In silico characterization and transcriptomic analysis of nif family genes from <Emphasis Type="Italic">Anabaena</Emphasis> sp. PCC7120

In silico approaches in conjunction with morphology, nitrogenase activity, and qRT-PCR explore the impact of selected abiotic stressor such as arsenic, salt, cadmium, copper, and butachlor on nitrogen fixing (nif family) genes of diazotrophic cyanobacterium Anabaena sp. PCC7120. A total of 19 nif genes are present within the Anabaena genome that is involved in the process of nitrogen fixation. Docking studies revealed the interaction between these nif gene-encoded proteins and the selected abiotic stressors which were further validated through decreased heterocyst frequency, fragmentation of filaments, and downregulation of nitrogenase activity under these stresses indicating towards their toxic impact on nitrogen fixation potential of filamentous cyanobacterium Anabaena sp. PCC7120. Another appealing finding of this study is even though having similar binding energy and similar interacting residues between arsenic/salt and copper/cadmium to nif-encoded proteins, arsenic and cadmium are more toxic than salt and copper for nitrogenase activity of Anabaena which is crucial for growth and yield of rice paddy and soil reclamation.     

Functional analysis of the two cyclophilin isoforms of <Emphasis Type="Italic">Sinorhizobium meliloti</Emphasis>

The nitrogen fixing Sinorhizobium meliloti possesses two genes, ppiA and ppiB, encoding two cyclophilin isoforms which belong to the superfamily of peptidyl prolyl cis/trans isomerases (PPIase, EC: 5.2.1.8). Here, we functionally characterize the two proteins and we demonstrate that both recombinant cyclophilins are able to isomerise the Suc-AAPF-pNA synthetic peptide but neither of them displays chaperone function in the citrate synthase thermal aggregation assay. Furthermore, we observe that the expression of both enzymes increases the viability of E. coli BL21 in the presence of abiotic stress conditions such as increased heat and salt concentration. Our results support and strengthen previous high-throughput studies implicating S. meliloti cyclophilins in various stress conditions.     

The effect of combined and separate inoculation of alfalfa plants with <Emphasis Type="Italic">Azospirillum lipoferum</Emphasis> and <Emphasis Type="Italic">Sinorhizobium meliloti</Emphasis> on denitrification and nitrogen-fixing activities

The effects of associative nitrogen fixer Azospirillum lipoferum strain 137 and root nodule bacteria Sinorhizobium meliloti after combined and separate inoculation of alfalfa seedlings on the background of mineral nitrogen applied at various times were studied. It was demonstrated that exudates of the alfalfa seedlings with the first pair of cotyledonary leaves already provide a high activity of these bacteria in the rhizosphere. To 74.6% of the introduced nitrate was transformed into N₂O when the binary preparation of these bacteria was used. In an extended experiment (30 days), an active reduction of nitrates to N₂O with inhibition of nitrogen fixation was observed in all of the experimental variants during the formation of legume-rhizobial and associative symbioses and simultaneous introduction of nitrates and bacteria. The most active enzyme fixation was observed in the case of a late (after 14 days) application of nitrates in the variants with both separate inoculations and inoculation with the binary preparation of A. lipoferum and S. meliloti. Separation in time of the application of bacterial preparations and mineral nitrogen assisted its preservation in all of the experimental variants. The variant of alfalfa inoculation with the binary preparation of A. lipoferum and S. meliloti and application of nitrates 2 weeks after inoculation was optimal for active nitrogen fixation (224.7 C₂H₄ nmol/flask · 24 h) and low denitrification activity (1.8 μmol N₂O/flask · 24 h). These results are useful in applied developments aimed at the use of bacterial and mineral fertilizers for leguminous plants.     

