Enzymes of ammonia assimilation in hyphae and vesicles of Frankia sp. strain CpI1.

Frankia spp. are filamentous actinomycetes that fix N2 in culture and in actinorhizal root nodules. In combined nitrogen-depleted aerobic environments, nitrogenase is restricted to thick-walled spherical structures, Frankia vesicles, that are formed on short stalks along the vegetative hyphae. The activities of the NH4(+)-assimilating enzymes (glutamine synthetase [GS], glutamate synthase, glutamate dehydrogenase, and alanine dehydrogenase) were determined in cells grown on NH4+ and N2 and in vesicles and hyphae from N2-fixing cultures separated on sucrose gradients. The two frankial GSs, GSI and GSII, were present in vesicles at levels similar to those detected in vegetative hyphae from N2-fixing cultures as shown by enzyme assay and two-dimensional polyacrylamide gel electrophoresis. Glutamate synthase, glutamate dehydrogenase, and alanine dehydrogenase activities were restricted to the vegetative hyphae. Vesicles apparently lack a complete pathway for assimilating ammonia beyond the glutamine stage.     

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 N(2)-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.     

Study on Infectivity of the Nodule Endophyte of Some Non-Leguminous N2-Fixing Plants

Cross inoculations were made with Frankia spp. from the nodules of non-leguminous plants belonging to different families, genera and species. The results showed that there are no apparent host specificity in these strains. Under general cases, many strains can nodulate plants in different families, genera and species, but there also are some special results. Both infective ability of the same strains on diffierent host and the different strains on the same host are different. Different isolates from the same host plant were found in certain cases to have various degrees of infectivity. If the original host plant was replaced by others, both these Frankia infective ability and nitrogenase activity in new symbiotic system were lower. The strains that are higher N2-fixing activity in the nodules of the original host also possess stronger N2-fixing activity in the nodules of other hosts. Under test condition, there are positive correlation between the number of the nodules of host plants and N2-fixing activity of the root nodules. The morphology of the spores of the strains in the nodules of new host plant also change more or less.     

Effect of inoculation and leaf litter amendment on establishment of nodule-forming Frankia populations in soil

High-N(2)-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 [(15)N]NO(3) 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 N(2) 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 N(2) 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 N(2) fixation rates by (15)N dilution revealed that both the indigenous and the inoculated Frankia populations of group IV had a higher specific N(2)-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 N(2)-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.     

Developmental potential of Frankia vesicles.

The ability of nitrogenase-containing Frankia sp. strain CpI1 vesicles to regrow vegetative hyphae is demonstrated. Vesicles attached to hyphae in N2-fixing CpI1 cultures and sucrose gradient-isolated vesicles exhibited hyphal outgrowths when incubated in certain defined liquid media. Single or multiple hyphal extensions grew out from the vesicles.     

Significant natural product biosynthetic potential of actinorhizal symbionts of the genus frankia, as revealed by comparative genomic and proteomic analyses

Bacteria of the genus Frankia are mycelium-forming actinomycetes that are found as nitrogen-fixing facultative symbionts of actinorhizal plants. Although soil-dwelling actinomycetes are well-known producers of bioactive compounds, the genus Frankia has largely gone uninvestigated for this potential. Bioinformatic analysis of the genome sequences of Frankia strains ACN14a, CcI3, and EAN1pec revealed an unexpected number of secondary metabolic biosynthesis gene clusters. Our analysis led to the identification of at least 65 biosynthetic gene clusters, the vast majority of which appear to be unique and for which products have not been observed or characterized. More than 25 secondary metabolite structures or structure fragments were predicted, and these are expected to include cyclic peptides, siderophores, pigments, signaling molecules, and specialized lipids. Outside the hopanoid gene locus, no cluster could be convincingly demonstrated to be responsible for the few secondary metabolites previously isolated from other Frankia strains. Few clusters were shared among the three species, demonstrating species-specific biosynthetic diversity. Proteomic analysis of Frankia sp. strains CcI3 and EAN1pec showed that significant and diverse secondary metabolic activity was expressed in laboratory cultures. In addition, several prominent signals in the mass range of peptide natural products were observed in Frankia sp. CcI3 by intact-cell matrix-assisted laser desorption-ionization mass spectrometry (MALDI-MS). This work supports the value of bioinformatic investigation in natural products biosynthesis using genomic information and presents a clear roadmap for natural products discovery in the Frankia genus.     

