Oligonucleotide Probes That Hybridize with rRNA as a Tool To Study Frankia Strains in Root Nodules

Oligonucleotide probes that hybridize with specific sequences in variable regions of the 16S rRNA of the nitrogen-fixing actinomycete Frankia were used for the identification of Frankia strains in nodules. Frankia cells were released from plant tissue by grinding glutaraldehyde-fixed root nodules in guanidine hydrochloride solution. rRNA was obtained after sonication, precipitation with ethanol, and purification by phenolchloroform extraction. Degradation of rRNA, evident in Northern blots, did not affect hybridization with the oligonucleotides. Nodules of about 1 mg (fresh weight) provided sufficient rRNA for reliable detection of the Frankia strain. The utility of this rRNA extraction method was tested in a competition experiment between two effective Frankia strains on cloned Alnus glutinosa plants.     

Identification of Frankia Strains by Direct DNA Hybridization of Crushed Nodules

A hybridization procedure was developed to identify Frankia strains inside actinorhizae by direct probing of crushed root nodules. The probe consisted of an indigenous cryptic plasmid. This well-conserved, 8-kilobase plasmid was detected in Frankia isolates that were very close taxonomically (they possessed a very high DNA sequence homology). The probe did not hybridize to the DNA of Frankia isolates which did not carry the plasmid. Endophyte DNA was extracted by a modification of a technique originally developed for the detection of plasmids in Frankia isolates. The hybridization procedure applied to nodules collected in a stand of alder permitted determination of a distribution map of the plasmid-bearing Frankia strains.     

Identification and Localization of Frankia Strains in Alnus Nodules by In Situ Hybridization of nif H mRNA with Strain-Specific Oligonucleotide Probes

In situ hybridization of Frankia mRNA with specific probes wasused to localize the strains Arl3 and AcoN24d in Alnus nodulesobtained after inoculation with one or both strains. The probesconsisted of 18-mer oligonucleotides, complementary to strain-specificsequences located within the nif H gene. Sections of nodulesinoculated with only one strain revealed a specific hybridizationbetween the probe and the corresponding Frankia strain mRNA.In sections of dually-inoculated nodules the presence of thestrain AcoN24d in the nodule was clearly shown whereas thoseof the strain Arl3 could not be detected. This suggests thatthe strain Arl3 is less infective than the strain AcoN24d andis not present within the nodule. Key words: Nitrogen fixation, actinorhizae, autoradiography, histochemistry     

Activities, occurrence, and localization of hydrogenase in free-living and symbiotic frankia

Symbiotic and free-living Frankia were investigated for correlation between hydrogenase activities (in vivo/in vitro assays) and for occurrence and localization of hydrogenase protein by Western blots and immuno-gold localization, respectively. Freshly prepared nodule homogenates from the symbiosis between Alnus incana and a local source of Frankia did not show any detectable in vivo or in vitro hydrogenase uptake activity, as also has been shown earlier. However, a free-living Frankia strain originally isolated from these nodules clearly showed both in vivo and in vitro hydrogenase activity, with the latter being approximately four times higher. Frankia strain Cpl1 showed hydrogen uptake activity both in symbiosis with Alnus incana and in a free-living state. Western blots on the different combinations of host plants and Frankia strains used in the present study revealed that all the Frankia sources contained a hydrogenase protein, even the local source where no in vivo or in vitro activity could be measured. The 72 kilodalton protein found in the symbiotic Frankia as well as in the free-living Frankia strains were immunologically related to the large subunit of a dimeric hydrogenase purified from Alcaligenes latus. Recognitions to polypeptides with molecular masses of about 41 and 19.5 kilodaltons were also observed in Frankia strain UGL011101 and in the local source of Frankia, respectively. Immunogold localization of the protein demonstrated that in both the symbiotic state and the free-living nitrogen-fixing Frankia, the protein is located in vesicles and in hyphae. The inability to measure any uptake hydrogenase activity is therefore not due to the absence of hydrogenase enzyme. However, the possibility of an inactive hydrogenase enzyme cannot be ruled out.     