A nodule endophytic plant growth-promoting <Emphasis Type="Italic">Pseudomonas</Emphasis> and its effects on growth,nodulation and metal uptake in <Emphasis Type="Italic">Medicago lupulina</Emphasis> under copper stress

The aim of this study was to determine the plant growth-promoting potential of the nodule endophytic Pseudomonas brassicacearum strain Zy-2-1 when used as a co-inoculant of Medicago lupulina with Sinorhizobium meliloti under copper (Cu) stress conditions. Strain Zy-2-1 was capable of producing ACC deaminase activity, IAA and siderophores, and was able to grow in the presence of Cu²⁺ up to 2.0 mmol/L. Co-inoculation of S. meliloti with Zy-2-1 enhanced M. lupulina root fresh weight, total plant dry weight, number of nodules, nodule fresh weight and nitrogen content in the presence of 100 or 300 mg/kg Cu²⁺. In the presence of 500 mg/kg Cu²⁺, co-inoculation with S. meliloti and strain Zy-2-1 increased plant height, number of nodules, nodule fresh weight and nitrogen content in comparison to S. meliloti inoculation alone. Furthermore, a higher amount of Cu accumulation in both shoots and roots and a higher level of Cu translocation to shoots were observed in co-inoculated plants. These results demonstrate that co-inoculation of M. lupulina with S. meliloti and P. brassicacearum Zy-2-1 improves plant growth, nitrogen nutrition and metal extraction potential. This can be of practical importance in the remediation of heavy metal-contaminated soils.     

Use of RNA Immunoprecipitation Method for Determining <Emphasis Type="Italic">Sinorhizobium meliloti</Emphasis> RNA<Emphasis Type="Italic">-</Emphasis>Hfq Protein Associations In Vivo

Background

Soil bacterium Sinorhizobium meliloti (S. meliloti) forms an endosymbiotic partnership with Medicago truncatula (M. truncatula) roots which results in root nodules. The bacteria live within root nodules where they function to fix atmospheric N₂ and supply the host plant with reduced nitrogen. The bacterial RNA-binding protein Hfq (Hfq) is an important regulator for the effectiveness of the nitrogen fixation. RNA immunoprecipitation (RIP) method is a powerful method for detecting the association of Hfq protein with specific RNA in cultured bacteria, yet a RIP method for bacteria living in root nodules remains to be described.

Results

A modified S. meliloti gene encoding a His-tagged Hfq protein (Hfq^His) was placed under the regulation of the native Hfq gene promoter (P_hfqsm). The trans produced Hfq^His protein was accumulated at its nature levels during all stages of the symbiosis, allowing RNAs that associated with the given protein to be immunoprecipitated with the anti-His antibody against the protein from root nodule lysates. RNAs that associated with the protein were selectively enriched in the immunoprecipitated sample. The RNAs were recovered by a simple method using heat and subsequently analyzed by RT-PCR. The nature of PCR products was determined by DNA sequencing. Hfq association with specific RNAs can be analyzed at different conditions (e. g. young or older root nodules) and/or in wild-type versus mutant strains.

Conclusions

This article describes the RIP method for determining Sinorhizobium meliloti RNA-Hfq associations in vivo. It is also applicable to other rhizobia living in planta, although some tissue-specific modification related to sample disruption and homogenization may be needed.

     

The effects of gibberellins and mepiquat chloride on nitrogenase activity in <Emphasis Type="Italic">Bradyrhizobium japonicum</Emphasis>

Application of plant growth regulators (PGRs) to soybean plants is known to induce changes in nitrogenase activity in root nodules, and this led us to hypothesize that PGRs would affect nitrogenase activity in free-living rhizobia cultures. Little is known about the molecular basis of the effects of PGRs on nitrogenase activity in free-living rhizobia cultures. Therefore, a comparative study was conducted on the effects of gibberellins (GA₃) and mepiquat chloride (PIX), which regulate plant growth, on the nitrogenase activity of the nitrogen-fixing bacterium Bradyrhizobium japonicum. Fix and nif gene regulation and protein expression in free-living cultures of B. japonicum were investigated using real-time PCR and two-dimensional electrophoresis after treatment with GA₃ or PIX. GA₃ treatment decreased nitrogenase activity and the relative expression of nifA, nifH, and fixA genes, but these effects were reversed by PIX treatment. As expected, several proteins involved in nitrogenase synthesis were down-regulated in the GA₃-treated group. Conversely, several proteins involved in nitrogenase synthesis were up-regulated in the PIX-treated group, including bifunctional ornithine acetyltransferase/N-acetylglutamate synthase, transaldolase, ubiquinol-cytochrome C reductase iron-sulfur subunit, electron transfer flavoprotein subunit beta, and acyl-CoA dehydrogenase. Two-pot experiments were conducted to evaluate the effects of GA₃ and PIX on nodulation and nitrogenase activity in Rhizobium-treated legumes. Interestingly, GA₃ treatment increased nodulation and depressed nitrogenase activity, but PIX treatment decreased nodulation and enhanced nitrogenase activity. Our data show that the nif and fix genes, as well as several proteins involved in nitrogenase synthesis, are up-regulated by PIX and down-regulated by GA₃, respectively, in B. japonicum.     

