Endophytic Fungi Pezicula radicicola in the Root Nodules of Actinorhizal Plants

Microbiology - Based on analysis of the nucleotide sequences of the ITS regions, LSU gene fragments of rDNA, and cultural and morphological characteristics, species affiliation of six strains of...     

Purification of Hemoglobin from the Actinorhizal Root Nodules of Myrica gale L

Hemoglobins are generally absent or present in low concentrations in the nodules of actinorhizal plants. An exception is Casuarina, where a hemoglobin occurs at relatively high concentration. However, this plant is unique in that Frankia, the microsymbiont, lacks the vesicles that are normally the site of nitrogen fixation. The present paper shows that a hemoglobin also occurs at high concentrations in Myrica gale L., an actinorhizal plant in which Frankia does form vesicles. Hemoglobin was extracted from root nodules under anaerobic conditions using a buffer containing CO, detergent, and a reducing agent. Carboxyhemoglobin was purified using gel filtration followed by aerobic ion-exchange chromatography. The optical absorption spectra of the oxy-, deoxy-, and carboxyhemoglobins were similar to those of other hemoglobins. The molecular mass of the native hemoglobin estimated by gel filtration was 38,500 D. The molecular mass of the subunits estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis was 16,200 D, consistent with the mass of other hemoglobin subunits. Thus, the native hemoglobin is probably a dimer.     

Natural Diversity of Frankia Strains in Actinorhizal Root Nodules from Promiscuous Hosts in the Family Myricaceae

Actinorhizal plants invade nitrogen-poor soils because of their ability to form root nodule symbioses with N₂-fixing actinomycetes known as Frankia. Frankia strains are difficult to isolate, so the diversity of strains inhabiting nodules in nature is not known. To address this problem, we have used the variability in bacterial 16S rRNA gene sequences amplified from root nodules as a means to estimate molecular diversity. Nodules were collected from 96 sites primarily in northeastern North America; each site contained one of three species of the family Myricaceae. Plants in this family are considered to be promiscuous hosts because several species are effectively nodulated by most isolated strains of Frankia in the greenhouse. We found that strain evenness varies greatly between the plant species so that estimating total strain richness of Frankia within myricaceous nodules with the sample size used was problematical. Nevertheless, Myrica pensylvanica, the common bayberry, was found to have sufficient diversity to serve as a reservoir host for Frankia strains that infect plants from other actinorhizal families. Myrica gale, sweet gale, yielded a few dominant sequences, indicating either symbiont specialization or niche selection of particular ecotypes. Strains in Comptonia peregrina nodules had an intermediate level of diversity and were all from a single major group of Frankia.     

Alfalfa Root Nodule Carbon Dioxide Fixation : II. Partial Purification and Characterization of Root Nodule Phosphoenolpyruvate Carboxylase

A nonphotosynthetic phosphoenolpyruvate carboxylase (EC 4.1.1.31) was partially purified from the cytosol of root nodules of alfalfa. The enzyme was purified 86-fold by ammonium sulfate fractionation, DEAE-cellulose, hydroxylapatite chromatography, and reactive agarose with a final yield of 32%. The enzyme exhibited a pH optimum of 7.5 with apparent K_m values for phosphoenolpyruvate and magnesium of 210 and 100 micromolar, respectively. Two isozymes were resolved by nondenaturing polyacrylamide disc gel electrophoresis. Subsequent electrophoresis of these isozymes in a second dimension by sodium dodecyl sulfate slab gel electrophoresis yielded identical protein patterns for the isozymes with one major protein band at molecular weight 97,000. Malate and AMP were slightly inhibitory (about 20%) to the partially purified enzyme. Phosphoenolpyruvate carboxylase comprised approximately 1 to 2% of the total soluble protein in actively N₂-fixing alfalfa nodules.     

Alfalfa Root Nodule Carbon Dioxide Fixation : III. Immunological Studies of Nodule Phosphoenolpyruvate Carboxylase

Antiserum was prepared in rabbits against purified alfalfa (Medicago sativa L.) nodule phosphoenolpyruvate carboxylase (PEPC). Immunotitration assays revealed that the antiserum recognized the enzyme from alfalfa nodules, uninoculated alfalfa roots, and from soybean nodules. Tandem-crossed immunoelectrophoresis showed that the PEPC protein from alfalfa roots and nodules was immunologically indistinguishable. The 101 kilodalton polypeptide subunit of alfalfa nodule PEPC was identified on Western blots. The PEPC polypeptide was detected in low quantities in young alfalfa roots and nodules but was present at increased levels in mature nodules. Senescent nodules appeared to contain a reduced amount of the PEPC polypeptide. PEPC was also detected by western blot in some plant- and bacterially-conditioned ineffective alfalfa nodules but was not detected in bacteroids isolated from effective nodules. Alfalfa nodule PEPC is constitutively expressed in low levels in roots. In nodules, expression of PEPC polypeptide increases several-fold, resulting in increased PEPC activity. Antiserum prepared against the C₄ PEPC from maize leaves recognized the PEPC enzyme in all legume nodules and roots tested, while the antiserum prepared against alfalfa nodule PEPC also recognized the leaf PEPC of several C₄ plant species. Neither antiserum reacted strongly with any C₃ leaf proteins. The molecular weight of the PEPC polypeptide from C₄ leaves and legume nodules appears to be similar.     