Effect of Inoculation and Leaf Litter Amendment on Establishment of Nodule-Forming Frankia Populations in Soil

High-N₂-fixing activities of Frankia populations in root nodules on Alnus glutinosa improve growth performance of the host plant. Therefore, the establishment of active, nodule-forming populations of Frankia in soil is desirable. In this study, we inoculated Frankia strains of Alnus host infection groups I, IIIa, and IV into soil already harboring indigenous populations of infection groups (IIIa, IIIb, and IV). Then we amended parts of the inoculated soil with leaf litter of A. glutinosa and kept these parts of soil without host plants for several weeks until they were spiked with [¹⁵N]NO₃ and planted with seedlings of A. glutinosa. After 4 months of growth, we analyzed plants for growth performance, nodule formation, specific Frankia populations in root nodules, and N₂ fixation rates. The results revealed that introduced Frankia strains incubated in soil for several weeks in the absence of plants remained infective and competitive for nodulation with the indigenous Frankia populations of the soil. Inoculation into and incubation in soil without host plants generally supported subsequent plant growth performance and increased the percentage of nitrogen acquired by the host plants through N₂ fixation from 33% on noninoculated, nonamended soils to 78% on inoculated, amended soils. Introduced Frankia strains representing Alnus host infection groups IIIa and IV competed with indigenous Frankia populations, whereas frankiae of group I were not found in any nodules. When grown in noninoculated, nonamended soil, A. glutinosa plants harbored Frankia populations of only group IIIa in root nodules. This group was reduced to 32% ± 23% (standard deviation) of the Frankia nodule populations when plants were grown in inoculated, nonamended soil. Under these conditions, the introduced Frankia strain of group IV was established in 51% ± 20% of the nodules. Leaf litter amendment during the initial incubation in soil without plants promoted nodulation by frankiae of group IV in both inoculated and noninoculated treatments. Grown in inoculated, amended soils, plants had significantly lower numbers of nodules infected by group IIIa (8% ± 6%) than by group IV (81% ± 11%). On plants grown in noninoculated, amended soil, the original Frankia root nodule population represented by group IIIa of the noninoculated, nonamended soil was entirely exchanged by a Frankia population belonging to group IV. The quantification of N₂ fixation rates by ¹⁵N dilution revealed that both the indigenous and the inoculated Frankia populations of group IV had a higher specific N₂-fixing capacity than populations belonging to group IIIa under the conditions applied. These results show that through inoculation or leaf litter amendment, Frankia populations with high specific N₂-fixing capacities can be established in soils. These populations remain infective on their host plants, successfully compete for nodule formation with other indigenous or inoculated Frankia populations, and thereby increase plant growth performance.     

The improvement and utilization in forestry of nitrogen fixation by actinorhizal plants with special reference toAlnus in Scotland

Summary Alnus species are used widely in Britain for land reclamation, forestry and other purposes. Rapid juvenile growth of the AmericanAlnus rubra makes it an attractive species for planting on N-deficient soils, particularly those of low organic content. In small plot trials, this species is nodulated by indigenous soil frankiae as effectively asAlnus glutinosa. Over a three year period both species return similar amounts of N to the ecosystem, estimated at up to 10–12 kg N ha^–1. Several strains ofFrankia have been isolated from local (Lennox Forest)A. rubra nodules. These differ morphologically and in their growth on different culture media, both from each other and fromA. glutinosa nodule isolates. AllAlnus isolates, however, have a total cellular fatty acid composition qualitatively similar to some other Group B frankiae. Glasshouse tests in N free culture suggest thatA. rubra nodules formed after inoculation of seedlings with American spore (–) isolates are three times more effective in N fixation than those inoculated with LennoxA. rubra spore (+) nodule homogenates. By contrast, the early growth of seedlings inoculated with spore (–)Frankia strains suggests at best a 35% improvement in N fixing activity over seedlings inoculated with LennoxA. rubra nodule isolates. Nevertheless, this improvement in activity, together with the better performance of seedlings inoculated with isolates compared with those treated with crushed nodule preparations, suggest that it would be worthwhile commercially to inoculate nursery stock with a spore (–)Frankia strain.     