Structural Polymorphism of <Emphasis Type="Italic">Sinorhizobium meliloti</Emphasis> Genes Related to Virulence and Salt Tolerance

Analysis of the structural polymorphism of eight genes in Sinorhizobium meliloti (nodA, nodB, nodC, and nodH, as well as betA, betB, betC, and betB2) involved in virulence control and salt tolerance, respectively, was carried out in native populations from two geographically distant areas of alfalfa diversity. These areas are located in the North Caucasian gene center of cultivated plants (NCG) and in the modern center of introgressive hybridization of alfalfa located next to the Aral Sea area (PAG) subjected to salinization. RFLP types (alleles) of the nod and bet genes, similar to those in the reference strain Rm1021 (A-type) and different from them (divergent, or D-type alleles) were revealed. The combinations for A- and D-type alleles of the aforementioned genes (analysis of the linkage disequilibrium, LD) were studied in both populations. It was shown that D-type alleles of the nod genes were two times more frequent in the NCG population, while D-type alleles of the bet genes were predominantly identified in the PAG population. At the same time, different combinations of D-type alleles of both the nod and bet genes prevailed in populations. For instance, in the case of the glycine betaine metabolism pathway, these were the betC and betB2 genes in NCG population and betB and betA genes in PAG population. The state of linkage disequilibrium was shown for 60.7% of combinations of alleles of the nod and bet genes in the S. meliloti strains from NCG and more than twice less in strains from the PAG population. It was concluded that clonal lines prevailed in NCG, while the PAG population of S. meliloti had a panmictic structure with revealed single clonal lines.     

Evolution of <Emphasis Type="Italic">fixNOQP</Emphasis> genes encoding cytochrome oxidase with high affinity to oxygen in rhizobia and related bacteria

Many bacteria belonging to the order Rhizobiales have fixNOQP genes which encode cytochrome oxidase with high affinity to oxygen required for oxidative phosphorylation in microaerophilic conditions. There is one copy of the identified fixNOQP operon in ancestral forms of rhizobia (Bradyrhizobium), as well as in their putative evolutionary predecessors (bacteria related to Rhodopseudomonas). At the same time, forms deeply specialized in symbiosis (Rhizobium leguminosarum, Sinorhizobium meliloti) have multiple (2–3) copies, some of them have a high similarity (>90%) to fixNOQP genes of Bradyrhizobium and Rhodopseudomonas, and others have only 30–50% similarity. Two divergent copies fixNOQP are detected in Tardiphaga, which is a representative of the Bradyrhizobiaceae family, lacking the ability to fix N₂ (lack of nif genes encoding the synthesis of nitrogenase) and to induce the formation of nodules on legumes roots (lack of nod genes encoding the synthesis of signal Nod factors activating symbiosis development). The presence of Tardiphaga in nodule bacterial communities from a range of legumes, including Vavilovia formosa (relic representative of the tribe Fabeae, for which R. leguminosarum bv. viciae is the main microsymbiont), suggests that the ancestral gene duplication and subsequent divergence of fixNOQP operon in bacteria related to Tardiphaga opened the possibility of wide dissemination of functionally different copies of this cluster among symbiotically active forms of Rhizobiales. It is possible that the acquisition of fixNOQP genes determines adaptation of bacteria to microaerophilic niches not only in plants nodules but also in their environment (the rhizosphere, rhizoplane, internal portions of soil aggregates).     

