Structural and functional analysis of the fixLJ genes of Rhizobium leguminosarum biovar phaseoli CNPAF512

The fixLJ genes of Rhizobium leguminosarum biovar phaseoli CNPAF512 were identified by DNA hybridization of a genomic library with an internal fragment of the Rhizobium meliloti fixJ gene. The nucleotide sequence was determined and the corresponding amino acid sequence was aligned with the amino acid sequences of the FixL proteins of R. meliloti, Bradyrhizobium japonicum and Azorhizobium caulinodans. While the FixJ protein and the carboxy-terminal part of the FixL protein are highly homologous to the other FixL and FixJ proteins, the homology in the central heme-binding, oxygen-sensing domain and in the amino-terminal domain of FixL is very low. The R. leguminosarum bv. phaseoli FixL protein does not contain the heme-binding motif defined for the previously described FixL proteins. R. leguminosarum bv. phaseoli fixLJ and fixJ mutants were constructed. These mutants can still fix nitrogen, albeit at a reduced level. Expression analysis of nifA-gusA and nifH-gusA fusions in the constructed mutants revealed that the R. leguminosarum bv. phaseoli fixLJ genes are involved in microaerobic nifH expression but not in nifA expression.The nucleotide sequence data reported will appear in the EMBL, Genbank and DDBJ Nucleotide Sequence Databases under the accession number U27314     

The dnaJ (hsp40) locus in Rhizobium leguminosarum bv. phaseoli is required for the establishment of an effective symbiosis with Phaseolus vulgaris

P121R25 is a Tn5-induced mutant of the effective Rhizobium leguminosarum bv. phaseoli strain P121R that is unable to use glutamate as the sole carbon and nitrogen source and is defective in symbiotic nitrogen fixation. Enzymatic analysis showed that three enzymes implicated in glutamate metabolism (glutamate dehydrogenase, 2-oxoglutarate dehydrogenase, and glutamate synthase) were affected by this mutation. Sequencing of the chromosomal locus bordering the Tn5 in P121R25 indicated the presence of the dnaK and dnaJ genes in an arrangement similar to that described in R. leguminosarum bv. viciae (GenBank accession number Y14649). The mutation was located in the dnaJ (hsp40) gene.     

Some properties of carbohydrate and C4-dicarboxylic acid utilization negative mutants ofRhizobium leguminosarum biovarphaseoli strain P121

After NTG treatment of the very effective wild type strain P121 ofRhizobium leguminosarum biovarphaseoli, mutants defective in the utilization of sugars or organic acids were obtained. All the mutants nodulated the cultivar Goldie ofPhaseolus vulgaris. The arabinose, fructose, glucose and pyruvate utilization mutants formed nodules similar in shape and size to the nodules formed by the wild type strain. These mutants exhibited an acetylene reduction activity significantly lower than the activity observed with the wild type strain. All the C₄-dicarboxylic acid utilization mutatns, formed ineffective nodules that did not show a significant acetylene reduction activity. The C₄-dicarboxylic acids uptake system is apparently inducible in the free-living bacteria of strain P121. When P121 cells were grown on glucose in the presence of 2.5 mM malate, the rate of glucose-dependent O₂ consumption significantly decreased suggesting the presence of a catabolite repression-like phenomenon. Isolated bacteroids of strain P121, under the experimental conditions used, were able to oxidize succinate, fumarate or malate but did not oxidize pyruvate, glucose, fructose or sucrose.     

Tolerance ofRhizobium leguminosarum bv.phaseoli to acidity and drought

Ten strains ofRhizobium leguminosarum bv.phaseoli isolated from soils of Morocco were more tolerant than three culture collection strains to acid conditions in culture media or in sterile soil. The survival rate of a tolerant strain in a sandy acid soil was greater than a sensitive strain at different humidity levels. These properties should give locally selected strains an advantage in nodulatingPhaseolus vulgaris roots in soils similar to those used here.     

How does l-glutamate transport relate to selection of mixed nitrogen sources in Rhizobium leguminosarum biovar trifolii MNF1000 and cowpea Rhizobium MNF2030?

