The Rhizobium etli FixL protein differs in structure from other known FixL proteins

The central heme-binding domain in the FixL proteins of Sinorhizobium meliloti, Bradyrhizobium japonicum, Rhizobium leguminosarum biovar viciae and Azorhizobium caulinodans, is highly conserved. The similarity with the corresponding domain in the Rhizobium etli FixL protein is considerably less. This observation prompted us to analyze the heme-binding capacities of the R. etli FixL protein. The R. etlifixL gene was overexpressed in Escherichia coli. In the presence of S. meliloti FixJ, the overexpressed R. etli FixL protein was able to enhance FixJ-mediated activation of an S. meliloti pnifA-lacZ fusion, indicating that the R.?etli FixL protein possesses an active conformation in E. coli. Subsequently, using a non-denaturing gel assay for heme, we analyzed the heme-binding capacity of the R.?etli FixL protein expressed in E. coli, taking the S.?meliloti FixL protein as a positive control. The R. etli FixL protein expressed in E. coli does not contain a heme group, in contrast to the S. meliloti FixL protein. Therefore we conclude that the R. etli FixL is a non-heme protein in the nif regulatory cascade.     

Spin-state equilibria and axial ligand bonding in FixL hydroxide: a resonance raman study

 Vibrational assignments for the Fe-OH unit of ferric alkaline forms of two deletion derivatives of Rhizobium meliloti FixL, FixL*, a functional O₂-sensing heme kinase, and FixLN, which contains only the heme domain, are made. Appearance of ²H- and ¹⁸O-sensitive Raman bands indicates that the heme group of FixL binds hydroxide as a distal ligand to form a six-coordinate complex. The alkaline FixLs are distributed between high- and low-spin states. The high- and low-spin bands corresponding to the ν (Fe-OH) modes occur at 479 and 539 cm^–1, respectively. Low temperature favors formation of the low-spin complex, indicative of a thermal spin-state equilibrium. The ν (Fe-OH) frequencies of FixLN and FixL* are 11 to 18 cm^–1 lower than those observed for the respective vibrations in alkaline myoglobin and hemoglobin. The weaker Fe-OH bond in the FixLs is attributed to a lack of hydrogen bonding on the distal side of the heme pocket. Received: 20 November 1997 / Accepted: 2 March 1998     

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     

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.     

A Proteomic Approach Towards the Analysis of Salt Tolerance in Rhizobium etli and Sinorhizobium meliloti Strains

Soluble proteins from the salt-tolerant Rhizobium etli strain EBRI 26 were separated by two-dimensional (2D) gel electrophoresis and visualised by Commassie staining. Six proteins are highly expressed after induction by 4% NaCl compared to the non-salt-stressed cells. These proteins have pI between 5 and 5.5 and masses of approximately 22, 25, 40, 65, 70, and 95 kDa. These proteins were analysed by Matrix-assisted laser adsorption ionization time of flight (MALDI-TOF) after digestion with trypsin. Despite having very good peptide mass fingerprint data, these proteins could not be identified, because the genome sequence of R. etli is not yet published. In a second approach, soluble proteins from salt-induced or non-salt-induced cultures from R. etli strain EBRI 26 were separately labelled with different fluorescent cyano-dyes prior to 2D difference in gel electrophoresis. Results revealed that 49 proteins are differentially expressed after the addition of sodium chloride. Fourteen proteins are overexpressed and 35 were downregulated. The genome of Sinorhizobium meliloti, a closely related species to R. etli, has been published. Similar experiments using Sinorhizobium meliloti strain 2011 identified four overexpressed and six downregulated proteins. Among the overexpressed protein is a carboxynospermidin decarboxylase, which plays an important role in the biosynthesis of spermidin (polyamine). The enzyme catalase is among the downregulated proteins. These proteins may play a role in salt tolerance.     

Interspecies regulation of the recA gene of gram-negative bacteria lacking an E. coli-like SOS operator

The recA genes of Agrobacterium tumefaciens, Rhizobium meliloti, Rhizobium phaseoli and Rhodobacter sphaeroides, species belonging to the alpha-group bacteria of the Proteobacteria class, have been fused in vitro to the lacZ gene of Escherichia coli. By using a mini-Tn5 transposon derivative, each of these recA-lacZ fusions was introduced into the chromosome of each of the four species, and into that of E. coli. The recA genes of three of the alpha bacteria are induced by DNA damage when inserted in A. tumefaciens, R. phaseoli or R. meliloti chromosomes. The expression of the recA gene of R. sphaeroides is DNA damage-mediated only when present in its own chromosome; none of the genes is induced in E. coli. Likewise, the recA gene of E. coli is not induced in any of the four alpha species. These data indicate that A. tumefaciens, R. meliloti and R. phaseoli possess a LexA-like repressor, which is able to block the expression of their recA genes, as well as that of R. sphaeroides, but not the recA gene of E. coli. The LexA repressor of R. sphaeroides does not repress the recA gene of A. tumefaciens, R. meliloti, R. phaseoli or E. coli.     

