Overexpression and Purification of Rhizobium etli Glutaminase A by Recombinant and Conventional Procedures: A Comparative Study of Enzymatic Properties

Rhizobium etli glutaminase A was purified to homogeneity by conventional procedures that included ammonium sulfate differential precipitation, ion-exchange chromatography, hydrophobic interaction chromatography, gel filtration, and dye-ligand chromatography. Alternatively, the structural glsA gene that codifies for glutaminase A was amplified by PCR and cloned in the expression vector pTrcHis. The recombinant protein was purified to homogeneity by affinity chromatography. This protein showed the same kinetic properties as native glutaminase A (K_m for glutamine of 1.5 mM and V_max of 80 μmol ammonium min⁻¹ mg protein⁻¹). Physicochemical and biochemical properties of native and recombinant glutaminase were identical. The molecular mass of recombinant glutaminase A (M_r 106.8 kDa) and the molecular mass of the subunits (M_r 26.9 kDa) were estimated by mass spectrometry. These results suggest that R. etli glutaminase A is composed of four identical subunits. The high-level production of recombinant glutaminase A elevates the possibilities for determination of its three-dimensional structure through X-ray crystallography.     

Conservation of Plasmid-Encoded Traits among Bean-Nodulating Rhizobium Species

Rhizobium etli type strain CFN42 contains six plasmids. We analyzed the distribution of genetic markers from some of these plasmids in bean-nodulating strains belonging to different species (Rhizobium etli, Rhizobium gallicum, Rhizobium giardinii, Rhizobium leguminosarum, and Sinorhizobium fredii). Our results indicate that independent of geographic origin, R. etli strains usually share not only the pSym plasmid but also other plasmids containing symbiosis-related genes, with a similar organization. In contrast, strains belonging to other bean-nodulating species seem to have acquired only the pSym plasmid from R. etli.     

Prevalence of the Rhizobium etli-Like Allele in Genes Coding for 16S rRNA among the Indigenous Rhizobial Populations Found Associated with Wild Beans from the Southern Andes in Argentina

A collection of rhizobial isolates from nodules of wild beans, Phaseolus vulgaris var. aborigineus, found growing in virgin lands in 17 geographically separate sites in northwest Argentina was characterized on the basis of host range, growth, hybridization to a nifH probe, analysis of genes coding for 16S rRNA (16S rDNA), DNA fingerprinting, and plasmid profiles. Nodules in field-collected wild bean plants were largely dominated by rhizobia carrying the 16S rDNA allele of Rhizobium etli. A similar prevalence of the R. etli allele was observed among rhizobia trapped from nearby soil. Intragroup diversity of wild bean isolates with either R. etli-like or Rhizobium leguminosarum bv. phaseoli-like alleles was generally found across northwest Argentina. The predominance of the R. etli allele suggests that in this center of origin of P. vulgaris the coevolution of Rhizobium spp. and primitive beans has resulted in this preferential symbiotic association.     

The calcium-stimulated lipid A 3-O deacylase from Rhizobium etli is not essential for plant nodulation

The lipid A component of lipopolysaccharide from the nitrogen-fixing plant endosymbiont, Rhizobium etli, is structurally very different from that found in most enteric bacteria. The lipid A from free-living R. etli is structurally heterogeneous and exists as a mixture of species which are either pentaacylated or tetraacylated. In contrast, the lipid A from R. etli bacteroids is reported to consist exclusively of tetraacylated lipid A species. The tetraacylated lipid A species in both cases lack a β-hydroxymyristoyl chain at the 3-position of lipid A. Here, we show that the lipid A modification enzyme responsible for 3-O deacylation in R. etli is a homolog of the PagL protein originally described in Salmonella enterica sv. typhimurium. In contrast to the PagL proteins described from other species, R. etli PagL displays a calcium dependency. To determine the importance of the lipid A modification catalyzed by PagL, we isolated and characterized a R. etli mutant deficient in the pagL gene. Mass spectrometric analysis confirmed that the mutant strain was exclusively tetraacylated and radiochemical analysis revealed that 3-O deacylase activity was absent in membranes prepared from the mutant. The R. etli mutant was not impaired in its ability to form nitrogen-fixing nodules on Phaseolus vulgaris but it displayed slower nodulation kinetics relative to the wild-type strain. The lipid A modification catalyzed by R. etli PagL, therefore, is not required for nodulation but may play other roles such as protecting bacterial endosymbionts from plant immune responses during infection.     

