Solution NMR structure of CHU_1110 from Cytophaga hutchinsonii,an AHSA1 protein potentially involved in metal ion stress response

We report the solution nuclear magnetic resonance (NMR) structure of CHU_1110 from Cytophaga hutchinsonii. CHU_1110 contains three α-helices and one antiparallel β-sheet, forming a large cavity in the center of the protein, which are consistent with the structural characteristics of AHSA1 protein family. This protein shows high structural similarities to the prokaryotic proteins RHE_CH02687 from Rhizobium etli and YndB from Bacillus subtilis, which can bind with flavinoids. Unlike these two homologs, CHU_1110 shows no obvious interaction with flavonoids in NMR titration experiments. In addition, no direct interaction has been observed between CHU_1110 and ATP, although many homologous sequences of CHU_1110 have been annotated as ATPase. Combining the analysis of structural similarity of CHU_1110 and genomic context of its encoding gene, we speculate that CHU_1110 may be involved in the stress response of bacteria to heavy metal ions, even though its specific biological functions that need to be further investigated.     

Solution structure and function of YndB,an AHSA1 protein from Bacillus subtilis

The solution structure of the Bacillus subtilis protein YndB has been solved using NMR to investigate proposed biological functions. The YndB structure exhibits the helix‐grip fold, which consists of a β‐sheet with two small and one long α‐helix, forming a hydrophobic cavity that preferentially binds lipid‐like molecules. Sequence and structure comparisons with proteins from eukaryotes, prokaryotes, and archaea suggest that YndB is very similar to the eukaryote protein Aha1, which binds to the middle domain of Hsp90 and induces ATPase activity. On the basis of these similarities, YndB has been classified as a member of the activator of Hsp90 ATPase homolog 1‐like protein (AHSA1) family with a function that appears to be related to stress response. An in silico screen of a compound library of ～18,500 lipids was used to identify classes of lipids that preferentially bind YndB. The in silico screen identified, in order of affinity, the chalcone/hydroxychalcone, flavanone, and flavone/flavonol classes of lipids, which was further verified by 2D ¹H‐¹⁵N HSQC NMR titration experiments with trans‐chalcone, flavanone, flavone, and flavonol. All of these compounds are typically found in plants as precursors to various flavonoid antibiotics and signaling molecules. The sum of the data suggests an involvement of YndB with the stress response of B. subtilis to chalcone‐like flavonoids released by plants due to a pathogen infection. The observed binding of chalcone‐like molecules by YndB is likely related to thesymbiotic relationship between B. subtilis and plants. Proteins 2010. © 2010 Wiley‐Liss, Inc.     

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. Received: 22 August 1997 / Accepted: 20 October 1997     

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.     

Lipopolysaccharide core components of Rhizobium etli reacting with a panel of monoclonal antibodies

Monoclonal antibodies that react with Rhizobium leguminosarum lipopolysaccharide core antigens (LPS-2) have been used to investigate LPS-2 structure in Rhizobium etli. The panel of antibodies (JIM 32 - JIM 35, JIM 37, JIM 38) specific for LPS-2 of R. leguminosarum strain 3841 and its core components displays similar reactivities towards isolated LPS-2 from R. etli CE109 (a mutant of wild-type strain R. etli CE3 that displays LPS-2 as its main LPS form on the cell surface). This result suggests the antibodies bind to similar epitopes on both strains and, hence, that R. leguminosarum and R. etli have very similar LPS core and lipid A antigen structures. More detailed analysis of the antibody binding sites with isolated LPS-2 and lipid A from R. etli suggests that some of the antibodies (JIM 32, 33, 34, and MASM-I) bind some part of the core oligosaccharides, while others (JIM 35 and JIM 38) involve lipid A. These antibodies have already proven useful in the biochemical analysis of the LPS antigen forms. For example, the loss of reactivity of certain LPS forms with antibody JIM 37 has led to the discovery of a hitherto unnoticed form of the LPS antigen in a precipitate formed during the phenol/water extraction procedure. This new form reacts with the JIM 37 antibody. Furthermore, the positive reaction of some of the antibodies with only sonicated wild-type R. etli cells suggests that either an effective way of masking the display of core antigens on whole bacterial cells is occurring or that core forms of the LPSs are never displayed on the surface of the bacterial cells. Either possibility, once confirmed, could be important for our picture of the Rhizobium cell surface and could also have some bearing on symbiotic nodule infection and development.Abbreviations LPS lipopolysaccharide     

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.     