Low genetic diversity among Frankia spp. strains nodulating sympatric populations of actinorhizal species of Rosaceae, Ceanothus (Rhamnaceae) and Datisca glomerata (Datiscaceae) west of the Sierra Nevada (California)

Frankia spp. strains typically induce N2-fixing root nodules on actinorhizal plants. The majority of host plant taxa associated with the uncultured Group 1 Frankia strains, i.e., Ceanothus of the Rhamnaceae, Datisca glomerata (Datiscaceae), and all actinorhizal members of the Rosaceae except Dryas, are found in California. A study was conducted to determine the distribution of Frankia strains among root nodules collected from both sympatric and solitary stands of hosts. Three DNA regions were examined, the 5' end of the 16S rRNA gene, the internal transcribed spacer region between the 16S and 23S rRNA genes, and a portion of the glutamine synthetase gene (glnA). The results suggest that a narrow range of Group 1 Frankia spp. strains dominate in root nodules collected over a large area of California west of the Sierra Nevada crest with no apparent host-specificity. Comparisons with Group 2 Frankia strain diversity from Alnus and Myrica within the study range suggest that the observed low diversity is peculiar to Group 1 Frankia strains only. Factors that may account for the observed lack of genetic variability and host specificity include strain dominance over a large geographical area, current environmental selection, and (or) a past evolutionary bottleneck.     

Non-Frankia actinomycetes isolated from surface-sterilized roots of Casuarina equisetifolia fix nitrogen

Based on partial 16S sequences, we previously described a novel group of nonsymbiotic, acetylene reduction activity-positive actinomycetes which were isolated from surface-sterilized roots of Casuarina equisetifolia growing in Mexico. An amplified rRNA restriction analysis confirmed that these actinomycetes are distinct from Frankia, a finding substantiated by a 16S rRNA gene phylogenetic analysis of two of the Mexican isolates. Further support for these actinomycetes being separate from Frankia comes from the very low DNA-DNA homology that was found. Nevertheless, the Mexican isolates may be diazotrophs based not only on their ability to grow in N-free medium and reduce acetylene to ethylene but also on the results from (15)N isotope dilution analysis and the finding that a nifH gene was PCR amplified. A comparison of the nifH sequences from the various isolates showed that they are closely related to nifH from Frankia; the similarity was 84 to 98% depending on the host specificity group. An analysis of complete 16S rRNA gene sequences demonstrated that the two strains analyzed in detail are most closely related to actinobacteria in the Thermomonosporaceae and the Micromonosporaceae.     

几种非豆科植物根瘤内生菌侵染特征的研究

自不同科、属、种的非豆科植物根瘤分离内生菌,对其寄主植物进行了交叉侵染,结果表明,这些Frankia菌对不同寄主的侵染没有明显的专一性,供试菌可以进行跨越科、属、种的侵染,但有的菌株对于某些植物的侵染,可能存在一些特殊情况,相同菌株对不同植物的侵染能力,以及不同菌株对同一寄主的侵染能力是有差异的。从同一种植物根瘤中分离的不同菌株,侵染能力也有高低之分,供试菌随寄主植物的改变,侵染能力及所建立的共生系统固氮活性有所降低,侵染原寄主植物所形成的根瘤固氮活性较高的菌株,在改变寄主后所形成的根瘤固氮活性也比较高,在一定条件下,寄主植物的结瘤量与根瘤固氮活性呈正相关,而侵染不同寄主后,根瘤中菌体孢子的表面结构也发生了一定变化。     

Molecular cloning, sequencing, and expression of the glutamine synthetase II (glnII) gene from the actinomycete root nodule symbiont Frankia sp. strain CpI1.