Occurrence of Choline and Glycine Betaine Uptake and Metabolism in the Family Rhizobiaceae and Their Roles in Osmoprotection

The role of glycine betaine and choline in osmoprotection of various Rhizobium, Sinorhizobium, Mesorhizobium, Agrobacterium, and Bradyrhizobium reference strains which display a large variation in salt tolerance was investigated. When externally provided, both compounds enhanced the growth of Rhizobium tropici, Sinorhizobium meliloti, Sinorhizobium fredii, Rhizobium galegae, Agrobacterium tumefaciens, Mesorhizobium loti, and Mesorhizobium huakuii, demonstrating their utilization as osmoprotectants. However, both compounds were inefficient for the most salt-sensitive strains, such as Rhizobium leguminosarum (all biovars), Agrobacterium rhizogenes, Rhizobium etli, and Bradyrhizobium japonicum. Except for B. japonicum, all strains exhibit transport activity for glycine betaine and choline. When the medium osmolarity was raised, choline uptake activity was inhibited, whereas glycine betaine uptake was either increased in R. leguminosarum and S. meliloti or, more surprisingly, reduced in R. tropici, S. fredii, and M. loti. The transport of glycine betaine was increased by growing the cells in the presence of the substrate. With the exception of B. japonicum, all strains were able to use glycine betaine and choline as sole carbon and nitrogen sources. This catabolic function, reported for only a few soil bacteria, could increase competitiveness of rhizobial species in the rhizosphere. Choline dehydrogenase and betaine-aldehyde dehydrogenase activities were present in the cells of all strains with the exception of M. huakuii and B. japonicum. The main physiological role of glycine betaine in the family Rhizobiaceae seems to be as an energy source, while its contribution to osmoprotection is restricted to certain strains.     

Altered lipid A structures and polymyxin hypersensitivity of Rhizobium etli mutants lacking the LpxE and LpxF phosphatases

The lipid A of Rhizobium etli, a nitrogen-fixing plant endosymbiont, displays significant structural differences when compared to that of Escherichia coli. An especially striking feature of R. etli lipid A is that it lacks both the 1- and 4′-phosphate groups. The 4′-phosphate moiety of the distal glucosamine unit is replaced with a galacturonic acid residue. The dephosphorylated proximal unit is present as a mixture of the glucosamine hemiacetal and an oxidized 2-aminogluconate derivative. Distinct lipid A phosphatases directed to the 1 or the 4′-positions have been identified previously in extracts of R. etli and Rhizobium leguminosarum. The corresponding structural genes, lpxE and lpxF, respectively, have also been identified. Here, we describe the isolation and characterization of R. etli deletion mutants in each of these phosphatase genes and the construction of a double phosphatase mutant. Mass spectrometry confirmed that the mutant strains completely lacked the wild-type lipid A species and accumulated the expected phosphate-containing derivatives. Moreover, radiochemical analysis revealed that phosphatase activity was absent in membranes prepared from the mutants. Our results indicate that LpxE and LpxF are solely responsible for selectively dephosphorylating the lipid A molecules of R. etli. All the mutant strains showed an increased sensitivity to polymyxin relative to the wild-type. However, despite the presence of altered lipid A species containing one or both phosphate groups, all the phosphatase mutants formed nitrogen-fixing nodules on Phaseolus vulgaris. Therefore, the dephosphorylation of lipid A molecules in R. etli is not required for nodulation but may instead play a role in protecting the bacteria from cationic antimicrobial peptides or other immune responses of plants.     

