Soil bacteria that also form mutualistic symbioses in plants encounter two major levels of select... more Soil bacteria that also form mutualistic symbioses in plants encounter two major levels of selection. One occurs during adaptation to and survival in soil, and the other occurs in concert with host plant speciation and adaptation. Actinobacteria from the genus Frankia are facultative symbionts that form N2-fixing root nodules on diverse and globally distributed angiosperms in the “actinorhizal” symbioses. Three closely related clades of Frankia sp. strains are recognized; members of each clade infect a subset of plants from among eight angiosperm families. We sequenced the genomes from three strains; their sizes varied from 5.43 Mbp for a narrow host range strain (Frankia sp. strain HFPCcI3) to 7.50 Mbp for a medium host range strain (Frankia alni strain ACN14a) to 9.04 Mbp for a broad host range strain (Frankia sp. strain EAN1pec.) This size divergence is the largest yet reported for such closely related soil bacteria (97.8%–98.9% identity of 16S rRNA genes). The extent of gene del...
The localization and expression of the hydrogenase in free-living Frankia KB5 was investigated im... more The localization and expression of the hydrogenase in free-living Frankia KB5 was investigated immunologically and by monitoring activity, focusing on its relationships with nitrogenase and H2. Immunological studies revealed that the large subunit of the hydrogenase in Frankia KB5 was modified post-translationally, and transferred into the membrane after processing. The large subunit was constitutively expressed and no correlation was found between hydrogenase activity and synthesis. Although H2 was not needed for induction of hydrogenase synthesis, exogenously added H2 triggered hydrogen uptake in medium containing nitrogen, i.e., in the hyphae. A correlation between nitrogenase activity and hydrogen uptake was found in cultures grown in media without nitrogen, but interestingly the two enzymes showed no co-regulation.
DNase activities in different protein fractions of Frankia strain R43 were studied. The extracell... more DNase activities in different protein fractions of Frankia strain R43 were studied. The extracellular and the cell wall-associated fractions revealed the presence of exo- and endonucleolytic enzymes, but none was detected in the cytoplasmic fraction. The strongest DNase hydrolysis was found in the extracellular fraction, in which six DNases were detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis.
Moench were inoculated and grown without combined nitrogen for seven weeks. The effects of ammoni... more Moench were inoculated and grown without combined nitrogen for seven weeks. The effects of ammonium on the function and structure of the root nodules were studied by adding 20 mM NH4C1 (20 mM KC1 = control) for four days. Nitrogenase activity decreased to ca. 50% after one day and to less than 10% after two days in ammonium treated plants, but was unaffected in control plants. The results were similar at photon flux densities of 200 and 50 gmol m -2 s-1. At the higher light level the effect was concentration dependent between 2 and 20 mM NH4C1. The recovery was slow, and more than 11 d were needed for plants treated with 20 mM ammonium to reach initial activity. The distribution of 14C to the root nodules after assimilation of 14CO2 by the plants was not changed by the ammonium treatment. Microscopical studies of root nodules showed high frequencies of endophyte vesicles being visually damaged in nodules from ammonium-treated plants, but not in nodules from control plants. When nitrogenase activity was restored, visually damaged vesicles were again few, whereas young developing vesicles were numerous. The slow recovery, the 14C-translocation pattern, and the structural changes of the endophyte indicate a more complex mechanism of ammonium influence than simply a short-term reduction in supply of carbon compounds to the nodules.
ABSTRACT A monofunctional catalase and a bifunctional catalase-peroxidase were revealed by activi... more ABSTRACT A monofunctional catalase and a bifunctional catalase-peroxidase were revealed by activity staining of non-denaturing PAGE in Frankia strain R43. Both enzymes were shown to be cytoplasmatic, growth regulated and expressed mainly during the stationary growth phase. Nevertheless, low levels of constitutive expression could also be detected during the early stages of growth. Immunoblot analyses using a polyclonal antibody raised against a catalase-peroxidase purified from Streptomyces reticuli showed a band of 83.2 kDa, with the same growth dependent pattern as obtained by the non-denaturing PAGE analyses. Induction studies revealed that both enzymes were strongly induced by raising the intracellular concentration of H2O2 with paraquat, but not with exogenous H2O2. In addition, no acquisition of tolerance to exogenous H2O2 was observed whatever the pretreatment of the inocula, i.e. despite the expression level of both hydroperoxidases.
Enzymes involved in hydrogen metabolism were immunologically investigated for occurrence and loca... more Enzymes involved in hydrogen metabolism were immunologically investigated for occurrence and localization. Western immunoblots on free-living and symbiotic Frankia KB revealed that a 36 kDa protein found both in symbiotic and free-living Frunkia was immunologically related to the Fe-protein of nitrogenase purified from Rhodospiriilum rubrum. Also, a 63 kDa protein immunologically related to the large subunit of a dimeric hydrogenase from Akaligenes lam was found in free-living Frankia KB. Reversible hydrogenase could not be detected.