When growing on a mixture of ammonia and l-glutamate as nitrogen sources, Rhizobium leguminosarum biovar trifolii MNF1000 utilizes ammonia exclusively, while cowpea Rhizobium MNF2030 utilizes both compounds at similar rates. l-Glutamate transport in both strain MNF1000 and MNF2030 is active, giving rise to a 60-fold concentration gradient across the membrane of cells of strain MNF2030. Both strains produce two kinetically distinguishable glutamate transport systems under all conditions of growth — a high affinity system with an apparent K _m of 0.06–0.17 M but of relatively low V _max, and a low affinity system with a K _m of 1.2–6.7\ M, but of higher overall capacity. l-Glutamate transport activity in cells of MNF2030 was relatively insensitive to the presence of ammonia in the growth medium. By contrast, ammonia in the growth medium resulted in low activities of glutamate transport in cells of MNF1000 which were provided with a carbon source, offering one explanation for the failure of this strain to use glutamate in the presence of ammonia. However, in cells of MNF1000 growing on glutamate as sole source of carbon and nitrogen, the glutamate transport system is synthesized, even in the presence of accumulated or added ammonia. This suggests that the regulation of the glutamate permease also depends on availability of carbon source.Abbreviations CCCP carbonyl cyanide m-chlorophenyl hydrazone - HEPES N-hydroxyethylpiperazine-N-2-ethanesulphonic acid     

Rhizobium tropici nodulates field-grown Phaseolus vulgaris in France

Two hundred and eighty seven isolates of Rhizobium nodulating Phaseolus vulgaris L. were sampled in France from four geographically distant field populations. They were characterized by their colony morphology and by plasmid profiles. A representative sample was further characterized: a) by the ability of each isolate to nodulate a potential alternative host Leucaena leucocephala and to grow on specific media, and b) by RFLP analysis of PCR amplified 16S rRNA genes. On the basis of their phenotypic and genetic characteristics the isolates could be assigned either to Rhizobium leguminosarum bv phaseoli, or to R. tropici. The two species co-occurred at three sites. R. leguminosarum bv phaseoli represented 2%, 4%, 72% and 100% of the population at the four different sites. Eighteen and 22 different plasmid profiles were identified within R. tropici and R. leguminosarum bv phaseoli, respectively. Some of them were conserved between distant geographical regions. The fact that R. tropici was found in France shows that this species is not limited to tropical regions and gives additional evidence of the multi-specific nature of the Phaseolus microsymbiont, even over a geographically limited area.     

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     

Characterization of the Rhizobium leguminosarum biovar phaseoli nifA gene,a positive regulator of nif gene expression

We report the isolation, mutational analysis and the nucleotide sequence of the Rhizobium leguminosarum bv. phaseoli nifA gene. Comparison of the deduced amino acid sequence with other NifA sequences indicated the presence of the conserved central activator and the C-terminal DNA-binding domains. Nodules elicited by a R. leguminosarum bv. phaseoli nifA mutant were symbiotically ineffective. The expression of a nifA-gusA fusion was shown to be independent on the oxygen status of the cell. We cloned the three nifH copies of R. leguminosarum bv. phaseoli and determined the nucleotide sequence of their promoter regions. The expression of nifH-gusA fusions is induced under microaerobic conditions and is dependent on the presence of NifA.Abbreviations bp base pair(s) - kb kilobase(s) - ORF open reading frame     

Analysis of N-acyl homoserine-lactone quorum-sensing molecules made by different strains and biovars of Rhizobium leguminosarum containing different symbiotic plasmids

Strains of Rhizobium leguminosarum use a cell density-dependent gene regulatory system to assess their population density. This is achieved by the accumulation of N-acyl-homoserine lactones (AHLs) in the environment during growth of the bacteria and these AHLs stimulate the induction of various bacterial genes that are up-regulated in the late-exponential and stationary phases of growth. A genetically well-characterised strain of R. leguminosarum biovar viciae was found to have four genes, whose products synthesise different AHLs. We have analysed AHL production by four genetically distinct isolates of R. leguminosarum, three of bv. viciae and one of bv. phaseoli. Distinct differences were seen in the pattern of AHLs produced by the bv. viciae strains compared with bv. phaseoli and the increased levels and diversity of AHLs found in bv. viciae strains can be attributed to the rhiI gene, which is located on the symbiotic (Sym) plasmid and is up-regulated when the bacteria are grown in the rhizosphere. Additional complexity to the profile of AHLs is found to be associated with highly transmissible plasmid pRL1JI of R. leguminosarum bv. viciae, but this is not observed with some other strains, including those carrying different transmissible plasmids. In addition to AHLs produced by the products of genes on the symbiotic plasmid, there is clear evidence for the presence of other AHL production loci. Expression levels and patterns of AHLs can change markedly in different growth media. These results indicate that there is a network of quorum-sensing loci in different strains of R. leguminosarum and these loci may play a role in adapting to rhizosphere growth and plasmid transfer.     