Novel species of Frankia,Frankia gtarii sp. nov. and Frankia tisai sp. nov., isolated from a root nodule of Alnus glutinosa

The status of four Frankia strains isolated from a root nodule of Alnus glutinosa was established in a polyphasic study. Taxogenomics and phenotypic features show that the isolates belong to the genus Frankia. All four strains form extensively branched substrate mycelia, multilocular sporangia, vesicles, lack aerial hyphae, but contain meso-diaminopimelic acid as the diamino acid of the peptidoglycan, galactose, glucose, mannose, ribose, xylose and traces of rhamnose as cell wall sugars, iso-C_16:0 as the predominant fatty acid, diphosphatidylglycerol, phosphatidylglycerol, phosphatidylinositol as the major polar lipids, have comparable genome sizes to other cluster 1, Alnus-infective strains with structural and accessory genes associated with nitrogen fixation. The genome sizes of the isolates range from 7.0 to 7.7 Mbp and the digital DNA G + C contents from 71.3 to 71.5 %. The four sequenced genomes are rich in biosynthetic gene clusters predicted to express for novel specialized metabolites, notably antibiotics. 16S rRNA gene and whole genome sequence analyses show that the isolates fall into two lineages that are closely related to the type strains of Frankia alni and Frankia torreyi. All of these taxa are separated by combinations of phenotypic properties and by digital DNA:DNA hybridization scores which indicate that they belong to different genomic species. Based on these results, it is proposed that isolates Agncl-4^T and Agncl-10, and Agncl-8^T and Agncl-18, be recognised as Frankia gtarii sp. nov. and Frankia tisai sp. nov. respectively, with isolates Agncl-4^T (=DSM 107976^T = CECT 9711^T) and Agncl-8^T (=DSM 107980^T = CECT 9715^T) as the respective type strains.     

Comparative physiology of nitrogenase activity and vesicle development for Frankia strains CpI1, ACN1 ag,EAN1pec and EUN1f

The relationship between nitrogen fixation and development of a specialized cell structure, called the vesicle, was studied using four Frankia isolates. Nitrogenase activity was repressed in all four strains during growth with ammonia. Strain CpI1 formed no vesicles during NH₄ growth. Strains ACN1 ^ag , EAN1_pec and EUN1_f produced low numbers of vesicles in the presence of ammonia. Following transfer to nitrogen-free media, a parallel increase in nitrogenase activity and vesicle numbers occurred with all four isolates. Appearance of nitrogenase activity was more rapid in those strains that possessed some vesicles at the time of shift to N₂ as a nitrogen source. The ratio of vesicle numbers to level of nitrogenase activity varied widely among the four strains and in response to different growth conditions and culture age of the individual strains. Optimum conditions of temperature, carbon and energy source, nitrogen source and availability of iron and molybdenum were different for each of the four strains. Those conditions that significantly reduced nitrogenase activity were always associated with decreased numbers of vesicles.     

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.     

Detection of pectolytic activity andpel homologous sequences inFrankia

Using a cup-plate pectin agar assay, pectolytic activity was detected in nodule filtrates obtained fromAlnus rugosa (DuRoi) Spreng,A. glutinosa (L.) Gaertn andA. crispa (Ait.) Pursh seedlings after infection with twoFrankia strains (ACN1 ^AG , CpI1). Pectolytic activity was also detected in cultures filtrates of the same twoFrankia isolates afterin vitro-cultivation on Qmod pectin liquid medium. When Southern blots of Frankia total DNAs from 3 isolates ofF. alni subsp.Pommerii (ACN1 ^AG , ArI3, and CPX32b) and 3 isolates ofF. elaeagni (EUN1 pec, SCN 10a and TX31e ^HR ) were hybridized withPelBDA probes fromErwinia chrysanthemi, positive signals were found in all 7 Frankiae tested.     

Molecular ecology ofFrankia: Advantages and disadvantages of the use of DNA probes

Ecological studies on the actinomyceteFrankia are often influenced by the difficulty to isolate and identify this microorganism. The application of molecular biological techniques offers possibilities to detect microbes without isolation and cultivation.Nif genes or whole plasmids can serve as targets for the design of specific probes. Alternatively, ribosomal RNA (rRNA), commonly used in modern phylogenetic studies, can be used as a target molecule in ecological studies. This paper gives an overview of new developments on the use of 16S rRNA as a target molecule for oligonucleotide probes. Group-specific sequences in the 16S rRNA ofFrankia can be used as targets for oligonucleotide probes that a) recognize ineffectiveFrankia strains onAlnus, b) recognize effective strains onAlnus, c) recognize allFrankia strains tested so far. The present paper summarizes the essential steps needed for the use of these probes for the detection ofFrankia strains in soil without isolation and cultivation.     

Diversity of Frankia strains isolated from single nodules of Alnus glutinosa

Diversity of Frankia isolates originating from lobes of single nodules collected on Alnus glutinosa root systems has been analyzed using isozyme electrophoresis method. Analysis of isozyme patterns showed no divergence among strains isolated from the same nodule. Each nodule (among 10 assayed) was inhabited by a single Frankia strain.     