A single clone of Alnus incana (L.) Moench was grown in a controlled-environment chamber. The pla... more A single clone of Alnus incana (L.) Moench was grown in a controlled-environment chamber. The plants were either inoculated with Frankia and fixed atmospheric nitrogen or were left uninoculated but received ammonium at the same rate as the first group fixed their nitrogen. Nitrogen fixation was calculated from frequent measurements of acetylene reduction and hydrogen evolution. The diurnal variation of acetylene reduction was also taken into account. The relative efficiency of nitrogenase could be used in the calculations of fixed nitrogen since the Frankia used did not show any detectable hydrogenase activity. Alders fixing nitrogen developed more biomass, longer shoots, larger leaf areas and contained more nitrogen than alders receiving ammonium. In one experiment, almost all ammonium given to the non-nodulated alders was taken up and 15% of the nitrogen taken up was excreted. In the other experiment, 34% of the ammonium was left in the nutrient solution and 8% of the nitrogen taken up was excreted. Alders inoculated with Frankia did not excrete any detectable amount of nitrogen. It seems that the energy demand for nitrogen fixation is not so high that biomass production in alders is retarded. The symbiotic system of A. incana and Frankia seems to be more efficient in utilizing its nitrogen than non-symbiotic A. incana receiving ammonium.
Acetylene reduction, 15N 2 reduction and H2 evolution were measured in root systems of intact pla... more Acetylene reduction, 15N 2 reduction and H2 evolution were measured in root systems of intact plants of grey alder (Alnus ineana (L.) Moench) in symbiosis with Frankia. The ratios of C2H2 : 15N 2 were compared with CzHz:N 2 ratios calculated from C2H2 reduction and H 2 evolution, and with C2H2: N2 ratios calculated from accumulated C2H4 production and nitrogen content. It was possible to calculate C2H2:N2 ratios from C2H2 reduction and H 2 evolution because this source of Frankia did not show any hydrogenase activity. The ratios obtained using the different methods ranged from 2.72 to 4.42, but these values were not significantly different. It was also shown that enriched tSN could be detected in the shoot after a 1-h incubation of the root-system. It is concluded that the measurement of H2 evolution in combination with C2H2 reduction represents a nondestructive assay for nitrogen fixation in a Frankia symbiosis which shows no detectable hydrogenase activity.
Acetylene reduction assays were shown to inactivate uptake hydrogenase activity to different exte... more Acetylene reduction assays were shown to inactivate uptake hydrogenase activity to different extents in one Casuarina and two Alnus symbioses. Inactivation was found to be caused by C2H2 and not by C2H4. Acetylene completely inactivated the hydrogenase activity of intact root systems of Alnus incana inoculated with Frankia strain Avcll in 90 minutes, as shown by a drop in the relative efficiency of nitrogenase from 1.0 to 0.73. The hydrogenase of Frankia preparations (containing vesicles) and of cell-free extracts (not containing vesicles) from the same symbiosis was much more susceptible to acetylene inactivation. Cellfree extracts lost all hydrogenase activity after 5 minutes of exposure to acetylene. The hydrogenase activity of intact root systems of Casuarina obesa was less sensitive to acetylene than that of root systems of A. incana, since the relative efficiency of nitrogenase changed only from 1.0 to 0.95 over 90 minutes. Frankia preparations and cell-free extracts of C. obesa still retained hydrogenase activity after a 10 minute-exposure to acetylene.
Symbiotic and free-living Frankia were investigated for correlation between hydrogenase activitie... more 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 809 www.plant.org on March 1, 2016 -Published by www.plantphysiol.org Downloaded from
The effect of different carbon sources on the growth of Frankia isolates for Casuarina sp. was st... more The effect of different carbon sources on the growth of Frankia isolates for Casuarina sp. was studied.
Nitrogenase activity, hydrogen evolution, biomass production and nodulation were studied in three... more Nitrogenase activity, hydrogen evolution, biomass production and nodulation were studied in three Casuarina species, C. equisetifolia Forst., C. glauca Sieber ex Spreng and C. obesa Miq., either inoculated with a crushed nodule inoculum prepared from C. glauca nodules or inoculated with the pure culture HFP Cci3. Nodulation was also studied in C. cr&tata Miq. inoculated with the above mentioned Frankia sources.
... Microbiol. 85: 193-201. - 1987. Relative efficiency of nitrogen fixation and the occur-rence ... more ... Microbiol. 85: 193-201. - 1987. Relative efficiency of nitrogen fixation and the occur-rence ofhydrogenase in pea root nodules. - Plant Soil. 100: 149-156. - , Blunden, EAG & Searle, JW 1981. Intercellular space and hydrogen diffusion in pea lupin root nodules. - Plant Sci. Lett. ...
