced sucrose uptake. Sucrose transport was also sensitive to a functional sulfhydryl reagent but was much less sensitive to EDTA and arsenate. We obtained nonlinear Lineweaver-Burk plots for the uptake of sucrose (by sucrose-grown bacteria), and this implied the existence of at least two uptake mechanisms. Invertase (EC 3.2.1.26) is the main enzyme for sucrose hydrolysis in this organism. This enzyme was induced by sucrose and had high activity in mid-log phase cells when sucrose was the sole carbon source (0.2%). Invertase activity was not detected in growth medium. In general, the results obtained support the idea, thatR. tropici is adapted to sucrose utilization and to multicarbon nutrition during its interaction with plants.     

Nitrogen-fixing nodules induced by Agrobacterium tumefaciens harboring Rhizobium phaseoli plasmids.

Rhizobium phaseoli CFN299 forms nitrogen-fixing nodules in Phaseolus vulgaris (bean) and in Leucaena esculenta. It has three plasmids of 185, 225, and 410 kilobases. The 410-kilobase plasmid contains the nitrogenase structural genes. We have transferred these plasmids to the plasmid-free strain Agrobacterium tumefaciens GMI9023. Transconjugants containing different combinations of the R. phaseoli plasmids were obtained, and they were exhaustively purified before nodulation was assayed. Only transconjugants harboring the 410-kilobase plasmid nodulate P. vulgaris and L. esculenta. Nodules formed by all such transconjugants are able to reduce acetylene. Transconjugants containing the whole set of plasmids from CFN299 nodulate better and fix more nitrogen than the transconjugants carrying only the Sym plasmid. Microscopic analysis of nodules induced by A. tumefaciens transconjugants reveals infected cells and vascular bundles. None of the A. tumefaciens transconjugants, not even the one with the whole set of plasmids from CFN299, behaves in symbiosis like the original R. phaseoli strain; the transconjugants produce fewer nodules and have lower acetylene reduction (25% as compared to the original R. phaseoli strain) and more amyloplasts per nodule. More than 2,000 bacterial isolates from nodules of P. vulgaris and L. esculenta formed by the transconjugants were analyzed by different criteria. Not a single rhizobium could be detected. Our results show that R. phaseoli plasmids may be expressed in the A. tumefaciens background and direct the formation of effective, differentiated nodules.     

Nodulating ability of Rhizobium tropici is conditioned by a plasmid-encoded citrate synthase

Rhizobium species elicit the formation of nitrogen-fixing root nodules through a complex interaction between bacteria and plants. Various bacterial genes involved in the nodulation and nitrogen-fixation processes have been described and most have been localized on the symbiotic plasmids (pSym). We have found a gene encoding citrate synthase on the pSym plasmid of Rhizobium tropici, a species that forms nitrogen-fixing nodules on the roots of beans (PhasBoius vuigaris) and trees (Leucaena spp.). Citrate synthase is a key metabolic enzyme that incorporates carbon into the tricarboxylic acid cycle by catalysing the condensation of acetyl-CoA and oxalo-acetic acid to form citrate. R. tropici pcsA (the plasmid citrate synthase gene) is closely related to the corresponding genes of Proteobacteria. pcsA inactivation by a Tn5-mob insertion causes the bacteria to form fewer nodules (30–50% of the original strain) and to have a decreased citrate synthase activity in minimal medium with sucrose. A clone carrying the pcsA gene complemented ail the phenotypic alterations of the pcsA mutant, and conferred Rhizobium iegumino-sarum bv. phaseoli (which naturally lacks a plasmid citrate synthase gene) a higher nodulation and growth capacity in correlation with a higher citrate synthase activity. We have also found that pcsA gene expression is sensitive to iron availability, suggesting a possible role of pcsA in iron uptake.     