Hormones in Plants Bearing Nitrogen-fixing Root Nodules: Cytokinin Transport from the Root Nodules of Alnus glutinosa (L.) Gaertn.

The movement and metabolism of [8-¹⁴C]zeatin applied to theroot nodules of Alnus glutinosa (L.) Gaertn, was investigated.Twenty-four hours after the start of uptake, zeatin and a numberof its metabolites were detected in all parts of the plant.The major radioactive compounds present in a cationic fractionof different plant parts at this time co-chromatographed onSephadex LH20 with zeatin (in nodules, stems, and leaves) andwith zeatin riboside (in roots, stems, and buds). In the roots,in addition to the peak co-chromatographing with zeatin riboside,there was also a prominent unidentified polar peak. The presence of zeatin and zeatin riboside in the stems andleaves was indicated also by chromatographic behaviour in othersystems, effects of permanganate oxidation, and cocrystallisationwith the authentic unlabelled compounds. Biological activitywas exhibited by both peaks in the soybean callus bioassay.Other metabolites in the shoot, possibly active as cytokinins,had the characteristics of dihydrozeatin, zeatin or dihydrozeatin-5'-nucleotide(s),and zeatin or dihydrozeatin glucosides. The gradual disappearancewith time of zeatin and its riboside from the shoot was accompaniedby an increase in the proportion of more polar metabolites. These results are discussed in relation to the possible exportof endogenous cytokinins by the nodules.     

Candidatus Frankia Datiscae Dg1, the Actinobacterial Microsymbiont of Datisca glomerata,Expresses the Canonical nod Genes nodABC in Symbiosis with Its Host Plant

Frankia strains are nitrogen-fixing soil actinobacteria that can form root symbioses with actinorhizal plants. Phylogenetically, symbiotic frankiae can be divided into three clusters, and this division also corresponds to host specificity groups. The strains of cluster II which form symbioses with actinorhizal Rosales and Cucurbitales, thus displaying a broad host range, show suprisingly low genetic diversity and to date can not be cultured. The genome of the first representative of this cluster, Candidatus Frankia datiscae Dg1 (Dg1), a microsymbiont of Datisca glomerata, was recently sequenced. A phylogenetic analysis of 50 different housekeeping genes of Dg1 and three published Frankia genomes showed that cluster II is basal among the symbiotic Frankia clusters. Detailed analysis showed that nodules of D. glomerata, independent of the origin of the inoculum, contain several closely related cluster II Frankia operational taxonomic units. Actinorhizal plants and legumes both belong to the nitrogen-fixing plant clade, and bacterial signaling in both groups involves the common symbiotic pathway also used by arbuscular mycorrhizal fungi. However, so far, no molecules resembling rhizobial Nod factors could be isolated from Frankia cultures. Alone among Frankia genomes available to date, the genome of Dg1 contains the canonical nod genes nodA, nodB and nodC known from rhizobia, and these genes are arranged in two operons which are expressed in D. glomerata nodules. Furthermore, Frankia Dg1 nodC was able to partially complement a Rhizobium leguminosarum A34 nodC::Tn5 mutant. Phylogenetic analysis showed that Dg1 Nod proteins are positioned at the root of both α- and β-rhizobial NodABC proteins. NodA-like acyl transferases were found across the phylum Actinobacteria, but among Proteobacteria only in nodulators. Taken together, our evidence indicates an Actinobacterial origin of rhizobial Nod factors.     

Bacteriology of Manganese Nodules: I. Bacterial Action on Manganese in Nodule Enrichments

Bacteria, found in manganese nodules from the Atlantic Ocean, enhance the adsorption of Mn from sea water by crushed manganese nodules in the presence of peptone. When bacterial outgrowth from crushed manganese nodules was experimentally delayed, peptone did not enhance Mn adsorption by nodular substance, but hindered it in some cases. A mechanism to explain the role of bacteria in enhancing Mn adsorption by manganese nodules is presented. Oyster shells were shown to adsorb Mn in the absence of bacteria. Peptone did not enhance the rate of Mn adsorption. Adsorbed Mn was not visibly oxidized during experimental observation. These results suggest one way whereby nodule formation may be initiated in the oceans. Some bacteria in the nodules were found to release manganese from them in the presence of glucose and peptone. Bacteria may, therefore, play a role not only in nodule buildup but also in nodule breakdown.     