Role of conserved Fα-helix residues in the native fold and stability of the kinase-inhibited oxy state of the oxygen-sensing FixL protein from Sinorhizobium meliloti

The oxygen-sensing FixL protein from Sinorhizobium meliloti is part of the heme-PAS family of gas sensors that regulate many important signal transduction pathways in a wide variety of organisms. We examined the role of the conserved F_α-9 arginine 200 and several other conserved residues on the proximal F_α-helix in the heme domain of SmFixL* using site-directed mutagenesis in conjunction with UV-visible, EPR, and resonance Raman spectroscopy. The F_α-helix variants R200A, E, Q, H, Y197A, and D195A were expressed at reasonable levels and purified to homogeneity. The R200I and Y201A variants did not express in observable quantities. Tyrosine 201 is crucial for forming the native protein fold of SmFixL* while Y197 and R200 are important for stabilizing the kinase-inhibited oxy state. Our results show a clear correlation between H-bond donor ability of the F_α-9 side chain and the rate of heme autoxidation. This trend in conjunction with crystal structures of liganded BjFixL heme domains, show that H-bonding between the conserved F_α-9 arginine and the heme-6-propionate group contributes to the kinetic stability of the kinase-inactivated, oxy state of SmFixL*.     

Cloning and Functional Expression of an MscL Ortholog from Rhizobium etli: Characterization of a Mechanosensitive Channel

Rhizobium etli is equipped with several systems to handle both hyper- and hypo-osmotic stress. For adaptation to hypo-osmotic stress, R. etli possesses a single gene with clear homology to MscS, four MscS-like channels and one ortholog of MscL (ReMscL, identity ≈ 44% compared to Escherichia coli MscL). We subcloned and expressed the ReMscL channel ortholog from R. etli in E. coli to examine its activity by patch clamp in giant spheroplasts and characterized it at the single-channel level. We obtained evidence that ReMscL prevents the lysis of E. coli null mutant log-phase cells upon a rapid, osmotic downshock and identified a slight pH dependence for ReMscL activation. Here, we describe the facilitation of ReMscL activation by arachidonic acid (AA) and a reversible inhibitory effect of Gd³⁺. The results obtained in these experiments suggest a stabilizing effect of micromolar AA and traces of Gd³⁺ ions in the partially expanded conformation of the protein. Finally, we discuss a possible correlation between the number of gene paralogs for MS channels and the habitats of several microorganisms. Taken together, our data show that ReMscL may play an important role in free-living rhizobacteria during hypo-osmotic shock in the rhizosphere.     

Nitrosyl adducts of FixL as probes of heme environment

This report presents a spectroscopic investigation of the nitrosyl adducts of FixL, the sensor in the signal transduction system responsible for regulating nitrogen fixation in Rhizobium meliloti. Variable-temperature resonance Raman (RR), electron spin resonance (ESR), and variable-temperature UV-visible absorption data are presented for the ferrous NO adducts of two FixL deletion derivatives, FixLN (the heme-containing domain) and FixL* (a functional heme-kinase). A temperature-dependent equilibrium is observed between the five-coordinate (5-c) and six-coordinate (6-c) ferrous nitrosyl adducts, with lower temperatures favoring formation of the 6-c nitrosyl adduct. This equilibrium is perturbed as the solution freezes, and the amount of 5-c FixL-NO increases sharply until a nearly constant ratio of 6-c to 5-c adducts is obtained. Complexation between the heme domain of FixL and its response regulator, FixJ, is revealed through specfic FixJ-induced increase in the energy separation between 5-c and 6-c FixL-NO. Ferric nitrosyl adducts of FixL* and FixLN autoreduce to their corresponding ferrous nitrosyl adducts. The kinetic behavior of this reduction is monophasic for FixL*-NO, while the reaction for ferric FixLN-NO is biphasic. These results suggest conformational inhomogeneity in the heme pocket of FixLN and conformational homogeneity in that of FixL*. Hence the kinase domain plays a role in distal pocket conformational stability. Implications for the signal transduction mechanism are discussed.     

Iron coordination structures of oxygen sensor FixL characterized by Fe K-edge extended x-ray absorption fine structure and resonance raman spectroscopy.