In common with other plant symbionts, Frankia spp., the actinomycete N2-fixing symbionts of certain nonleguminous woody plants, synthesize two glutamine synthetases, GSI and GSII. DNA encoding the Bradyrhizobium japonicum gene for GSII (glnII) hybridized to DNA from three Frankia strains. B. japonicum glnII was used as a probe to clone the glnII gene from a size-selected KpnI library of Frankia strain CpI1 DNA. The region corresponding to the Frankia sp. strain CpI1 glnII gene was sequenced, and the amino acid sequence was compared with that of the GS gene from the pea and glnII from B. japonicum. The Frankia glnII gene product has a high degree of similarity with both GSII from B. japonicum and GS from pea, although the sequence was about equally similar to both the bacterial and eucaryotic proteins. The Frankia glnII gene was also capable of complementing an Escherichia coli delta glnA mutant when transcribed from the vector lac promoter, but not when transcribed from the Frankia promoter. GSII produced in E. coli was heat labile, like the enzyme produced in Frankia sp. strain CpI1 but unlike the wild-type E. coli enzyme.     

Comparative genetics and evolutionary morphology of symbiosis formed by plants with nitrogen-fixing microbes and endomycorrhizal fungi

Results of comparative morphological and genetic analyses are described for two major plant-microbe endosymbioses: N2-fixing nodules (with rhizobia or actinomycetes Frankia) and arbuscular mycorrhiza (with Glomales fungi). Development from the primordia formed de novo in root tissues is common for all known types of N2-fixing nodules. However, their structure varies greatly with respect to: (i) tissue topology (location of vascular bundles is peripheral in legumes but central in non-legumes); (ii) position of nodule primordium (inner or outer cortex in legumes, whereas pericycle in non-legumes); (iii) stability of apical meristem (persistent in the indeterminate nodules, transient in the determinate ones). In addition, legumes vary in ability to form compartments harboring endosymbiotic rhizobia that can be located intercellularly (infection threads) and intracellularly (symbiosomes). Using pea (Pisum sativum) symbiotic mutants, the nodule developmental program is dissected into a range of spatially and temporarily differentiated steps composing four sub-programs (development of endosymbiotic compartments; nodule histogenesis; autoregulation of nodulation; bacteroid differentiation). The developmental mutations are suggested in some cases to reverse the endosymbiotic system into the morphologically simpler forms some of which may correspond to the ancestral stages of nodule evolution. Origination of legume-rhizobial and actinorhizal symbioses is suggested to be based on a set of preadaptations many of which had been evolved in angiosperms during coevolution with arbuscular mycorrhizal fungi (e.g. inter- and intracellular maintenance of symbionts, their control via defence-like reactions and recognition of chitin-like molecules). Analysis of parallel morphological variation in symbiotic mutants and wild-growing legume species enables us to reconstruct the major stages of evolution for N2-fixing symbioses. This evolution proceeded to a sufficient degree independently from the basic physiological function of nodules (symbiotic N2-fixation) and possibly a recruiting of plant genes that initially fulfilled various "non-symbiotic" functions into the genetic networks monitoring plant-microbe interactions.     

Frankia alni proteome under nitrogen-fixing and nitrogen-replete conditions

Frankia alni induces root nodules on Alnus , in which the bacterium differentiates into nitrogen (N)-fixing cells called vesicles. In culture, F. alni also undergoes major morphological changes as it alternates between N-replete and N-fixing conditions. Lack of biologically available N induces the synthesis of vesicles in which nitrogenase is protected from molecular oxygen by a thick lipid hopanoid envelope. Very little is known about the molecular basis of Frankia –host interaction as well as Frankia cell differentiation. The recent determination of the complete genome sequence of F. alni strain ACN14a has permitted us to characterize its proteome, particularly in the extracellular compartment, which could be involved in Frankia –host interaction, and in the switch from N-replete to N-fixing conditions. To that end, 126 bacterial proteins were analyzed by two-dimensional protein gel electrophoresis and identified by matrix-assisted laser desorption/ionization time of flight fingerprinting using a F. alni proteome database. Interestingly, the extracellular fraction contains some glycolytic enzymes lacking secretion signals, already reported to be extracellularly localized in some streptococci, as well as some abundant stress-resistance proteins. As expected, several proteins involved in N assimilation and oxidative defense system were upregulated in F. alni grown under N-fixing vs N-replete conditions. Furthermore, two Raf kinase inhibitor protein homologs that could play a role in cellular signaling, and a hemoglobin-like protein HbN that could be involved in detoxification of nitric oxide were also upregulated. More surprising, a succinate dehydrogenase was strongly downregulated, which could be linked to the need of pyruvate for the biosynthesis of hopanoids or to reduced oxygen diffusion in vesicles.     