HmuS and HmuQ of <Emphasis Type="Italic">Ensifer</Emphasis>/<Emphasis Type="Italic">Sinorhizobium meliloti</Emphasis> degrade heme in vitro and participate in heme metabolism in vivo

Ensifer meliloti is a nitrogen-fixing symbiont of the alfalfa legume able to use heme as an iron source. The transport mechanism involved in heme acquisition in E. meliloti has been identified and characterized, but the fate of heme once inside the cell is not known. In silico analysis of E. meliloti 1021 genome revealed no canonical heme oxygenases although two genes encoding putative heme degrading enzymes, smc01518 and hmuS, were identified. SMc01518 is similar to HmuQ of Bradyrhizobium japonicum, which is weakly homologous to the Staphylococcus aureus IsdG heme-degrading monooxygenase, whereas HmuS is homolog to Pseudomonas aeruginosa PhuS, a protein reported as a heme chaperone and as a heme degrading enzyme. Recombinant HmuQ and HmuS were able to bind hemin with a 1:1 stoichiometry and displayed a K_d value of 5 and 4 µM, respectively. HmuS degrades heme in vitro to the biliverdin isomers IX-β and IX-δ in an equimolar ratio. The HmuQ recombinant protein degrades heme to biliverdin IX-δ only. Additionally, in this work we demonstrate that humS and hmuQ gene expression is regulated by iron and heme in a RirA dependent manner and that both proteins are involved in heme metabolism in E. meliloti in vivo.     

Site of Nitrogenase Activity in the Blue-Green Alga <Emphasis Type="Italic">Anabaena</Emphasis> sp. L-31

MANY blue-green algae fix nitrogen, assimilate carbon dioxide and evolve oxygen and as algal nitrogenase is inhibited^1–3 by high oxygen pressure, enhanced nitrogen fixation accompanying photosynthesis is surprising. Heterocysts do not contain⁴ or have comparatively less amounts^4–7 of photosystem II (PS II) pigments, which are responsible for the evolution of oxygen. This tends to favour the suggestion of Fay et al.⁸ that these cells are the sites of nitrogenase activity. Until now, however, attempts at obtaining unequivocal evidence for heterocysts as principal loci for nitrogenase activity have yielded conflicting results. Stewart et al.⁷ first demonstrated nitrogenase activity in heterocysts incubated aerobically, a finding confirmed by Wolk and Wojciuch⁹ and Van Gorkom and Donze¹⁰. By contrast, Smith and Evans^3,11 and Kurz and La Rue¹² reported results favouring vegetative cells as the major site of nitrogenase activity. Other evidence^2,13 showed high nitrogenase activity in cell-free preparations of Anabaena cylindrica and the non-heterocystous alga Plectonema boryanum strain 594.     

Rhizobial diversity associated with the spontaneous legume <Emphasis Type="Italic">Genista saharae</Emphasis> in the northeastern Algerian Sahara

Genista saharae is an indigenous shrub legume that spontaneously grows in the northeastern Algerian Sahara. It is known for efficient dune fixation and soil preservation against desertification, due to its drought tolerance and its contribution to sustainable nitrogen resources implemented by biological N₂-fixation. In this study, the root nodule bacteria of G. saharae were investigated using phenotypic and phylogenetic characterization. A total of 57 rhizobial strains were isolated from nodules from several sites in the hyper-arid region of Metlili and Taibet (east Septentrional Sahara). They all nodulate G. saharae species but they differed in their symbiotic efficiency and effectiveness. The genetic diversity was assessed by sequencing three housekeeping genes (atpD, recA and 16S rRNA). The majority of isolates (81 %) belonged to the genus Ensifer (previously Sinorhizobium), represented mainly by the species Ensifer meliloti. The next most abundant genera were Neorhizobium (17 %) with 3 different species: N. alkalisoli, N. galegae and N. huautlense and Mesorhizobium (1.75 %) represented by the species M. camelthorni. Most of the isolated strains tolerated up to 4 % (w/v) NaCl and grew at 45 °C. This study is the first report on the characterization of G. saharae microsymbionts in the Algerian Sahara.     