FixL is a heme-based O(2) sensor protein involved in a two-component system of a symbiotic bacterium. In the present study, the iron coordination structure in the heme domain of Rhizobium meliloti FixLT (RmFixLT, a soluble truncated FixL) was examined using Fe K-edge extended x-ray absorption fine structure (EXAFS) and resonance Raman spectroscopic techniques. In the EXAFS analyses, the interatomic distances and angles of the Fe-ligand bond and the iron displacement from the heme plane were obtained for RmFixLT in the Fe(2+), Fe(2+)O(2), Fe(2+)CO, Fe(3+), Fe(3+)F(-), and Fe(3+)CN(-) states. An apparent correlation was found between the heme-nitrogen (proximal His-194) distance in the heme domain and the phosphorylation activity of the histidine kinase domain. Comparison of the Fe-CO coordination geometry between RmFixLT and RmFixLH (heme domain of RmFixL), based on the EXAFS and Raman results, has suggested that the kinase domain directly or indirectly influences steric interaction between the iron-bound ligand and the heme pocket. Referring to the crystal structure of the heme domain of Bradyrhizobium japonicum FixL (Gong, W., Hao, B., Mansy, S. S., Gonzalez, G., Gilles-Gonzalez, M. A., and Chan, M. K. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 15177-15182), we discussed details of the iron coordination structure of RmFixLT and RmFixLH in relation to an intramolecular signal transduction mechanism in its O(2) sensing.     

Sensitivity of selected bacterial species to UV radiation

The effect of exposure of bacterial suspensions to UV radiation by means of the dose-response curves was assessed. The D₃₇ and D₁₀ values were used for subsequent statistical analysis of the results. The aim of this article is to evaluate the sensitivity to UV radiation of several microorganisms of different habitats (Rhizobium meliloti, Rhodobacter sphaeroides, Escherichia coli, and Deinococcus radiodurans), two mutants with nonfunctional SOS DNA repair system (R.meliloti recA ^- and E. coli recA ^-), and a mutant in the synthesis of carotenoids (R. sphaeroides crtD). The results reveal that D. radiodurans was an extremely resistant bacterium, R. meliloti was more resistant than R. sphaeroides, and E. coli was the most sensitive bacterium tested. The high sensitivity of recA ^- mutants was also verify. Moreover, it seems that the possession of pigments had no important effect in the sensitivity of R. sphaeroides to UV radiation.     

Interspecies regulation of the recA gene of gram-negative bacteria lacking an E. coli-like SOS operator

The recA genes of Agrobacterium tumefaciens, Rhizobium meliloti, Rhizobium phaseoli and Rhodobacter sphaeroides, species belonging to the alpha-group bacteria of the Proteobacteria class, have been fused in vitro to the lacZ gene of Escherichia coli. By using a mini-Tn5 transposon derivative, each of these recA-lacZ fusions was introduced into the chromosome of each of the four species, and into that of E. coli. The recA genes of three of the alpha bacteria are induced by DNA damage when inserted in A. tumefaciens, R. phaseoli or R. meliloti chromosomes. The expression of the recA gene of R. sphaeroides is DNA damage-mediated only when present in its own chromosome; none of the genes is induced in E. coli. Likewise, the recA gene of E. coli is not induced in any of the four alpha species. These data indicate that A. tumefaciens, R. meliloti and R. phaseoli possess a LexA-like repressor, which is able to block the expression of their recA genes, as well as that of R. sphaeroides, but not the recA gene of E. coli. The LexA repressor of R. sphaeroides does not repress the recA gene of A. tumefaciens, R. meliloti, R. phaseoli or E. coli.     

Analysis of a chemotaxis operon in Rhizobium meliloti*

Genes controlling chemotaxis towards L-amino acids and d -mannitol in Rhizobium meliloti have been identified by Tn5 insertions that lead to chemotaxis-deficient mutants. The tagged genes span an 8.7 kbp region that has been sequenced. These genes are part of a large operon containing three novel open reading frames, orf1, orf2 and orf9, and six familiar chemotaxis (che) genes, cheY1-cheA-cheW-cheR-cheB-cheY2, that have been assigned by their similarity to known Escherichia coli genes. The second copy of cheY may be part of a second signalling chain; orf1 and orf2 encode sequence motifs that resemble the signalling domain of E. coli MCPs (methyl-accepting chemotaxis proteins), while the product of orf9 may contain a transmembrane domain. No protein methylation has been observed in Rhizobium meliloti in response to l -amino acids. However, the presence of cheR (methyltransferase gene) and cheB (methyl-esterase gene) suggested that MCPs are likely components of the chemotactic response in R. meliloti. Therefore, it is postulated that two chemotaxis pathways are functional in R meliloti: one responds to l -amino acids via ORF1-ORF2, whereas the other (probably responding to specific plant exudates) acts via MCP-like receptors, and both interact with the central components CheW-CheA-CheY1 and/or CheY2.     