Siderophore and organic acid production in root nodule bacteria

Nineteen strains of root nodule bacteria were grown under various iron regimes (0.1, 1.0 and 20 M added iron) and tested for catechol and hydroxamate siderophore production and the excretion of malate and citrate. The growth response of the strains to iron differed markedly. For 12 strains (Bradyrhizobium strains NC92B and 32H1, B. japonicum USDA110 and CB1809, B. lupini WU8, cowpea Rhizobium NGR234, Rhizobium meliloti strains U45 and CC169, Rhizobium leguminosarum bv viciae WU235 and Rhizobium leguminosarum bv trifolii strains TA1, T1 and WU95) the mean generation time showed no variation with the 200-fold increase in iron concentration. In contrast, in Bradyrhizobium strains NC921, CB756 and TAL1000, B. japonicum strain 61A76 and R. leguminosarum bv viciae MNF300 there was a 2–5 fold decrease in growth rate at low iron. R. meliloti strains WSM419 and WSM540 showed decreased growth at high iron.All strains of root nodule bacteria tested gave a positive CAS (chrome azurol S) assay for siderophore production. No catechol-type siderophores were found in any strain, and only R. leguminosarum bv trifolii T1 and bv viciae WU235 produced hydroxamate under low iron (0.1 and 1.0 M added iron).Malate was excreted by all strains grown under all iron regimes. Citrate was excreted by B. japonicum USDA110 and B. lupini WU8 in all iron concentrations, while Bradyrhizobium TAL1000, R. leguminosarum bv viciae MNF300 and B. japonicum 61A76 only produced citrate under low iron (0.1 and/or 1.0 M added iron) during the stationary phase of growth.Abbreviations CAS chrome azurol S - HDTMA hexadecyltrime-thylammonium bromide     

Discrete differences between strains of different Rhizobium spp. for competitive nodule occupancy on beans

Rhizobium etli strain TAL182 and R. leguminosarum bv phaseoli strain 8002, both of which produce melanin pigment, were tested for their nodulation competitiveness on beans by paired inoculation with two strains which do not produce melanin: R. tropici strain CIAT899 and Rhizobium sp. strain TAL1145. An assay was developed to distinguish nodules formed by the melanin-producing and non-producing strains. Strain TAL182 had discrete competitive superiority over CIAT899 and TAL1145 for nodulation of beans. Nodulation competitiveness was not correlated with the ability to produce melanin pigment or the host range of the Rhizobium strains tested.The authors are with the Department of Plant Molecular Physiology, University of Hawaii, 3050 Maile Way, Gillmore 402, Honolulu, HI 96822, USA     

Enhancement of symbiotic nitrogen fixation by vitamin-secreting fluorescent Pseudomonas

Fluorescent Pseudomonas sp. strain 267 promotes growth of nodulated clover plants under gnotobiotic conditions. In the growth conditions (60 M FeCl₃), the production of siderophores of the pseudobactin-pyoverdin group was repressed. Plant growth enhancement results from secretion of B vitamins by Pseudomonas sp. strain 267. This was proven by stimulation of clover growth by naturally auxotrophic strains of Rhizobium leguminosarum bv. trifolii and marker strains E. coli thi^- and R. meliloti pan^- in the presence of the supernatant of Pseudomonas sp. strain 267. The addition of vitamins to the plant medium increased symbiotic nitrogen fixation by the clover plants.     

Characterization of recA genes and recA mutants of Rhizobium meliloti and Rhizobium leguminosarum biovar viciae