Rhizobium etli cycHJKL gene locus involved in c-type cytochrome biogenesis: sequence analysis and characterization of two cycH mutants

The cycHJKL gene locus was cloned from Rhizobium etli by the rescue of a Tn5mob insertion of a mutant (IFC01) which was affected in the production of c-type cytochromes. The cycH, cycJ, cycK and cycL genes are proposed to code for different subunits of a haem lyase complex involved in the attachment of haem to cytochrome c apoproteins. CycH of 365 aa shared 27, 36, 47 and 63% identity with CycH from Paracoccus denitrificans, Bradyrhizobium japonicum, R. meliloti , and R. leguminosarum, respectively. CycJ of 153 aa shared 52, 71, and 85% identity to the cycJ gene product of B. japonicum, R. meliloti, R. leguminosarum, respectively. CycK of 666 aa shared 62, 73, and 90% homology with CycK from B. japonicum, R. meliloti , and R. leguminosarum, respectively, while CycL of 151 aa shared 57, 67 and 86% homology with CycL from the abovementioned species. The Tn5mob insertion present in the IFC01 strain was located in the cycH gene. This strain was able to infect bean plants, but unable to fix nitrogen during symbiosis. A previously described R. etli cytochrome c-deficient MuD1lac-induced mutant (CFN4202) that induced empty nodules on Phaseolus vulgaris, also have lesions in cycH. Complementation analysis suggested that the MuD1lac insertion of the CFN4202 strain was polar on expression of genes downstream of cycH in contrast with the Tn5mob insertion present in IFC01, which showed no polarity on cycJKL. Our data suggest that CycH may not be essential for the infection process, but is necessary for nitrogen fixation.     

Effect of divalent cations on succinate transport in Rhizobium tropici,R. leguminosarum bv phaseoli and R. loti

Rhizobium tropici, R. leguminosarum bv phaseoli and R. loti each have an active C₄-dicarboxylic acid transport system dependent on an energized membrane. Free thiol groups are probably involved at the active site. Since EDTA inhibited succinate transport in R. leguminosarum bv phaseoli and R. loti, divalent cations may participate in the process; the activity was reconstituted by the addition of Ca²⁺ or Mg²⁺. However, EDTA had no effect on succinate transport in R. tropici, R. meliloti or R. trifolii strains. Ca²⁺ or Mg²⁺ had a similar effect on the growth rates of R. tropici and R. leguminosarum bv phaseoli; R. tropici did not require Ca²⁺ to grow on minimal medium supplemented with succinate but R. leguminosarum bv phaseoli required either or both of the divalent cations Ca²⁺ and Mg²⁺. A R. tropici Mu-dI (lacZ) mutant defective in dicarboxylic acid transport, was isolated and found unable to form effective bean nodules.The authors are with the Division of Biochemistry, Instituto de Investigaciones Biológicas Clemente Estable, Avda, Italia 3318, 11.600 Montevideo, Uruguay     

Multiple copies of nodD in Rhizobium tropici CIAT899 and BR816.

Rhizobium tropici strains are able to nodulate a wide range of host plants: Phaseolus vulgaris, Leucaena spp., and Macroptilium atropurpureum. We studied the nodD regulatory gene for nodulation of two R. tropici strains: CIAT899, the reference R. tropici type IIb strain, and BR816, a heat-tolerant strain isolated from Leucaena leucocephala. A survey revealed several nodD-hybridizing DNA regions in both strains: five distinct regions in CIAT899 and four distinct regions in BR816. Induction experiments of a nodABC-uidA fusion in combination with different nodD-hybridizing fragments in the presence of root exudates of the different hosts indicate that one particular nodD copy contributes to nodulation gene induction far more than any other nodD copy present. The nucleotide sequences of both nodD genes are reported here and show significant homology to those of the nodD genes of other rhizobia and a Bradyrhizobium strain. A dendrogram based on the protein sequences of 15 different NodD proteins shows that the R. tropici NodD proteins are linked most closely to each other and then to the NodD of Rhizobium phaseoli 8002.     

<Emphasis Type="Italic">Rhizobium etli</Emphasis> maize populations and their competitiveness for root colonization

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.     