An Fnr-like protein encoded in Rhizobium leguminosarum biovar viciae shows structural and functional homology to Rhizobium meliloti FixK

Summary A 1.9 kb DNA region of Rhizobium leguminosarum biovar viciae strain VF39 capable of promoting microaerobic and symbiotic induction of the Rhizobium meliloti fixN gene was identified by heterologous complementation. Sequence analysis of this DNA region revealed the presence of two complete open reading frames, orf240 and orf114. The deduced amino acid sequence of orf240 showed significant homology to Escherichia coli Fnr and R. meliloti FixK. The major difference between ORF240 and FixK is the presence of 21 N-terminal amino acids in ORF240 that have no counterpart in FixK. A similar protein domain is also present in E. coli Fnr and is essential for the oxygen-regulated activity of this protein. Analysis of the nucleotide sequence upstream of orf240 revealed a motif similar to the NtrA-dependent promoter consensus sequence, as well as two DNA regions resembling the Fnr consensus binding sequence. A Tn5-generated mutant in orf240 lost the ability to induce the R. meliloti fixN-lacZ fusion. Interestingly, this mutant was still capable of nitrogen fixation but showed reduced nitrogenase activity.     

Ammonia regulation of the Rhizobium meliloti nitrogenase structural and regulatory genes under free-living conditions: Involvement of the fixL gene product?

Summary The expression under microaerobic conditions of the Rhizobium meliloti nifA and consequently the nifHDK genes was found to be negatively regulated by ammonia and nitrate. Assimilation of the ammonia to glutamate and glutamine is not required for this regulation to occur. This indicates that ammonia itself, and not a product of its metabolism, may be regulating nif expression. Unlike the situation in Klebsiella pneumoniae, NtrC is apparently not involved in mediating the ammonia effect on nifA expression in R. meliloti. Neither does the fixK gene product, which is known to regulate nifA in R. meliloti, appear to be involved in mediating the ammonia effect. The regulation of nifA by ammonia is shown to be mediated through the FixL protein. A truncated fixJ gene, the product of which has been shown to induce nifA expression irrespective of the oxygen status of the cell, also circumvented the repressive effect of ammonia on nifA expression. This suggests that the ammonia effect is mediated through the FixLJ regulatory cascade. Interstingly no effect of ammonia on fixK expression was observed.     

Cloning and characterization of a Rhizobium meliloti nonspecific acid phosphatase

Nodulated legumes require high levels of phosphorus for optimal symbiotic performance. However, the basis for this elevated phosphorus requirement is poorly understood, and very little information regarding bacteroid phosphorus metabolism is available. To develop an understanding of the relative importance of organic and inorganic phosphorus sources for bacteroids, we investigated phosphatase activity in Rhizobium meliloti. An R. meliloti plasmid library clone that complemented an Escherichia coli phosphatase mutant was isolated, and the clone was sequenced. The complementing fragment contained a 337-amino-acid open reading frame that has a potential leader sequence and processing sites characteristic of periplasmic proteins. The phosphatase activity was located in the periplasm of R. meliloti and of E. coli containing the cloned gene. The subunit molecular mass of the cloned phosphatase was 33 kDa, and gel filtration indicated the active enzyme was a 66-kDa homodimer. Lack of substrate specificity suggests the cloned gene, napD, encodes a nonspecific acid phosphatase with a pH optimum of approximately 6.5. An R. meliloti napD transposon-insertion mutant was constructed, and its symbiotic phenotype was determined to be Fix⁺ regardless of the level of phosphorus provided to the host plant. Received: 26 August 1997 / Accepted: 4 February 1998     

Bacterial Growth Rates and Competition Affect Nodulation and Root Colonization by Rhizobium meliloti

The addition of streptomycin to nonsterile soil suppressed the numbers of bacterial cells in the rhizosphere of alfalfa (Medicago sativa L.) for several days, resulted in the enhanced growth of a streptomycin-resistant strain of Rhizobium meliloti, and increased the numbers of nodules on the alfalfa roots. A bacterial mixture inoculated into sterile soil inhibited the colonization of alfalfa roots by R. meliloti, caused a diminution in the number of nodules, and reduced plant growth. Enterobacter aerogenes, Pseudomonas marginalis, Acinetobacter sp., and Klebsiella pneumoniae suppressed the colonization by R. meliloti of roots grown on agar and reduced nodulation by R. meliloti, the suppression of nodulation being statistically significant for the first three species. Bradyrhizobium sp. and “Sarcina lutea” did not suppress root colonization nor nodulation by R. meliloti. The doubling times in the rhizosphere for E. aerogenes, P. marginalis, Acinetobacter sp., and K. pneumoniae were less and the doubling times for Bradyrhizobium sp. and “S. lutea” were greater than the doubling time of R. meliloti. Under the same conditions, Arthrobacter citreus injured alfalfa roots. We suggest that competition by soil bacteria reduces nodulation by rhizobia in soil and that the extent of inhibition is related to the growth rates of the rhizosphere bacteria.