Response of common bean lines to inoculation: comparison between the <Emphasis Type="Italic">Rhizobium tropici</Emphasis> CIAT899 and the native <Emphasis Type="Italic">Rhizobium etli</Emphasis> 12a3 and their persistence in Tunisian soils

Since Phaseolus vulgaris (L) is poorly nodulated in all regions of Tunisia where this crop is grown, the response of common-bean lines CocoT and Flamingo to inoculation with reference Rhizobium tropici CIAT 899 or native rhizobia, namely Sinorhizobium fredii 1a6, Rhizobium etli 12a3, and Rhizobium gallicum 8a3, was studied in a field station. Since R. etli 12a3 was found to be the most effective native rhizobium, it was subsequently compared with R. tropici CIAT 899 in a broader study in two stations over 3 years. A significant interaction between bean and rhizobia was observed for nodule number, shoot dry weight, grain yield, and contents of nitrogen and chlorophyll. The native rhizobia was more efficient than CIAT899 for Flamingo, though not for CocoT. The Enzyme-linked immunosorbent assay technique was used with polyclonal antibody to assess the occupancy in nodule and persistence in soil of the inoculated rhizobia. For both stations the nodule occupancy was 100% during the first year for each rhizobium, but during the next 2 years, between 7 and 15% of nodules were formed by the rhizobia inoculated in the neighboring plot. It is concluded that the first-year inoculation is sufficient to maintain an adequate rate of nodulation during three growth cycles, and that the native R etli can be recommended for the common-bean inoculation in similar soils of Tunisia.     

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.     

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.     

Ability of selected accessions of Phaseolus vulgaris L. to restrict nodulation by particular rhizobia

Plant genotypes that limit nodulation by indigenous rhizobia while nodulating normally with inoculant-strain nodule occupancy in Phaseolus vulgaris. In this study, eight of nine Rhizobium tropici strains and six of 15 Rhizobium etli strains examined, showed limited ability to nodulate and fix nitrogen with the two wild P. vulgaris genotypes G21117 and G10002, but were effective in symbiosis with the cultivated bean genotypes Jamapa and Amarillo Gigante. Five of the R. etli strains restricted in nodulation by G21117 and G10002 produced an alkaline reaction in yeast mannitol medium. In a competition experiment in which restricted strains were tested in 1:1 mixtures with the highly effective R. etli strain CIAT632, the restricted strains produced a low percentage of the nodules formed on G2117, but produced over 40% of the nodules formed on Jamapa. The interaction of the four Rhizobium strains with the two bean genotypes, based on the percentage of nodules formed, was highly significant (P<0.001).     

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.     

<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.     

Ethiopian soils harbor natural populations of rhizobia that form symbioses with common bean (<Emphasis Type="Italic">Phaseolus vulgaris</Emphasis> L.)

<p>The diversity and taxonomic relationships of 83 bean-nodulating rhizobia indigenous to Ethiopian soils were characterized by PCR-RFLP of the internally transcribed spacer (ITS) region between the 16S and 23S rRNA genes, 16S rRNA gene sequence analysis, multilocus enzyme electrophoresis (MLEE), and amplified fragment-length polymorphism. The isolates fell into 13 distinct genotypes according to PCR-RFLP analysis of the ITS region. Based on MLEE, the majority of these genotypes (70%) was genetically related to the type strain of Rhizobium leguminosarum. However, from analysis of their 16S rRNA genes, the majority was placed with Rhizobium etli. Transfer and recombination of the 16S rRNA gene from presumptively introduced R. etli to local R. leguminosarum is a possible theory to explain these contrasting results. However, it seems unlikely that bean rhizobia originating from the Americas (or Europe) extensively colonized soils of Ethiopia because Rhizobium tropici, Rhizobium gallicum, and Rhizobium giardinii were not detected and only a single ineffective isolate of R. etli that originated from a remote location was identified. Therefore, Ethiopian R. leguminosarum may have acquired the determinants for nodulation of bean from a low number of introduced bean-nodulating rhizobia that either are poor competitors for nodulation of bean or that failed to survive in the Ethiopian environment. Furthermore, it may be concluded from the genetic data presented here that the evidence for separating R. leguminosarum and R. etli into two separate species is inconclusive.     

Early responses to Nod factors and mycorrhizal colonization in a non-nodulating Phaseolus vulgaris mutant

Legumes can acquire nitrogen through a symbiotic interaction with rhizobial bacteria. The initiation of this process is determined by a molecular dialogue between the two partners. Legume roots exude flavonoids that induce the expression of the bacterial nodulation genes, which encode proteins involved in the synthesis and secretion of signals called Nod factors (NFs). NFs signal back to the plant root and trigger several responses, leading to bacterial invasion and nodule formation. Here, we describe the molecular and cellular characterization of a Phaseolus vulgaris non-nodulating mutant (NN-mutant). Root hair cells of the NN-mutant plant respond with swelling and branching when inoculated with Rhizobium etli, albeit without curling induction. Furthermore, neither initiation of cell division in the outer cortex, nor entrapment of bacteria nor infection thread formation was observed. Both the bean wild-type and the NN-mutant responded with elevated intracellular calcium changes in the root hairs. Although the NN-mutant is deficient in early nodulin gene expression when inoculated with R. etli, it can be effectively colonized by arbuscular mycorrhizal fungi (Glomus intraradices). Our data indicate that the P. vulgaris NN-mutant is not blocked at the NFs early perception stage, but at later downstream stages between Ca²⁺ signaling and early nodulin induction. This supports the idea that both microsymbionts are perceived and trigger different downstream pathways in the host plant.     

