Strain ArI3 Research Articles

Effect of different Alnus taxa on abundance and diversity of introduced and indigenous Frankia in soils and root nodules.

The effect of host plants on the abundance and distribution of introduced and indigenous Frankia populations was assessed in soils and root nodules of four alder species, Alnus glutinosa,Alnus cordata,Alnus rubra and Alnus viridis. Plants were grown in microcosms with either a sandy soil without detectable frankiae, with or without inoculation of a mixture of Frankia isolates, or a silty clay loam soil with indigenous Frankia. The presence of frankiae in soils increased plant height and root nodule formation, with significant increases in the presence of indigenous frankiae. Abundance in soils increased significantly for both introduced and indigenous Frankia populations independent of alder species, with generally largest increases in cluster 1b frankiae. Root nodules formed by introduced frankiae did not reflect the diversity of strains inoculated, with nodules generally only formed by strain ArI3 representing cluster 1a/d. All indigenous Frankia populations detected in soil were also found in A. glutinosa nodules, while A. cordata or A. rubra nodules contained different subsets of frankiae with unique abundances dependent on plant species. These results demonstrate the intrageneric differences of host plants in the selection of specific Frankia populations in soils for root nodule formation.

Contiguous Genome Sequence of Frankia sp. Strain ArI3, Isolated from Root Nodules of Alnus rubra Bong.

ABSTRACTWe report the genome sequence of Frankia sp. strain ArI3, recovered as a single contig from one run of the Oxford Nanopore Technologies (ONT) MinION instrument. The genome has a G+C content of 72%, is 7,541,222 bp long, and contains 5,427 predicted protein-coding genes.

Saprophytic growth of inoculated Frankia sp. in soil microcosms

The potential of two Frankia strains to grow saprophytically was studied in nonsterile soil microcosms with ground leaf litter of Alnus glutinosa as the sole carbon and nitrogen sources. Strains Ag45/Mut15 and ArI3, which represent two taxonomic subgroups within the Alnus host infection group were inoculated alone, or together to investigate potential competition. Their growth was analyzed by in situ and dot-blot hybridization. A significant increase in cell numbers and filament length was observed during the first 6 weeks after inoculation for strain Ag45/Mut15, both alone and in mixed culture with strain ArI3, followed by a decrease until the end of the study after 12 weeks. The number of filaments remained unchanged. In contrast, the cell numbers and filament length of strain ArI3 were reduced significantly during the first 2 weeks and were undetectable for the remainder of the study. These results were comparable with those obtained in sterile mineral medium amended with leaf litter of A. glutinosa, although reductions in cell numbers and filament length were less pronounced than in soil microcosms. In concomitant control studies without leaf litter amendments for both experimental setups, filaments of both strains could only be detected immediately after inoculation. These results were matched in all experimental setups by concomitant shifts in the rRNA content of both strains, i.e., an immediate decline in the rRNA content for strain ArI3 after inoculation, and an increase in the rRNA content, followed by a late decline during incubation for strain Ag45/Mut15. These results demonstrated that Frankia strain Ag45/Mut15 could grow saprophytically in soil with complex carbon and nitrogen sources such as leaf litter, while the growth of strain ArI3 was not supported.

DNA sequence and genetic characterization of plasmid pFQ11 from Frankia alni strain CpI1

An 8551-bp plasmid, pFQ11, from Frankia alni strain CpI1 was sequenced. Its sequence was found to be very similar to that presented for pFQ31 from strain ArI3. Six potential protein-encoding open reading frames (ORFs) were identified, and transcriptional activity was shown within four of those regions of the plasmid by reverse transcriptase-polymerase chain reaction (RT-PCR) analysis. An earlier study reported that ORF E F of pFQ31, which is nearly identical to the 3′ 45% of ORF1 of pFQ11, is significantly similar to RepF. We found no such similarity. ORF2 and ORF3 predict products that are similar to a repressor protein and a partition protein, respectively. We found inverted repeats within and covering the start codon of ORF3; palindromic sequences and direct repeats between ORF3 and ORF4; and 3′ from ORF3, an AT-rich sequence that extensively overlaps the promoter region of a uvrB homolog in strain ArI3.

DNA sequence and genetic characterization of plasmid pFQ11 from Frankia alni strain CpI1.

An 8551-bp plasmid, pFQ11, from Frankia alni strain CpI1 was sequenced. Its sequence was found to be very similar to that presented for pFQ31 from strain ArI3. Six potential protein-encoding open reading frames (ORFs) were identified, and transcriptional activity was shown within four of those regions of the plasmid by reverse transcriptase-polymerase chain reaction (RT-PCR) analysis. An earlier study reported that ORF E(F) of pFQ31, which is nearly identical to the 3' 45% of ORF1 of pFQ11, is significantly similar to RepF. We found no such similarity. ORF2 and ORF3 predict products that are similar to a repressor protein and a partition protein, respectively. We found inverted repeats within and covering the start codon of ORF3; palindromic sequences and direct repeats between ORF3 and ORF4; and 3' from ORF3, an AT-rich sequence that extensively overlaps the promoter region of a uvrB homolog in strain ArI3.