Phylogenetic multilocus sequence analysis of native rhizobia nodulating faba bean (Vicia faba L.) in Egypt

The taxonomic diversity of forty-two Rhizobium strains, isolated from nodules of faba bean grown in Egypt, was studied using 16S rRNA sequencing, multilocus sequence analyses (MLSA) of three chromosomal housekeeping loci and one nodulation gene (nodA). Based on the 16S rRNA gene sequences, most of the strains were related to Rhizobium leguminosarum, Rhizobium etli, and Rhizobium radiobacter (syn. Agrobacterium tumefaciens). A maximum likelihood (ML) tree built from the concatenated sequences of housekeeping proteins encoded by glnA, gyrB and recA, revealed the existence of three distinct genospecies (I, II and III) affiliated to the defined species within the genus Rhizobium/Agrobacterium. Seventeen strains in genospecies I could be classified as R. leguminosarum sv. viciae. Whereas, a single strain of genospecies II was linked to R. etli. Interestingly, twenty-four strains of genospecies III were identified as A. tumefaciens. Strains of R. etli and A. tumefaciens have been shown to harbor the nodA gene and formed effective symbioses with faba bean plants in Leonard jar assemblies. In the nodA tree, strains belonging to the putative genospecies were closely related to each other and were clustered tightly to R. leguminosarum sv. viciae, supporting the hypothesis that symbiotic and core genome of the species have different evolutionary histories and indicative of horizontal gene transfer among these rhizobia.     

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.     

Characterization of Rhizobial Isolates of Phaseolus vulgaris by Staircase Electrophoresis of Low-Molecular-Weight RNA

Low-molecular-weight (LMW) RNA molecules were analyzed to characterize rhizobial isolates that nodulate the common bean growing in Spain. Since LMW RNA profiles, determined by staircase electrophoresis, varied across the rhizobial species nodulating beans, we demonstrated that bean isolates recovered from Spanish soils presumptively could be characterized as Rhizobium etli, Rhizobium gallicum, Rhizobium giardinii, Rhizobium leguminosarum bv. viciae and bv. trifolii, and Sinorhizobium fredii.     

Rhizobium etli USDA9032 Engineered To Produce a Phenazine Antibiotic Inhibits the Growth of Fungal Pathogens but Is Impaired in Symbiotic Performance

Phenazine production was engineered in Rhizobium etli USDA9032 by the introduction of the phz locus of Pseudomonas chlororaphis O6. Phenazine-producing R. etli was able to inhibit the growth of Botrytis cinerea and Fusarium oxysporum in vitro. Black bean inoculated with phenazine-producing R. etli produced brownish Fix⁻ nodules.     

Physiological role of glutaminase activity in Saccharomyces cerevisiae

The participation of glutaminase activity in glutamine degradation was studied in a wild-type strain (S288C) of Saccharomyces cerevisiae. Evidence is presented that this strain has two glutaminase activities, a readily extractable form (glutaminase B) and a membrane-bound enzyme (glutaminase A). Glutaminase A and B activities could also be distinguished by their thermostability, pyruvate sensitivity and pH optimum. Glutaminase B activity was negatively modulated by some 2-oxo acids, and in vivo pyruvate accumulation inhibited this activity. A mutant strain (CN10) with an altered glutaminase B activity was isolated and partially characterized. Its glutaminase B activity was more sensitive to inhibition by pyruvate and 2-oxoglutarate than the wild type, thus resulting in inactivation of this enzyme in vivo. The physiological role of glutaminase activity is discussed with regard to the phenotype shown by the mutant strain.     