Effects of symbiosis with Frankia and arbuscular mycorrhizal fungus on the natural abundance of 15N in four species of Casuarina

The effect of interactions between Casuarina species, Frankia strains and AMF on nitrogen isotope fractionation within the plant were determined under conditions where changes in source nitrogen were minimized by growing plants in mineral nitrogen-deficient conditions and without added organic N. Casuarina cunninghamiana, C. equisetifolia, C. glauca, and C. junghuniana were inoculated singly with three Frankia strains or were dual inoculated with Frankia and Glomus fasciculatum. The %N and delta 15N of separated parts of plants inoculated with the three Frankia strains or with Frankia + Glomus were not significantly different within Casuarina species. However, the slow-growing C. junghuniana differed in several variables from the other three species. There was a highly significant, linear relationship between the natural logarithms of cladode N content and delta 15N of plants of the four Casuarina species when inoculated with Frankia or with Frankia + Glomus, showing that nitrogen supply and the correlated variable, plant growth rate, were major determinants of delta 15N. Provision of small quantities of (NH4)2SO4 or KNO3 increased several-fold the growth of three of the Casuarina species when inoculated with Frankia alone or with Frankia + Glomus. Within species, mycorrhizal and non-mycorrhizal plants receiving supplementary soluble phosphate were of similar dry weights at harvest. delta 15N values for cladodes of C. cunninghamiana, C. equisetifolia and C. glauca were similar, but values for the poor growing C. junghuniana were more variable and, with the exception of plants receiving KNO3, were lower than those of the other three species. Reduced growth due to suboptimal availability of N or P had a major influence on delta 15N and, in these conditions where plants could not access significant amounts of organic N, outweighed any effects on cladode delta 15N of colonization by Glomus. delta 15N values of nodules were higher than other parts of Frankia or Frankia + Glomus inoculated Casuarinas, conceivably due to retention in nodules of fixed N, with delta 15N close to zero.     

Co-evolution between Frankia populations and host plants in the family Casuarinaceae and consequent patterns of global dispersal

Symbioses between the root nodule-forming, nitrogen-fixing actinomycete Frankia and its angiospermous host plants are important in the nitrogen economies of numerous terrestrial ecosystems. Molecular characterization of Frankia strains using polymerase chain reaction/restriction fragment length polymorphism (PCR/RFLP) analyses of the 16S rRNA-ITS gene and of the nifD-nifK spacer was conducted directly on root nodules collected worldwide from Casuarina and Allocasuarina trees. In their native habitats in Australia, host species contained seven distinctive sets of Frankia in seven different molecular phylogenetic groups. Where Casuarina and Allocasuarina trees are newly planted outside Australia, they do not normally nodulate unless Frankia is introduced with the host seedling. Nodules from Casuarina trees introduced outside Australia over the last two centuries were found to contain Frankia from only one of the seven phylogenetic groups associated with the host genus Casuarina in Australia. The phylogenetic group of Frankia found in Casuarina and Allocasuarina trees introduced outside Australia is the only group that has yielded isolates in pure culture, suggesting a greater ability to survive independently of a host. Furthermore, the Frankia species in this group are able to nodulate a wider range of host species than those in the other six groups. In baiting studies, Casuarina spp. are compatible with more Frankia microsymbiont groups than Allocasuarina host spp. adapted to drier soil conditions, and C. equisetifolia has broader microsymbiont compatibility than other Casuarina spp. Some Frankia associated with the nodular rhizosphere and rhizoplan, but not with the nodular tissue, of Australian hosts were able to nodulate cosmopolitan Myrica plants that have broad microsymbiont compatibility and, hence, are a potential host of Casuarinaceae-infective Frankia outside the hosts' native range. The results are consistent with the idea that Frankia symbiotic promiscuity and ease of isolation on organic substrates, suggesting saprophytic potential, are associated with increased microsymbiont ability to disperse and adapt to diverse new environments, and that both genetics and environment determine a host's nodular microsymbiont.     

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.     