Bacteriology of Manganese Nodules: II. Manganese Oxidation by Cell-free Extract from a Manganese Nodule Bacterium

A cell-free extract from Arthrobacter 37, isolated from a manganese nodule from the Atlantic Ocean, exhibited enzymatic activity which accelerated manganese accretion to synthetic Mn-Fe oxide as well as to crushed manganese nodule. The reaction required oxygen and was inhibited by HgCl₂ and p-chloromercuribenzoate but not by Atebrine dihydrochloride. The rate of enzymatic action depended on the concentration of cell-free extract used. The enzymatic activity had a temperature optimum around 17.5 C and was destroyed by heating at 100 C. The amount of heat required for inactivation depended on the amount of nucleic acid in the preparation. In the cell-free extract, unlike the whole-cell preparation, peptone could not substitute for NaHCO₃ in the reaction mixture. An enzyme-containing protein fraction and a nucleic acid fraction could be separated from cell extract by gel filtration, when prepared in 3% NaCl but not in seawater. The nucleic acid fraction was not required for enzymatic activity.     

The Composition of Lotus corniculatus Root Nodule Bacteria Depending on the Host Plant Vegetation Stage

Microbiology - The legume-Rhizobium symbiosis is a unique natural phenomenon responsible for providing a legume plant with mineral nitrogen fixed from the atmosphere. In the temperate climatic...     

Genetic Diversity and Phylogeny of Root Nodule Bacteria Isolated from Nodules of Plants of the Lupinaster Genus Inhabiting the Southern Urals

Russian Journal of Genetics - The genetic diversity and phylogeny of root nodule bacteria, microsymbionts of plants of the genus Lupinaster Adans. (L. albus Link and L. pentaphyllus Moench), were...     

Studies on the Physiology of Nodule Formation: III. Experiments on the Excision of Root-tips and Nodules

The excision of nodules and root-tips from red clover inoculatedwith an effective strain of nodule bacteria leads to an increasein the number of nodules subsequently formed. This is thoughtto be due to the removal by excision of the source of an inhibitor.In the nodule the inhibitory activity appears to be centredin the growing-point and not in the bacterial tissue, sincethe excision of the nodular meristem only will stimulate furthernodulation. The response to excision depends upon the numberof excisions made and is inversely related to the inherent susceptibilityof the individual plant. Excision of nodules from plants inoculated with ineffectivestrains of bacteria has no influence on further nodulation,presumably because the growing-points of the nodules are abortive.     

Carbon Dioxide Fixation by Lupin Root Nodules: I. Characterization, Association with Phosphoenolpyruvate Carboxylase, and Correlation with Nitrogen Fixation during Nodule Development

In vivo CO₂ fixation and in vitro phosphoenolpyruvate (PEP) carboxylase levels have been measured in lupin (Lupinus angustifolius L.) root nodules of various ages. Both activities were greater in nodule tissue than in either primary or secondary root tissue, and increased about 3-fold with the onset of N₂ fixation. PEP carboxylase activity was predominantly located in the bacteroid-containing zone of mature nodules, but purified bacteroids contained no activity. Partially purified PEP carboxylases from nodules, roots, and leaves were identical in a number of kinetic parameters. Both in vivo CO₂ fixation activity and in vitro PEP carboxylase activity were significantly correlated with nodule acetylene reduction activity during nodule development. The maximum rate of in vivo CO₂ fixation in mature nodules was 7.9 nmol hour⁻¹ mg fresh weight⁻¹, similar to rates of N₂ fixation and reported values for amino acid translocation.     

Glutamate Oxaloacetate Transaminase in Pea Root Nodules : Participation in a Malate/Aspartate Shuttle between Plant and Bacteroid

Glutamate oxaloacetate transaminase (l-glutamate: oxaloacetate aminotransferase, EC 2.6.1.1 [GOT]), a key enzyme in the flow of carbon between the organic acid and amino acid pools in pea (Pisum sativum L.) root nodules, was studied. By ion exchange chromatography, the presence of two forms of GOT in the cytoplasm of pea root nodule cells was established. The major root nodule form was present in only a small quantity in the cytoplasm of root cells. Fractionation of root nodule cell extracts demonstrated that the increase in the GOT activity during nodule development was due to the increase of the activity in the cytoplasm of the plant cells, and not to an increase in activity in the plastids or in the mitochondria. The kinetic properties of the different cytoplasmic forms of GOT were studied. Some of the K_m values differed, but calculations indicated that not the kinetic properties but a high concentration of the major root nodule form caused the observed increase in GOT activity in the pea root nodules. It was found that the reactions of the malate/aspartate shuttle are catalyzed by intact bacteroids, and that these reactions can support nitrogen fixation. It is proposed that the main function of the nodule-stimulated cytoplasmic form of GOT is participation in this shuttle.     