Morphology,physiology and infectivity of twoFrankia isolates An 1 and An 2 from root nodules ofAlnus nitida

Summary Two different strains, An 1 and An 2, were obtained from root nodules ofAlnus nitida Endl., collected from one locality in the area of its natural habitat near Bahrin, District Swat, Pakistan. The light and electron microscopy of the isolates revealed the occurrence of septate and branched hyphae bearing sporangia and vesicles. The strains differed in their growth requirements, nitrogen-fixing ability and production of extracellular pigments, thus indicating the existence of more than oneFrankia strain in the same locality. In the absence of combined nitrogen in the medium strain An 1 formed vesicles and fixed N₂ (up to 200 nmol C₂H₄. mg protein^–1.h^–1), while strain An 2 under the experimental conditions formed only few vesicles and fixed N₂ at a very low rate (ca 10 nmol C₂H₄. mg protein^–1 .h^–1). The nitrogenase activity of strain An 1 was strongly affected by the O₂ concentration.Frankia An 1 and An 2 were infective and effective onA. nitida andA. glutinosa but not onDatisca cannabina andElaeagnus umbellata. Both An 1 and An 2 strains were more infective and effective onA. glutinosa thanFrankia strains AvcIl and CpI1.     

Respiration and nitrogenase activity in nodules ofCasuarina cunninghamiana and cultures ofFrankia sp. HFP020203: Effects of temperature and partial pressure of O2

The effects of time after exposure to acetylene and of nodule excision were examined using a flow-through system. After a transient depression in the rate of acetylene reduction that began about 1.5 min after exposure to acetylene, the rate recovered to 98% of the initial maximum value after 40 min. After nodule excision the rate stabilized to 90% of the initial maximum value observed in the intact plant.Excised nodules, measured at 6-min intervals in a closed system, with frequent changes of the gas mixture, were used for the remaining experiments. Acetylene reduction by the nodules increased rapidly as temperature was increased between 6 and 26°C. Between 26 and 36°C there was relatively little effect of temperature on acetylene reduction.Nodules and cultures ofFrankia were compared with respect to the effect of temperature and pO₂ (partial pressure of oxygen) on oxygen uptake. Cultures ofFrankia were grown on a nitrogen-free medium at either 0.3 kPa O₂ (vesicles absent) or 20 kPa O₂ (vesicles present). Oxygen uptake by nodules (vesicles absent) and by vesicle-containing cultures was strongly dependent on pO₂ at values below 20 kPa. This suggests the presence of a barrier to oxygen diffusion. Oxygen uptake was dependent on temperature as well as on pO₂, but the Q₁₀ was much larger for the cultures than for the nodules. This suggests that vesicles or related structures are not the source of the diffusion barrier in Casuarina nodules. Respiration by cultures ofFrankia lacking vesicles became O₂-saturated at low pO₂ values. Thus these cultures did not have a significant diffusion barrier. From these results it is concluded that nodules ofCasuarina cunninghamiana have a barrier to oxygen diffusion supplied by the host tissue and not byFrankia.     

Extraction of ribosomal RNA from soil for detection of Frankia with oligonucleotide probes

Sequences of 16S rRNA of the nitrogen-fixing Frankia strain Ag45/Mut15 and the ineffective Frankia strain AgB1.9 were used to design a genus-specific oligonucleotide probe. Hybridization experiments of this Frankia probe and a second probe, specific for Nif⁺-Frankia strains only, were used to detect Frankia specific target sequences in RNA isolations from soil. A method is described for direct isolation of RNA from a loamy soil and a peat. Yields of about 10 ng RNA/g wet soil are obtained without detectable contamination with humic acids. Isolation of RNA after initial extraction of bacteria from soil resulted in significantly lower RNA yields, compared to the direct isolation procedure. Hybridization with both probes against rRNA isolations from Frankia-containing soil could detect target sequences within RNA isolations from 1 g wet soil with an estimated detection limit of 10⁴ cells.     

Morphological and molecular characterization of Frankia sp. isolates from nodules of Alnus nepalensis Don

Nodules collected from Alnus nepalensis growing in mixed forest stands at three different sites around Shillong, were crushed in various culture media to obtain isolates of Frankia. The isolates were found to have typical Frankia morphology as revealed by the scanning electron microscope. Seedlings inoculated with isolates or crushed nodules formed nitrogen fixing nodules. Frankia specific DNA probes amplified the DNA of the tested isolate AnpUS4. Partial nucleotide sequence of the 16S rRNA gene indicated that AnpUS4 was phylogenetically distinct from all other Frankia strains characterized so far.     