Genetic evidence for 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) as a negative effector of cytochrome terminal oxidase cbb 3 production in Rhizobium etli

A Rhizobium etli Tn5mob-induced mutant (CFN035) exhibits an enhanced capacity to oxidize N,N,N′,N′, tetramethyl-p -phenylenediamine (TMPD), a presumptive indicator of elevated cytochrome c terminal oxidase activity. Sequencing of the mutated gene in CFN035 revealed that it codes for the amidophosphoribosyl transferase enzyme (PurF) that catalyzes the first step in the purine biosynthetic pathway. Two c-type cytochromes with molecular weights of 32 and 27 kDa were produced in strain CFN035, which also produced a novel CO-reactive cytochrome (absorbance trough at 553 nm), in contrast to strain CE3 which produced a single 32 kDa c-type protein and did not produce the 553 nm CO-reactive cytochrome. A wild-type R. etli strain that expresses the Bradyrhizobium japonicum fixNOQP genes, which code for the symbiotic cytochrome terminal oxidase cbb ₃, produced similar absorbance spectra (a trough at 553 nm in CO-difference spectra) and two c -type proteins similar in size to those of strain CFN035, suggesting that CFN035 also produces the cbb ₃ terminal oxidase. The expression of a R. etli fixN-lacZ gene fusion was measured in several R. etli mutants affected in different steps of the purine biosynthetic pathway. Our analysis showed that purF, purD, purQ, purL, purY, purK and purE mutants expressed three-fold higher levels of the fixNOQP operon than the wild-type strain. The derepressed expression of fixN was not observed in a purH mutant. The purH gene product catalyzes the conversion of 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) to 5-formaminoimidazole-4-carboxamide ribonucleotide (FAICAR) and inosine. Supplementation with AICA riboside lowered the levels of fixN expression in the purF mutants. These data are consistent with the possibility that AICAR, or a closely related metabolite, is a negative effector of the production of the symbiotic terminal oxidase cbb ₃ in R. etli. Received: 21 November 1996 / Accepted: 22 January 1997     

Lipopolysaccharides of Rhizobium etli Strain G12 Act in Potato Roots as an Inducing Agent of Systemic Resistance to Infection by the Cyst Nematode Globodera pallida

Recent studies have shown that living and heat-killed cells of the rhizobacterium Rhizobium etli strain G12 induce in potato roots systemic resistance to infection by the potato cyst nematode Globodera pallida. To better understand the mechanisms of induced resistance, we focused on identifying the inducing agent. Since heat-stable bacterial surface carbohydrates such as exopolysaccharides (EPS) and lipopolysaccharides (LPS) are essential for recognition in the symbiotic interaction between Rhizobium and legumes, their role in the R. etli-potato interaction was studied. EPS and LPS were extracted from bacterial cultures, applied to potato roots, and tested for activity as an inducer of plant resistance to the plant-parasitic nematode. Whereas EPS did not affect G. pallida infection, LPS reduced nematode infection significantly in concentrations as low as 1 and 0.1 mg ml⁻¹. Split-root experiments, guaranteeing a spatial separation of inducing agent and challenging pathogen, showed that soil treatments of one half of the root system with LPS resulted in a highly significant (up to 37%) systemic induced reduction of G. pallida infection of potato roots in the other half. The results clearly showed that LPS of R. etli G12 act as the inducing agent of systemic resistance in potato roots.     

A COMPARISON OF THE PROPERTIES AND THE SOLUTION STRUCTURE FOR RNA AND DNA QUADRUPLEXES WHICH CONTAIN TWO GGAGG SEQUENCES JOINED WITH A TETRANUCLEOTIDE LINKER

Abstract

We have determined solution structure of r(GGAGGUUUUGGAGG) (R14) by NMR; the RNA 14-mer forms an intra-strand parallel quadruplex with a G-tetrad and a hexad, in which a G-tetrad core is augmented by association of two A residues. The quadruplex further forms a dimer through stacking interaction between the hexads. In order to obtain insight into the difference between RNA and DNA quadruplexes, we synthesized the corresponding DNA 14-mer, d(GGAGGTTTTGGAGG) (D14), and examined its properties and structure by CD, gel electrophoresis, and NMR. K⁺ ions increased the thermal stability of both R14 and D14 structures. The binding affinity of K⁺ ions to R14 was much higher than that to D14. The CD and gel electrophoretic studies suggest that D14 forms a quadruplex entirely different from that of R14 in the presence of K⁺ ions; two molecules of D14 form a quadruplex with both antiparallel and parallel strand alignments and with diagonal loops at both ends of the stacked G-tetrads. The NMR study also gave results that are consistent with such structure: alternate glycosidic conformation, 5′G(syn)-G(anti)3′, and characteristic chemical shift data observed for many quadruplexes containing diagonal TTTT loops.     

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.