Early interactions between Alnus glutinosa and Frankia strain ArI3. Production and specificity of root hair deformation factor(s)

Actinomycetes from the genus Frankia are able to form symbiotic associations with more than 200 different species of woody angiosperms, so called actinorhizal plants. Many actinorhizal plants are infected via deformed root hairs. Factor(s) eliciting root hair deformation in actinorhizal symbioses have been found to be released into the culture medium, but the factor(s) has (have) not yet been characterized. In the present work, we describe the constitutive production of factor(s) by Frankia strain ArI3 causing root hair deformation on Alnus glutinosa. Deformation was detected after 4–5 h of incubation with both Frankia cultures and their cell‐free culture filtrates. When culture filtrate was used, deformation was concentration dependent. A contact time of 2 min between culture filtrate and host roots was sufficient to induce subsequent root hair deformation. No root hair deformation on A. glutinosa could be detected with purified Nod factors from Rhizobium meliloti or R. leguminosarum biovar viciae. No correlation was found between Frankia strains belonging to different host specificity groups and their ability to deform root hairs on A. glutinosa. However, strains not able to deform root hairs on A. glutinosa were also unable to nodulate.

Early interactions between Alnus glutinosa and Frankia strain ArI3. Production and specificity of root hair deformation factor(s)*

Early interactions between Alnus glutinosa and Frankia strain ArI3. Production and specificity of root hair deformation factor(s)*

Effects of extracts of Alnus glutinosa seeds on growth of Frankia strain ArI3 under static and fermentor culture conditions

In the present study, the effect of Alnus glutinosa seed extract on the in vitro growth of Frankia strain ArI3 was investigated under various culture conditions. Frankia filaments grown in static cultures showed a very slow growth with doubling time of 12 days. However, under the same growth conditions but with seed extract added to the medium, the doubling time was reduced to 6 days. A further reduction in doubling time (2.5 days) was obtained without seed extract when Frankia strain ArI3 was grown in a fermentor with continuous stirring. Under these culture conditions, seed extract did not influence the Frankia growth rate. In all our studies, Na-propionate was used as the only carbon source. Gradual addition of Na-propionate to Frankia cultures in the fermentor sustained exponential growth of the filaments over a prolonged period. The complete consumption of Na-propionate was the limiting growth factor under fermentor growth conditions. The mechanism of growth stimulation by Alnus glutinosa seed extract under growth limiting conditions in static cultures and its possible ecological significance is discussed.

Molecular structure of the Frankia spp. nifD-K intergenic spacer and design of Frankia genus compatible primer.

The nifD-K intergenic spacer (IGS) of ArI3 and ACoN24d were found to have a length 265 and 199 nucleotides, respectively. They are markedly less conserved than the two neighbouring genes and have, in some instances, a repeated structure reminiscent of an insertion event. The repeated sequence and the IGSs have no detectable homology with sequences in DNA databanks. The IGS has a stem-loop structure with a low folding energy, lower than that between nifH and nifD. No convincing alignment of IGS sequences could be obtained among Frankia strains. Only between ACoN24d and ArI3, which belong to the same genomic species, was the alignment good enough to permit detection of a doubly repeated structure. No promoter could be detected in the IGSs. The putative nifK open reading frame (ORF) in Frankia strain ArI3 has a length of 1587 nucleotides, starting with a GTG codon, preceded by a ribosome binding site of a structure similar to that of nifH (GGAGGN7). The codon usage was similar to that of previously sequenced Frankia genes with a strong bias toward G- and C-ending codons except in the case of glycine where GGT is frequent. Alignment of the three Frankia nifK sequences (EUN1f; ArI3 and ACoN24d) with those of other nitrogen-fixing bacteria permitted detection of a sequence conserved among the three Frankia strains but absent in the other sequences. A primer targeted to that region in combination with FGPD807-85 amplified the nifD-KIGS sequences of all Frankia strains (except the non-nitrogen-fixing Frankia strains CN3 and AgB1-9) and yet failed to amplify DNA of all other nitrogen-fixing bacteria.(ABSTRACT TRUNCATED AT 250 WORDS)

Nucleotide sequence of nifD from Frankia alni strain ArI3: phylogenetic inferences.

The complete nucleotide sequence of the nifD gene encoding the alpha subunit of component I of nitrogenase from Frankia alni strain ArI3 was determined. The coding region is 1,458 bp in length and encodes a polypeptide of 486 residues with a predicted molecular weight of 53,500. Phylogenetic inferences with 12 complete published nifD sequences were drawn using a variety of approaches. Frankia nifD clusters with proteobacteria rather than with Clostridium pasteurianum, the other Gram-positive bacterium studied. Extant eubacterial nif genes seem to have at least three distinct evolutionary origins as a result of ancient gene duplications. Within the Gram-positive bacterial phylum, functional nif genes descend from different duplicates.