Summary DNA fragments carrying the recA genes of Rhizobium meliloti and Rhizobium leguminosarum biovar viciae were isolated by complementing a UV-sensitive recA ^– Escherichia coli strain. Sequence analysis revealed that the coding region of the R. meliloti recA gene consists of 1044 by coding for 348 amino acids whereas the coding region of the R. leguminosarum bv. viciae recA gene has 1053 bp specifying 351 amino acids. The R. meliloti and R. leguminosarum bv. viciae recA genes show 84.8% homology at the DNA sequence level and of 90.1% at the amino acid sequence level. recA ^– mutant strains of both Rhizobium species were constructed by inserting a gentamicin resistance cassette into the respective recA gene. The resulting recA mutants exhibited an increased sensitivity to UV irradiation, were impaired in their ability to perform homologous recombination and showed a slightly reduced growth rate when compared with the respective wild-type strains. The Rhizobium recA strains did not have altered symbiotic nitrogen fixation capacity. Therefore, they represent ideal candidates for release experiments with impaired strains.The accession numbers: X59956 R. LEGUMINOSARUM REC A ALAS-DNA; X59957 R. MELITOTI REC A ALAS-DNA     

The Effect of the Carbohydrate Components of Pea Roots on the Enzymatic Activity of the Surface Agglutinins of Rhizobium leguminosarum bv. viciae 252

The interaction of the surface agglutinins of Rhizobium leguminosarum bv. viciae 252 with the carbohydrate components of host pea roots alters the -glucosidase and proteolytic activities of the agglutinins.     

Tn5 insertion mutants of Pseudomonas sp. 267 defective in siderophore production and their effect on clover (Trifolium pratense) nodulated with Rhizobium leguminosarum bv. trifolii

Pseudomonas sp. strain 267 isolated from soil promoted growth of different plants under field conditions and enhanced symbiotic nitrogen fixation in clover under gnotobiotic conditions. This strain produced pyoverdine-like compound under low-iron conditions and secreted vitamins of the B group. The role of fluorescent siderophore production in the beneficial effect of strain 267 on nodulated clover plants was investigated. Several non-fluorescent (Pvd^-) Tn5 insertion mutants of Pseudomonas sp. strain 267 were isolated and characterized. The presence of Tn5 insertions was confirmed by Southern analysis of EcoRI digested genomic DNA of each derivative strain. The siderophore-negative mutants were compared to the parental strain with respect to their growth promotion of nodulated clover infected with Rhizobium leguminosarum bv. trifolii 24.1. We found that all isolated Pvd^- mutants stimulated growth of nodulated clover plants in a similar manner to the parental strain. No consistent differences were observed between strain 267 and Pvd^- derivatives strains with respect to their plant growth promotion activity under gnotobiotic conditions.Dr Deryto died in august 1994     

4-aminobutyrate is not available to bacteroids of cowpea Rhizobium MNF2030 in snake bean nodules

Laboratory cultures of cowpea Rhizobium MNF2030 grew on 4-aminobutyrate (GABA) as sole source of carbon and nitrogen. GABA transport was active since it was inhibited by carbonyl cyanide mchlorophenyl hydrazone and 2,4-dinitrophenol and cells developed a 400-fold concentration gradient across the cell membrane. Arsenite treatment of GABA-grown cells revealed stoichiometric conversion of GABA to pyruvate, indicating that 2-oxoglutarate is not an intermediate in GABA catabolism. GABA catabolism by cells of strain MNF2030 grown on GABA appreared to involve GABA transaminase, succinic semialdehyde dehydrogenase and malic enzyme; the first two enzymes were specifically induced by growth on GABA. The deamination process and removal of NH₃ in cells catabolizing GABA involved GABA: 2-oxoglutarate transaminase; glutamate: oxaloacetate aminotransferase; asparate: pyruvate aminotransferase and alanine dehydrogenase.Isolated snakebean bacteroids of strain MNF2030 transported only small amounts of GABA and had uninduced levels of GABA catabolic enzymes, even though the nodules contained significant levels of GABA. The data suggest that GABA is not available to snakebean nodule bacteroids, presumably because of a control imposed by the peribacteroid membrane.Abbreviations CCCP Carbonyl cyanide m-chlorophenyl hydrazone - HEPES N-hydroxyethylpiperazine-N-2-ethanesulphonic acid - DTT dithiothreitol - SSAD succinic semialdehyde dehydrogenase - GABAT 4-aminobutyrate transaminase - GABA 4-aminobutyrate     