Micromonospora is a normal occupant of actinorhizal nodules

Actinorhizal plants have been found in eight genera belonging to three orders (Fagales, Rosales and Cucurbitales). These all bear root nodules inhabited by bacteria identified as the nitrogen-fixing actinobacterium Frankia. These nodules all have a peripheral cortex with enlarged cells filled with Frankia hyphae and vesicles. Isolation in pure culture has been notoriously difficult, due in a large part to the growth of fast-growing contaminants where, it was later found, Frankia was slow-growing. Many of these contaminants, which were later found to be Micromonospora, were obtained from Casuarina and Coriaria. Our study was aimed at determining if Micromonospora were also present in other actinorhizal plants. Nodules from Alnus glutinosa, Alnus viridis, Coriaria myrtifolia, Elaeagnus x ebbingei, Hippophae rhamnoides, Myrica gale and Morella pensylvanica were tested and were all found to contain Micromonospora isolates. These were found to belong to mainly three species: Micromonospora lupini, Micromonospora coriariae and Micromonospora saelicesensis. Micromonospora isolates were found to inhibit some Frankia strains and to be innocuous to other strains.     

Isolation of Frankia Strains from Alder Actinorhizal Root Nodules

A simple procedure, based on the rapid filtration and washing of Frankia vesicle clusters, was devised for the isolation of Frankia strains from alder actinorhizal root nodules. Of 46 Alnus incana subsp. rugosa nodules prepared, 42 yielded isolates. A simple medium containing mineral salts, Casamino Acids, and sodium pyruvate proved to be the most effective for isolation. In general, colonies appeared 6 to 20 days after inoculation. On the basis of hyphal morphology, two distinct types of Frankia strains were characterized. Randomly selected isolates were tested for infectivity, and all formed root nodules on A. glutinosa. Because of its simplicity and efficiency, the procedure is an improved method for the study of Frankia diversity in alder root nodules.     

Structural and gene expression analyses of uptake hydrogenases and other proteins involved in nitrogenase protection in Frankia

The actinorhizal bacterium Frankia expresses nitrogenase and can therefore convert molecular nitrogen into ammonia and the by-product hydrogen. However, nitrogenase is inhibited by oxygen. Consequently, Frankia and its actinorhizal hosts have developed various mechanisms for excluding oxygen from their nitrogen-containing compartments. These include the expression of oxygen-scavenging uptake hydrogenases, the formation of hopanoid-rich vesicles, enclosed by multi-layered hopanoid structures, the lignification of hyphal cell walls, and the production of haemoglobins in the symbiotic nodule. In this work, we analysed the expression and structure of the so-called uptake hydrogenase (Hup), which catalyses the in vivo dissociation of hydrogen to recycle the energy locked up in this ‘waste’ product. Two uptake hydrogenase syntons have been identified in Frankia: synton 1 is expressed under free-living conditions while synton 2 is expressed during symbiosis. We used qPCR to determine synton 1 hup gene expression in two Frankia strains under aerobic and anaerobic conditions. We also predicted the 3D structures of the Hup protein subunits based on multiple sequence alignments and remote homology modelling. Finally, we performed BLAST searches of genome and protein databases to identify genes that may contribute to the protection of nitrogenase against oxygen in the two Frankia strains. Our results show that in Frankia strain ACN14a, the expression patterns of the large (HupL1) and small (HupS1) uptake hydrogenase subunits depend on the abundance of oxygen in the external environment. Structural models of the membrane-bound hydrogenase subunits of ACN14a showed that both subunits resemble the structures of known [NiFe] hydrogenases (Volbeda et al. 1995), but contain fewer cysteine residues than the uptake hydrogenase of the Frankia DC12 and Eu1c strains. Moreover, we show that all of the investigated Frankia strains have two squalene hopane cyclase genes (shc1 and shc2). The only exceptions were CcI3 and the symbiont of Datisca glomerata, which possess shc1 but not shc2. Four truncated haemoglobin genes were identified in Frankia ACN14a and Eu1f, three in CcI3, two in EANpec1 and one in the Datisca glomerata symbiont (Dg).