Identification of atypical Frankia strains by fatty acid analysis

Abstract: Ineffective, non-infective actinomycetous isolates obtained from actinorhizal nodules of Coriaria nepalensis and Datisca cannabina were identified as Frankia using whole cell fatty acid analysis. The isolates exhibited fatty-acid patterns very similar to those of confirmed Frankia strains from other host plants ( Alnus, Casuarina, Colletia, Comptonia, Elaeagnus and Hippophae ). All Frankia strains, including Coriaria and Datisca isolates, showed fatty-acid profiles very distinct from those of other actinomycetes used as controls ( Actinomyces, Geodermatophilus, Nocardia, Mycobacterium and Streptomyces ). For the genus Frankia , a characteristic pattern of five fatty acids (15:0; 15:1; 16:0 iso; 17:0 and 17:1) was found. These fatty acids comprised 75% or more of the total content. All Frankia strains could be placed into three subgroups. Coriaria isolates were found in the largest subgroup which contained most Frankia strains from other hosts while ineffective strains from Alnus, Elaeagnus and Datisca were distributed in all three subgroups of Frankia .     

Developmental genetics and evolution of symbiotic structures in nitrogen-fixing nodules and arbuscular mycorrhiza.

Genetic and molecular mechanisms of development are compared for two major plant-microbe endosymbioses: N(2)-fixing nodules (with rhizobia or actinomycetes Frankia) and arbuscular mycorrhiza (with Glomales fungi). Development from the primordia formed de novo in root tissues is common for all known types of N(2)-fixing nodules. However, their structure varies greatly with respect to: (i) tissue topology (location of vascular bundles is peripherical in legumes or central in non-legumes); (ii) position of nodule primordium (inner or outer cortex in legumes, pericycle in non-legumes); (iii) stability of apical meristem (persistent in the indeterminate nodules, transient in the determinate ones). In addition, legumes vary in ability to form compartments harboring endosymbiotic rhizobia and located intercellularly (infection threads) and intracellularly (symbiosomes). Using pea (Pisum sativum) symbiotic mutants, the nodule developmental program is dissected into a range of spatially and temporarily differentiated steps comprising four sub-programs (development of endosymbiotic compartments; nodule histogenesis; autoregulation of nodulation; bacteroid differentiation). The developmental mutations are suggested in some cases to reverse the endosymbiotic system into the morphologically simpler forms some of which may correspond to the ancestral stages of nodule evolution. The origin of legume-rhizobial and actinorhizal symbioses is suggested to be based on a set of preadaptations many of which had been evolved in angiosperms during coevolution with arbuscular mycorrhizal fungi (e.g., inter- and intracellular maintenance of symbionts, their control via defence-like reactions and recognition of chitin-like molecules). An analysis of parallel morphological variation in symbiotic mutants and wild-growing legume species enables us to reconstruct the major stages of evolution for N(2)-fixing symbioses.     

Gas vesicles in actinomycetes: old buoys in novel habitats?

Gas vesicles are gas-filled prokaryotic organelles that function as flotation devices. This enables planktonic cyanobacteria and halophilic archaea to position themselves within the water column to make optimal use of light and nutrients. Few terrestrial microbes are known to contain gas vesicles. Genome sequences that have become available recently for many bacteria from non-planktonic habitats reveal gas vesicle gene clusters in members of the actinomycete genera Streptomyces, Frankia and Rhodococcus, which typically live in soils and sediments. Remarkably, there is an additional level of complexity in cluster number and gene content. Here, we discuss whether putative gas vesicle proteins in these actinomycetes might actually be involved in flotation or whether they might fulfil other cellular functions.     

Laser-based micromanipulation for separation and identification of individual Frankia vesicles

In studies of symbiotic efficiency it is of great importance to identify and separate individual Frankia strains from a nodule. Therefore, a new laser-based micromanipulation technique has been developed in which individual vesicles from root nodules of two Frankia-Alnus symbioses have been successfully cut loose and separated from clusters of vesicles in sterile conditions under light microscopy using a laser scalpel and optical tweezers. Vesicles from the Alnus incana-Frankia AvCI1 symbiosis were successfully isolated and grown in culture using this technique. The DNA from both Frankia sources was amplified by polymerase chain reaction (PCR). The work shows that a combination of laser-based manipulation techniques and PCR can be used for the separation and study of individual vesicles. This novel laser-based micromanipulation technique opens up various new possibilities, for instance, to study whether several Frankia strains can grow simultaneously in the same root nodule.