Relationships between nitrogen fixation and photosynthesis as the main components of the productivity in alfalfa

Alfalfa (Medicago sativa L.) plants were inoculated with Sinorhizobium meliloti Tn-5 mutants featuring various nitrogen-fixing effectiveness and then grown in sand culture to study relations between CO₂ exchange, plant productivity, and nitrogen fixation. At the flowering stage, the relationship between nitrogen fixation and photosynthesis of whole alfalfa plants was described with the logarithmic curve. At the same stage of plant development, a close relationship was observed between nitrogen fixation rate and plant weight; this relationship showed a trend toward saturation at high rates of nitrogen fixation. The increase in nitrogenase activity of root nodules was accompanied by stimulation of root respiration; the relation of respiration to nitrogen-fixing activity was manifested stronger than its relation to the total root weight. It is concluded that highly effective strains of root nodule bacteria can realize their potential only in combination with complementary plant genotypes featuring active photosynthesis that provides a balanced supply of assimilates for both the symbiotic apparatus and growth processes in the macrosymbiont.     

Biological fixation of nitrogen and growth of bacteria of the genus <Emphasis Type="Italic">Azotobacter</Emphasis> in liquid media in the presence of perfluorocarbons

The addition of perfluorocarbons (perfluorodecalin, carbogal, and perfluoromethyldecalin) to nitrogen-free liquid media during the submerged cultivation of bacteria of the genus Azotobacter was followed by (1) increases in biomass accumulation and nitrogenase activity and (2) fixation of molecular nitrogen. Addition of perfluorodecalin (5 vol %) to the culture medium of A. chroococcum ACB 121 contributed to increases in biomass accumulation, cell concentration (of more than by five times), nitrogenase activity (of 3.4 times), and total nitrogen content in the medium (of 4.5 times).     

The diversity of rhizobia nodulating the <Emphasis Type="Italic">Medicago</Emphasis>, <Emphasis Type="Italic">Melilotus</Emphasis> and <Emphasis Type="Italic">Trigonella</Emphasis> inoculation group in Egypt is marked by the dominance of two genetic types

Twenty four rhizobial strains were isolated from root nodules of Melilotus, Medicago and Trigonella plants growing wild in soils throughout Egypt. The nearly complete 16S rRNA gene sequence from each strain showed that 12 strains (50 %) were closely related to the Ensifer meliloti LMG6133^T type strain with identity values higher than 99.0 %, that 9 (37.5 %) strains were more than 99 % identical to the E. medicae WSM419^T type strain, and that 3 (12.5 %) strains showed 100 % identity with the type strain of N. huautlense S02^T. Accordingly, the diversity of rhizobial strains nodulating wild Melilotus, Medicago and Trigonella species in Egypt is marked by predominance of two genetic types, E. meliloti and E. medicae, although the frequency of isolation was slightly higher in E. meliloti. Sequencing of the symbiotic nodC gene from selected Medicago and Melilotus strains revealed that they were all similar to those of the E. meliloti LMG6133^T and E. medicae WSM419^T type strains, respectively. Similarly, nodC sequences of strains identified as members of the genus Neorhizobium were more than 99 % identical to that of N. galegae symbiovar officinalis HAMBI 114.     

Nitrogen-fixing cyanobacterium <Emphasis Type="Italic">Trichormus variabilis</Emphasis> of the Lake Baikal phytoplankton

A new filamentous cyanobacterial strain BAC 9610 was isolated from the lake Baikal pelagial. Data obtained by light, scanning, and transmission electron microscopy, along with 16S rRNA gene sequence analysis, allowed the bacterium identification as Trichormus variabilis, previously known as Anabaena variabilis. Trichormus is a cyanobacterial genus not presented in the list of Baikal plankton algae; A. variabilis also hasn’t been previously detected in Baikal phytoplankton. T. variabilis nitrogen fixation ability was demonstrated. The gene responsible for nitrogen fixation, nifH, was identified by PCR and was partially sequenced. No hepatotoxin synthesis genes were revealed in the strain.     