Reaction coupling through interdomain contacts in imidazole glycerol phosphate synthase

Imidazole glycerol phosphate (IGP) synthase, a triad glutamine amidotransferase, catalyzes the fifth step in the histidine biosynthetic pathway, where ammonia from glutamine is incorporated into N1-[(5'-phosphoribulosyl)-formimino]-5-aminoimidazole-4-carboxamide ribonucleotide (PRFAR) to yield IGP and 5'-(5-aminoimidazole-4-carboxamide) ribonucleotide (AICAR). The triad family of glutamine amidotransferases is formed by the coupling of two disparate subdomains, an acceptor domain and a glutamine hydrolysis domain. Each of the enzymes in this family share a common glutaminase domain for which the glutaminase activity is tightly regulated by an acceptor substrate domain. In IGP synthase the glutaminase and PRFAR binding sites are separated by 30 A. Using kinetic analyses of site-specific mutants and molecular dynamic simulations, we have determined that an interdomain salt bridge in IGP synthase between D359 and K196 (approximately 16 A from the PRFAR binding site) plays a key role in mediating communication between the two active sites. This interdomain contact modulates the glutaminase loop containing the histidine and glutamic acid of the catalytic triad to control glutamine hydrolysis. We propose this to be a general principle of catalytic coupling that may be applied to the entire triad glutamine amidotransferase family.     

Trifolitoxin Production Increases Nodulation Competitiveness of Rhizobium etli CE3 under Agricultural Conditions

A major barrier to the use of nitrogen-fixing inoculum strains for the enhancement of legume productivity is the inability of commercially available strains to compete with indigenous rhizobia for nodule formation. Despite extensive research on nodulation competitiveness, there are no examples of field efficacy studies of strains that have been genetically improved for nodulation competitiveness. We have shown previously that production of the peptide antibiotic trifolitoxin (TFX) by Rhizobium etli results in significantly increased nodule occupancy values in nonsterile soil in growth chamber experiments (E. A. Robleto, A. J. Scupham, and E. W. Triplett, Mol. Plant-Microbe Interact. 10:228–233, 1997). To determine whether TFX production by Rhizobium etli increases nodulation competitiveness in field-grown plants, seeds of Phaseolus vulgaris were inoculated with mixtures of Rhizobium etli strains at different ratios. The three nearly isogenic inoculum strains used included TFX-producing and non-TFX-producing strains, as well as a TFX-sensitive reference strain. Data was obtained over 2 years for nodule occupancy and over 3 years for assessment of the effect of the TFX production phenotype on grain yield. In comparable mixtures in which the test strain accounted for between 5 and 50% of the inoculum, the TFX-producing strain exhibited at least 20% greater nodule occupancy than the non-TFX-producing strain in both years. The TFX production phenotype had no effect on grain yield over 3 years; the average yields reached 2,400 kg/ha. These results show that addition of the TFX production phenotype significantly increases nodule occupancy under field conditions without adverse effects on grain yield. As we used common inoculation methods in this work, there are no practical barriers to the commercial adoption of the TFX system for agriculture.     

Glutamine metabolism in the kidney during induction of, and recovery from, metabolic acidosis in the rat

Experiments were carried out on rats to evaluate the possible regulatory roles of renal glutaminase activity, mitochondrial permeability to glutamine, phosphoenolpyruvate carboxykinase activity and systemic acid–base changes in the control of renal ammonia (NH₃ plus NH₄⁺) production. Acidosis was induced by drinking NH₄Cl solution ad libitum. A pronounced metabolic acidosis without respiratory compensation [pH=7.25; HCO₃⁻=16.9mequiv./litre; pCO₂=40.7mmHg (5.41kPa)] was evident for the first 2 days, but thereafter acid–base status returned towards normal. This improvement in acid–base status was accompanied by the attainment of maximal rates of ammonia excretion (onset phase) after about 2 days. A steady rate of ammonia excretion was then maintained (plateau phase) until the rats were supplied with tap water in place of the NH₄Cl solution, whereupon pCO₂ and HCO₃⁻ became elevated [55.4mmHg (7.37kPa) and 35.5mequiv./litre] and renal ammonia excretion returned to control values within 1 day (recovery phase). Renal arteriovenous differences for glutamine always paralleled rates of ammonia excretion. Phosphate-dependent glutaminase and phosphoenolpyruvate carboxykinase activities and the rate of glutamine metabolism (NH₃ production and O₂ consumption) by isolated kidney mitochondria all increased during the onset phase. The increases in glutaminase and in mitochondrial metabolism continued into the plateau phase, whereas the increase in the carboxykinase reached a plateau at the same time as did ammonia excretion. During the recovery phase a rapid decrease in carboxykinase activity accompanied the decrease in ammonia excretion, whereas glutaminase and mitochondrial glutamine metabolism in vitro remained elevated. The metabolism of glutamine by kidney-cortex slices (ammonia, glutamate and glucose production) paralleled the metabolism of glutamine in vivo during recovery, i.e. it returned to control values. The results indicate that the adaptations in mitochondrial glutamine metabolism must be regulated by extra-mitochondrial factors, since glutamine metabolism in vivo and in slices returns to control values during recovery, whereas the mitochondrial metabolism of glutamine remains elevated.     