Nitrogen-fixing nodules with Ensifer adhaerens harboring Rhizobium tropici symbiotic plasmids.

Ensifer adhaerens is a soil bacterium that attaches to other bacteria and may cause lysis of these other bacteria. Based on the sequence of its small-subunit rRNA gene, E. adhaerens is related to Sinorhizobium spp. E. adhaerens ATCC 33499 did not nodulate Phaseolus vulgaris (bean) or Leucaena leucocephala, but with symbiotic plasmids from Rhizobium tropici CFN299 it formed nitrogen-fixing nodules on both hosts. The nodule isolates were identified as E. adhaerens isolates by growth on selective media.     

Genetic diversity of rhizobia nodulating common bean (Phaseolus vulgaris L.) in the Central Black Sea Region of Turkey

We have analyzed 30 rhizobial isolates obtained from common bean (Phaseolus vulgaris L.) root nodules grown in the Middle Blacksea Region of Turkey, using ARDRA and nucleotide sequence data. ARDRA analysis with enzymes CfoI, HinfI, NdeII, MspI and PstI revealed three patterns. Based on sequence data from 16S rDNA, the patterns were identified as, Rhizobium leguminosarum bv. phaseoli (n = 16), R. etli bv. phaseoli (n = 8) and R. phaseoli (n = 6). On the other hand, nucleotide sequence phylogenies of housekeeping genes (recA, atpD and glnII) selected to confirm the 16S rDNA phylogeny revealed different evolutionary relationships. These results suggested the possibility of lateral transfers of these genes amongst different rhizobial species (including R. leguminosarum, R. etli and R. phaseoli) sharing the same ecological niche (nodulating P. vulgaris) which also indicates that there may be no true genetic barier among these species. Phylogenetic analysis based on DNA sequence data from the nodA and nifH genes showed that all rhizobial species obtained in this study were carrying nodA and nifH haplotypes which were the same or similar to those of CFN42 (R. etli type strain), suggesting a further support for the lateral transfer of CFN42 Sym plasmid, p42, amongst Turkish common bean nodulating rhizobial isolates.     

Carbon Metabolism Enzymes of Rhizobium tropici Cultures and Bacteroids

We determined the activities of selected enzymes involved in carbon metabolism in free-living cells of Rhizobium tropici CFN299 grown in minimal medium with different carbon sources and in bacteroids of the same strain. The set of enzymatic activities in sucrose-grown cells suggests that the pentose phosphate pathway, with the participation of the Entner-Doudoroff pathway, is probably the primary route for sugar catabolism. In glutamate- and malate-grown cells, high activities of the gluconeogenic enzymes (phosphoenolpyruvate carboxykinase, fructose-6-phosphate aldolase, and fructose bisphosphatase) were detected. In bacteroids, isolated in Percoll gradients, the levels of activity for many of the enzymes measured were similar to those of malate-grown cells, except that higher activities of glucokinase, glucose-6-phosphate dehydrogenase, and NAD-dependent phosphogluconate dehydrogenase were detected. Phosphoglucomutase and UDP glucose pyrophosphorylase showed high and constant levels under all growth conditions and in bacteroids.     

A Rhizobium tropici DNA region carrying the amino-terminal half of a nodD gene and a nod-box-like sequence confers host-range extension

Rhizobium tropici CIAT899 is a broad-host-range strain that, in addition to Phaseolus, nodulates other plant legumes such as Leucaena and Macroptilium. The narrow-host-range of Rhizobium leguminosarum biovars phaseoli (strain CE3) and trifolii (strain RS1051) can be extended to Leucaena esculents and Phaseolus vulgaris plants, respectively, by the introduction of a DNA fragment 521 bp long, which carries 128 amino acids of the amino-terminal region of a nodD gene from R. tropici, as well as a putative nod-box-like sequence, divergently oriented. The 521 bp fragment, in the presence of L. esculenta or P. vulgaris root exudates, induced a R. leguminosarum bv. viciae nodA-lacZ fusion in either a CE3 or RS1051 background, respectively.