我国蚕豆根瘤菌的数值分类及其16SrDNA PCR-RFLP研究

对分离自我国11个省24个地区49株蚕豆根瘤菌及11株参比菌株进行了唯一碳源、氮源、抗生素、耐逆性和酶活性等138个表型性状测定,并用MINTS软件进行聚类分析。结果表明,全部供试菌株在59％的相似水平上聚在一起,在80％的相似水平上可分为6个群。其中群4与参比菌株聚在一起,而其他5个群均由未知菌组成。进一步对36株菌进行了16S rDNA PCR—RFLP分析,在85％相似水平上供试菌可分为4个群和1个独立的分支,其聚群结果与数值分类结果有较好的一致性。表型及遗传型分析结果表明,我国蚕豆根瘤菌具有极大的多样性。     

我国蚕豆根瘤菌的数值分类及其16S rDNA PCR-RFLP研究*

对分离自我国11个省24个地区49株蚕豆根瘤菌及11株参比菌株进行了唯一碳源、氮源、抗生素、耐逆性和酶活性等138个表型性状测定,并用M INTS软件进行聚类分析。结果表明,全部供试菌株在59%的相似水平上聚在一起,在80%的相似水平上可分为6个群。其中群4与参比菌株聚在一起,而其他5个群均由未知菌组成。进一步对36株菌进行了16S rDNA PCR-RFLP分析,在85%相似水平上供试菌可分为4个群和1个独立的分支,其聚群结果与数值分类结果有较好的一致性。表型及遗传型分析结果表明,我国蚕豆根瘤菌具有极大的多样性。     

Two Isoenzymes of NADH-dependent Glutamate Synthase in Root Nodules of Phaseolus vulgaris L: Purification, Properties and Activity Changes during Nodule Development

The specific activity of plant NADH-dependent glutamate synthase (NADH-GOGAT) in root nodules of Phaseolus vulgaris L. is over threefold higher than the specific activity of ferredoxin-dependent GOGAT. The NADH-GOGAT is composed of two distinct isoenzymes (NADH-GOGAT I and NADH-GOGAT II) which can be separated from crude nodule extracts by ion-exchange chromatography. Both NADH-GOGAT isoenzymes have been purified to apparent homogeneity and shown to be monomeric proteins with similar M_rs of about 200,000. They are both specific for NADH as reductant. An investigation of their kinetic characteristics show slight differences in their K_ms for l-glutamine, 2-oxoglutarate, and NADH, and they have different pH optima, with NADH-GOGAT I exhibiting a broad pH optimum centering at pH 8.0 whereas NADH-GOGAT II has a much narrower pH optimum of 8.5. The specific activity of NADH-GOGAT in roots is about 27-fold lower than in nodules and consists almost entirely of NADH-GOGAT I. During nodulation both isoenzymes increase in activity but the major increase is due to NADH-GOGAT II which increases over a time course similar to the increase in nitrogenase activity. This isoenzyme is twice as active as NADH-GOGAT I in mature nodules. The roles and regulation of these two isoenzymes in the root nodule are discussed.     

Bacteriology of Manganese Nodules: III. Reduction of MnO2 by Two Strains of Nodule Bacteria1

MnO₂ reduction by aerobic growing cultures of Bacillus 29 and coccus 32, isolated from ferromanganese nodules, was assessed for 7 days. A 1-day lag was observed before the onset of MnO₂ reduction by either culture. Addition of HgCl₂ to a final concentration of about 10^-3 M caused a rapid cessation of MnO₂ reduction by the growing cultures. Neither culture reduced MnO₂ when grown under continued anaerobiosis from the start of an experiment. However, if conditions were made anaerobic after MnO₂ reduction was initiated, reduction continued at a rate only slightly lower than that under aerobic conditions. Resting-cell cultures reduced MnO₂ equally well aerobically and anaerobically, provided that ferricyanide was present to serve as electron carrier. These findings showed that oxygen is needed for culture adaptation to MnO₂ reduction, and that oxygen does not interfere with microbial MnO₂ reduction itself. Both cultures caused sharp drops in the pH of the medium during MnO₂ reduction: with coccus 32, during the entire incubation time; with Bacillus 29, for the first 3 days. The E_h of the medium fluctuated with either culture and never fell below 469 mv with Bacillus 29 and below 394 mv with coccus 32. The rates of glucose consumption and Mn²⁺ release by Bacillus 29 and coccus 32 were fairly constant, but the rates of lactate and pyruvate production were not. Although acid production undoubtedly helped in the reduction of pyrolusite (MnO₂) by the bacteria, it did not appear to be important in the reduction of manganese oxide in ferromanganese nodules, as shown by the results with a nodule enrichment.