The pathways of ammonium assimilation in Rhizobium meliloti

Two pathways of ammonium assimilation are known in bacteria, one mediated by glutamate dehydrogenase, the other by glutamine synthetase and glutamate synthase. The activities of these three enzymes were measured in crude extracts from four Rhizobium meliloti wild-type strains, 2011, M15S, 444 and 12. All the strains had active glutamine synthetase and NADP-linked glutamate synthase. Assimilatory glutamate dehydrogenase activity was present in strains 2011, M15S, 444, but not in strain 12. Three glutamate synthase deficient mutants were isolated from strain 2011. They were unable to use 1 mM ammonium as a sole nitrogen source. However, increased ammonium concentration allowed these mutants to assimilate ammonium via glutamate dehydrogenase. It was found that the sole mode of ammonium assimilation in strain 12 is the glutamine synthetase-glutamate synthase route; whereas the two pathways are functional in strain 2011.Abbreviations GS glutamine synthetase - GOGAT glutamate synthase - GDH glutamate dehydrogenase     

Effect of pH on competition for nodule occupancy by type I and type II strains ofRhizobium leguminosarum bv.phaseoli

The effect of soil pH on the competitive abilities of twoRhizobium leuminosarum bv.phaseoli type I and one type II strains was examined in a nonsterile soil system.Phaseolus vulgaris seedlings, grown in unlimed (pH 5.2) or limed (pH 7.6) soil, were inoculated with a single-strain inoculum containing 1 × 10⁶ cells mL^–1 of one of the three test strains or with a mixed inoculum (1:1, type I vs. type II) containing the type II strain CIAT 899 plus one type I strain (TAL 182 or CIAT 895). At harvest, nodule occupants were determined. In a separate experiment, a mixed suspension (1:1, type I vs. type II) of CIAT 899 paired with either TAL 182 or CIAT 895 was used to inoculateP. vulgaris seedlings grown in sterile, limed or unlimed soil. The numbers of each strain in the rhizosphere were monitored for 10 days following inoculation. The majority of nodules (> 60%) formed on plants grown in acidic soil were occupied by CIAT 899, the type II strain. This pattern of nodule occupancy changed in limed soil. When CIAT 899 was paired with TAL 182, the type I strain formed 78% of the nodules. The number of nodules formed by CIAT 899 and CIAT 895 (56% and 44%, respectively) were not significantly different. The observed patterns of nodule occupancy were not related to the relative numbers or specific growth rates of competing strains in the host rhizosphere prior to nodulation. The results indicate that soil pH can influence which symbiotype ofR. leguminosarum bv.phaseoli will competitively nodulateP. vulgaris.     

Presence of Rhizobium Etli bv . Phaseoli and Rhizobium Gallicum bv . Gallicum in Egyptian Soils

Seven bean rhizobial strains EBRI 2, 3, 21, 24, 26, 27 and 29 identified as Rhizobium etli, and EBRI 32 identified as Rhizobium gallicum, isolated from Egyptian soils and which nodulated Phaseolus vulgaris efficiently, were subjected to hybridization with a nifH probe in order to estimate the copy number of this gene. Seven strains (EBRI 2, 3, 21, 24, 26, 27 and 29) which were only able to nodulate Phaseolus vulgaris, contained three copies of the nifH gene, consistent with their identification as Rhizobium etli bv. phaseoli. Only one strain (EBRI 32) which nodulated both Phaseolus vulgaris and Leucaena leucocephala, had one copy of nifH gene. This confirmed the classification of this strain as Rhizobium gallicum bv. gallicum.     

Structural and functional analysis of the fixLJ genes of Rhizobium leguminosarum biovar phaseoli CNPAF512

The fixLJ genes of Rhizobium leguminosarum biovar phaseoli CNPAF512 were identified by DNA hybridization of a genomic library with an internal fragment of the Rhizobium meliloti fixJ gene. The nucleotide sequence was determined and the corresponding amino acid sequence was aligned with the amino acid sequences of the FixL proteins of R. meliloti, Bradyrhizobium japonicum and Azorhizobium caulinodans. While the FixJ protein and the carboxy-terminal part of the FixL protein are highly homologous to the other FixL and FixJ proteins, the homology in the central heme-binding, oxygen-sensing domain and in the amino-terminal domain of FixL is very low. The R. leguminosarum bv. phaseoli FixL protein does not contain the heme-binding motif defined for the previously described FixL proteins. R. leguminosarum bv. phaseoli fixLJ and fixJ mutants were constructed. These mutants can still fix nitrogen, albeit at a reduced level. Expression analysis of nifA-gusA and nifH-gusA fusions in the constructed mutants revealed that the R. leguminosarum bv. phaseoli fixLJ genes are involved in microaerobic nifH expression but not in nifA expression.