Identification of Frankia strains in nodules by hybridization of polymerase chain reaction products with strain-specific oligonucleotide probes

A set of oligonucleotides has been developed to study the competitivity of two Frankia strains in the nodulation of the roots of two host plant species: Alnus glutinosa and Alnus incana. Two 20 mer-oligonucleotides, complementary to highly conserved sequences inside the nifH gene, were used as primers for the polymerase chain reaction (PCR) system in order to amplify microsymbiont DNA extracted from actinorhizae. PCR products were analyzed using two strain-specific 15-mer oligonucleotides identified in the amplified region. Hybridization data indicate that strain ACoN24d is more competitive than strain ArI3 in the nodulation of both hosts.

Evaluating alder-endophyte (Alnus acuminata-Frankia-Mycorrhizae) interactions

The objective of this study was to compare the interaction betweenFrankia and vesicular-arbuscular mycorrhizae (VAM) on the growth and N-fixing response ofAlnus acuminata seedlings under three different phosphorus levels.A. acuminata is an actinorhizal tree, commonly associated with pastures on upland areas. Seedlings were grown in sterile vermiculite, and inoculated withFrankia strain ArI3 and/or VAM (asGlomus intra-radices) under three phosphorus levels (10, 50 and 100 ppm). After 120 days differences in growth were observed at the 50 ppm P level between nodulated and non-nodulated plants; either if inoculated withFrankia+VAM or just with VAM. Interaction betweenFrankia and VAM was positive on nodule weight at 50 ppm P level. Differences in acetylene reduction, per gram of fresh nodule, were observed between and within both groups:Frankia inoculated andFrankia+VAM inoculated seedlings. InFrankia inoculated seedlings differences were observed between seedlings receiving 50 ppm P which showed higher nitrogenase activity than seedlings treated with 100 ppm P. Plants inoculated withFrankia andGlomus intra-radices at low P level (10 ppm) showed the highest acetylene reduction. It was 150% higher than the mean of the other treatments within the group, and 87% higher than the general mean of the onlyFrankia inoculated plants.

Interaction between hydrogenase, nitrogenase, and respiratory activities in a Frankia isolate from Alnus rubra.

H2 uptake and H2-supported O2 uptake were measured in N2-fixing cultures of Frankia strain ArI3 isolated from root nodules of Alnus rubra. H2 uptake by intact cells was O2 dependent and maximum rates were observed at ambient O2 concentrations. No hydrogenase activity could be detected in NH4+-grown, undifferentiated filaments cultured aerobically indicating that uptake hydrogenase activity was associated with the vesicles, the cellular site of nitrogen fixation in Frankia. Hydrogenase activity was inhibited by acetylene but inhibition could be alleviated by pretreatment with H2. H2 stimulated acetylene reduction at supraoptimal but not suboptimal O2 concentrations. These results suggest that uptake hydrogenase activity in ArI3 may play a role in O2 protection of nitrogenase, especially under conditions of carbon limitation.

Purification and properties of trehalase in Frankia ArI3

Trehalase was purified from cultures of Frankia strain ArI3 grown on media with or without NH4Cl. The purified enzyme was specific for trehalose, exhibited a broad pH optimum of pH 4.5 to 5.3 and had a Km for trehalose of 4.2 mM. The trehalase was inhibited in vitro completely by sucrose, glucose and mannose and partially by mannitol and sorbitol. In addition to the specific trehalase, a mixture of non-specific α- and β-glucosidases which exhibited some activity with α,α-trehalose as a substrate were also partially purified in Frankia extracts made from nitrogen-fixing cells. These enzymes were not detected in the purifications of crude extracts made from non-nitrogen-fixing cells (grown on media supplemented with NH4Cl). Trehalase activity in crude extracts increased over time when cells were induced to fix nitrogen, and the maximum specific activity of trehalase from nitrogen-fixing cultures was 4 times the maximum activity from non-fixing cultures. Trehalase activity was also examined in crude extracts made from Frankia vesicle clusters isolated from Alnus rubra nitrogen-fixing nodules infected with ArI3. The maximum activity of trehalase in these clusters was 6–7 times greater than in the nitrogenfixing pure cultures of ArI3 and 26–33 times greater than the non-fixing pure cultures.

The role of diffusion in oxygen protection of nitrogenase in nodules of Alnus rubra

Endophyte respiration in actinorhizal nodules has two main functions: to provide ATP and reductant for nitrogenase and associated enzymes and to protect nitrogenase from inactivation by oxygen. Combined with a diffusion path which limits the flow of oxygen, the consumption of oxygen by respiration maintains a very low internal [Formula: see text]. We have used a continuous-flow root-nodule gas-exchange system to investigate the properties of such a diffusion path in intact, attached nodules of Alnus rubra infected with Frankia strain ArI3. When nodules were exposed to gas mixtures containing argon, oxygen, and acetylene, the resulting curves of ethylene production versus acetylene concentration showed diffusion-limited enzyme kinetics. Consequently, ethylene production at very low acetylene concentrations (<0.2% v/v) was used to characterize the diffusion path that both acetylene and oxygen must traverse to reach their active sites in the endophyte. The permeability of this path changed markedly with temperature and can account for the shape of the temperature response curve for total nitrogenase activity. Owing to the temperature-dependent barrier, energy production remains limited by oxygen diffusion, and nitrogenase can function over a wide temperature range at a relatively constant energy cost without inactivation by excess oxygen.