A metabolomic approach to characterize the acid-tolerance response in <Emphasis Type="Italic">Sinorhizobium meliloti</Emphasis>

Introduction

Sinorhizobium meliloti establishes a symbiosis with Medicago species where the bacterium fixes atmospheric nitrogen for plant nutrition. To achieve a successful symbiosis, however, both partners need to withstand biotic and abiotic stresses within the soil, especially that of excess acid, to which the Medicago-Sinorhizobium symbiotic system is widely recognized as being highly sensitive.

Objective

To cope with low pH, S. meliloti can undergo an acid-tolerance response (ATR(+)) that not only enables a better survival but also constitutes a more competitive phenotype for Medicago sativa nodulation under acid and neutral conditions. To characterize this phenotype, we employed metabolomics to investigate the biochemical changes operating in ATR(+) cells.

Methods

A gas chromatography/mass spectrometry approach was used on S. meliloti 2011 cultures showing ATR(+) and ATR(?) phenotypes. After an univariate and multivariate statistical analysis, enzymatic activities and/or reserve carbohydrates characterizing ATR(+) phenotypes were determined.

Results

Two distinctive populations were clearly defined in cultures grown in acid and neutral pH based on the metabolites present. A shift occurred in the carbon-catabolic pathways, potentially supplying NAD(P)H equivalents for use in other metabolic reactions and/or for maintaining intracellular-pH homeostasis. Furthermore, among the mechanisms related to acid resistance, the ATR(+) phenotype was also characterized by lactate production, envelope modification, and carbon-overflow metabolism.

Conclusions

Acid-challenged S. meliloti exhibited several changes in different metabolic pathways that, in specific instances, could be identified and related to responses observed in other bacteria under various abiotic stresses. Some of the observed changes included modifications in the pentose-phosphate pathway (PPP), the exopolysaccharide biosynthesis, and in the myo-inositol degradation intermediates. Such modifications are part of a metabolic adaptation in the rhizobia that, as previously reported, is associated to improved phenotypes of acid tolerance and nodulation competitiveness.

     

Characterization of surface motility in <Emphasis Type="Italic">Sinorhizobium meliloti</Emphasis>: regulation and role in symbiosis

Sinorhizobium meliloti can exhibit diverse modes of surface translocation whose manifestation depends on the strain. The mechanisms involved and the role played by the different modes of surface motility in the establishment of symbiosis are largely unknown. In this work, we have characterized the surface motility shown by two S. meliloti reference strains (Rm1021 and GR4) under more permissive conditions for surface spreading and analyzed the symbiotic properties of two flagella-less S. meliloti mutants with different behavior on surfaces. The use of Noble agar in semisolid minimal medium induces surface motility in GR4, a strain described so far as non-motile on surfaces. The motility exhibited by GR4 is swarming as revealed by the non-motile phenotype of the flagella-less flaAB mutant. Intriguingly, a flgK mutation which also abolishes flagella production, triggers surface translocation in GR4 through an as yet unknown mechanism. In contrast to GR4, Rm1021 moves over surfaces using mostly a flagella-independent motility which is highly reliant on siderophore rhizobactin 1021 production. Surprisingly, this motility is absent in a flagella-less flgE mutant. In addition, we found that fadD loss-of-function, known to promote surface motility in S. meliloti, exerts different effects on the two reference strains: while fadD inactivation promotes a flagella-independent type of motility in GR4, the same mutation interferes with the surface translocation exhibited by the Rm1021 flaAB mutant. The symbiotic phenotypes shown by GR4flaAB and GR4flgK, non-flagellated mutants with opposite surface motility behavior, demonstrate that flagella-dependent motility positively influences competitiveness for nodule occupation, but is not crucial for optimal infectivity.