Role of glutamine aminotransferase in glutamine catabolism by Saccharomyces cerevisiae under microaerophilic conditions

The involvement of glutamine aminotransferase activity in glutamine catabolism by Saccharomyces cerevisiae under microaerophilic conditions was studied. We were able to show that there are at least two different glutamine aminotransferase activities that are differentiated genetically, by their substrate specificity (pyruvate and glyoxylate dependence), and their different modes of regulation. The pyruvate-dependent glutamine aminotransferase activity plays a major role in glutamine catabolism under microaerophilic conditions since the wild-type strain S288C showed a 10-fold higher activity in static cultures than in agitated ones. The same strain also had 3-fold higher glutaminase B activity in agitated cultures than in static ones. Pyruvate-dependent glutamine aminotransferase activity is not regulated directly by O2 itself since a rho- strain showed a high activity regardless of the extent of aeration of cultures. Finally, we were able to isolate a mutant, strain CN20, derived from the rho- strain and unable to utilize glutamine as the sole nitrogen source, which was severely affected in pyruvate-dependent but not in glyoxylate-dependent aminotransferase activity.     

Engineering the nifH Promoter Region and Abolishing Poly-β-Hydroxybutyrate Accumulation in Rhizobium etli Enhance Nitrogen Fixation in Symbiosis with Phaseolus vulgaris

Rhizobium etli, as well as some other rhizobia, presents nitrogenase reductase (nifH) gene reiterations. Several R. etli strains studied in this laboratory showed a unique organization and contained two complete nifHDK operons (copies a and b) and a truncated nifHD operon (copy c). Expression analysis of lacZ fusion demonstrated that copies a and b in strain CFN42 are transcribed at lower levels than copy c, although this copy has no discernible role during nitrogen fixation. To increase nitrogenase production, we constructed a chimeric nifHDK operon regulated by the strong nifHc promoter sequence and expressed it in symbiosis with the common bean plant (Phaseolus vulgaris), either cloned on a stably inherited plasmid or incorporated into the symbiotic plasmid (pSym). Compared with the wild-type strain, strains with the nitrogenase overexpression construction assayed in greenhouse experiments had, increased nitrogenase activity (58% on average), increased plant weight (32% on average), increased nitrogen content in plants (15% at 32 days postinoculation), and most importantly, higher seed yield (36% on average), higher nitrogen content (25%), and higher nitrogen yield (72% on average) in seeds. Additionally, expression of the chimeric nifHDK operon in a poly-β-hydroxybutyrate-negative R. etli strain produced an additive effect in enhancing symbiosis. To our knowledge, this is the first report of increased seed yield and nutritional content in the common bean obtained by using only the genetic material already present in Rhizobium.     

Analysis of rhizobial strains nodulating Phaseolus vulgaris from Hispaniola Island,a geographic bridge between Meso and South America and the first historical link with Europe

Hispaniola Island was the first stopover in the travels of Columbus between America and Spain, and played a crucial role in the exchange of Phaseolus vulgaris seeds and their endosymbionts. The analysis of recA and atpD genes from strains nodulating this legume in coastal and inner regions of Hispaniola Island showed that they were almost identical to those of the American strains CIAT 652, Ch24-10 and CNPAF512, which were initially named as Rhizobium etli and have been recently reclassified into Rhizobium phaseoli after the analysis of their genomes. Therefore, the species R. phaseoli is more abundant in America than previously thought, and since the proposal of the American origin of R. etli was based on the analysis of several strains that are currently known to be R. phaseoli, it can be concluded that both species have an American origin coevolving with their host in its distribution centres. The analysis of the symbiovar phaseoli nodC gene alleles carried by different species isolated in American and European countries suggested a Mesoamerican origin of the α allele and an Andean origin of the γ allele, which is supported by the dominance of this latter allele in Europe where mostly Andean cultivars of common beans have been traditionally cultivated.     

Anaplerotic Function of Phosphoenolpyruvate Carboxylase in <Emphasis Type="Italic">Bradyrhizobium japonicum</Emphasis> USDA110

In bacteria, anaplerotic carbon fixation necessary for growth on carbon sources that are metabolized to three-carbon intermediates is provided by the activity of pyruvate carboxylase (PYC) and/or phosphoenolpyruvate carboxylase (PPC). In contrast to other rhizobia, which encode only one of these enzymes in their genomes, Bradyrhizobium japonicum USDA110 encodes both. Streptavidin-HRP western blot analysis of B. japonicum extracts demonstrated the presence of a biotin-containing protein whose molecular mass was indistinguishable from those of PYCs produced by Sinorhizobium meliloti and Rhizobium etli. Sequence analysis of the possible B. japonicum PYC revealed the lack of a pyruvate binding site as well as other characteristics indicating that the enzyme is non-functional, and PPC activity, but not PYC activity, was detectible in extracts prepared from strain USDA110. A B. japonicum cosmid genomic library was used to clone the ppc by functional complementation of S. meliloti pyc mutant RmF991. S. meliloti RmF991-carrying plasmids containing the B. japonicum ppc regained the ability to grow with glucose as a carbon source and produced PPC activity. The cloned ppc gene was inactivated by insertion mutagenesis and recombined into the USDA110 genome. The resulting ppc mutant was essentially devoid of PPC activity and grew poorly with glucose as carbon source in comparison to the wild-type strain. These data indicate that B. japonicum utilizes PPC, and not PYC, as an anaplerotic enzyme for growth on carbon sources metabolized to three-carbon intermediates.     

Kinetic characterization of ebselen,chelerythrine and apomorphine as glutaminase inhibitors

Glutaminase catalyzes the hydrolysis of glutamine to glutamate and plays a central role in the proliferation of neoplastic cells via glutaminolysis, as well as in the generation of excitotoxic glutamate in central nervous system disorders such as HIV-associated dementia (HAD) and multiple sclerosis. Both glutaminase siRNA and glutaminase inhibition have been shown to be effective in in vitro models of cancer and HAD, suggesting a potential role for small molecule glutaminase inhibitors. However, there are no potent, selective inhibitors of glutaminase currently available. The two prototypical glutaminase inhibitors, BPTES and DON, are either insoluble or non-specific. In a search for more drug-like glutaminase inhibitors, we conducted a screen of 1280 in vivo active drugs (Library of Pharmacologically Active Compounds (LOPAC¹²⁸⁰)) and identified ebselen, chelerythrine and (R)-apomorphine. The newly identified inhibitors exhibited 10 to 1500-fold greater affinities than DON and BPTES and over 100-fold increased efficiency of inhibition. Although non-selective, it is noteworthy that the affinity of ebselen for glutaminase is more potent than any other activity yet described. It is possible that the previously reported biological activity seen with these compounds is due, in part, to glutaminase inhibition. Ebselen, chelerythrine and apomorphine complement the armamentarium of compounds to explore the role of glutaminase in disease.