The plant responses to infection by pathogenic bacteria have been extensively reviewed in recent ... more The plant responses to infection by pathogenic bacteria have been extensively reviewed in recent years, including the spatial and temporal production of reactive oxygen species (ROS). The immediate and localized release of ROS upon infection, known as the oxidative burst, was shown not only to be part of the hypersensitive response but also likely responsible for mediating, directly or via signal transduction pathways, other plant defence strategies. This paradigm inspired studies in nitrogen-fixing root nodule symbioses, and a parallelism is unavoidable. In rhizobia-legume symbioses, histochemical data revealed the presence of ROS in the host infection threads and in the root nodules primordia. On the other hand, in actinorhizal infections, it has been shown that Alnus glutinosa root exudates induce several oxidative stress response-related proteins in compatible Frankia. These data suggest that the nitrogen-fixing microsymbionts must have had to evolve adaptations to overcome and possibly regulate an unfriendly environment. In this review, particular emphasis will be given to the bacteria antioxidant mechanisms at different developmental stages of the nitrogen-fixing root nodule symbioses.
Soil bacteria that also form mutualistic symbioses in plants encounter two major levels of select... more Soil bacteria that also form mutualistic symbioses in plants encounter two major levels of selection. One occurs during adaptation to and survival in soil, and the other occurs in concert with host plant speciation and adaptation. Actinobacteria from the genus Frankia are facultative symbionts that form N2-fixing root nodules on diverse and globally distributed angiosperms in the “actinorhizal” symbioses. Three closely related clades of Frankia sp. strains are recognized; members of each clade infect a subset of plants from among eight angiosperm families. We sequenced the genomes from three strains; their sizes varied from 5.43 Mbp for a narrow host range strain (Frankia sp. strain HFPCcI3) to 7.50 Mbp for a medium host range strain (Frankia alni strain ACN14a) to 9.04 Mbp for a broad host range strain (Frankia sp. strain EAN1pec.) This size divergence is the largest yet reported for such closely related soil bacteria (97.8%–98.9% identity of 16S rRNA genes). The extent of gene del...
The localization and expression of the hydrogenase in free-living Frankia KB5 was investigated im... more The localization and expression of the hydrogenase in free-living Frankia KB5 was investigated immunologically and by monitoring activity, focusing on its relationships with nitrogenase and H2. Immunological studies revealed that the large subunit of the hydrogenase in Frankia KB5 was modified post-translationally, and transferred into the membrane after processing. The large subunit was constitutively expressed and no correlation was found between hydrogenase activity and synthesis. Although H2 was not needed for induction of hydrogenase synthesis, exogenously added H2 triggered hydrogen uptake in medium containing nitrogen, i.e., in the hyphae. A correlation between nitrogenase activity and hydrogen uptake was found in cultures grown in media without nitrogen, but interestingly the two enzymes showed no co-regulation.
DNase activities in different protein fractions of Frankia strain R43 were studied. The extracell... more DNase activities in different protein fractions of Frankia strain R43 were studied. The extracellular and the cell wall-associated fractions revealed the presence of exo- and endonucleolytic enzymes, but none was detected in the cytoplasmic fraction. The strongest DNase hydrolysis was found in the extracellular fraction, in which six DNases were detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis.
Moench were inoculated and grown without combined nitrogen for seven weeks. The effects of ammoni... more Moench were inoculated and grown without combined nitrogen for seven weeks. The effects of ammonium on the function and structure of the root nodules were studied by adding 20 mM NH4C1 (20 mM KC1 = control) for four days. Nitrogenase activity decreased to ca. 50% after one day and to less than 10% after two days in ammonium treated plants, but was unaffected in control plants. The results were similar at photon flux densities of 200 and 50 gmol m -2 s-1. At the higher light level the effect was concentration dependent between 2 and 20 mM NH4C1. The recovery was slow, and more than 11 d were needed for plants treated with 20 mM ammonium to reach initial activity. The distribution of 14C to the root nodules after assimilation of 14CO2 by the plants was not changed by the ammonium treatment. Microscopical studies of root nodules showed high frequencies of endophyte vesicles being visually damaged in nodules from ammonium-treated plants, but not in nodules from control plants. When nitrogenase activity was restored, visually damaged vesicles were again few, whereas young developing vesicles were numerous. The slow recovery, the 14C-translocation pattern, and the structural changes of the endophyte indicate a more complex mechanism of ammonium influence than simply a short-term reduction in supply of carbon compounds to the nodules.
ABSTRACT A monofunctional catalase and a bifunctional catalase-peroxidase were revealed by activi... more ABSTRACT A monofunctional catalase and a bifunctional catalase-peroxidase were revealed by activity staining of non-denaturing PAGE in Frankia strain R43. Both enzymes were shown to be cytoplasmatic, growth regulated and expressed mainly during the stationary growth phase. Nevertheless, low levels of constitutive expression could also be detected during the early stages of growth. Immunoblot analyses using a polyclonal antibody raised against a catalase-peroxidase purified from Streptomyces reticuli showed a band of 83.2 kDa, with the same growth dependent pattern as obtained by the non-denaturing PAGE analyses. Induction studies revealed that both enzymes were strongly induced by raising the intracellular concentration of H2O2 with paraquat, but not with exogenous H2O2. In addition, no acquisition of tolerance to exogenous H2O2 was observed whatever the pretreatment of the inocula, i.e. despite the expression level of both hydroperoxidases.
Enzymes involved in hydrogen metabolism were immunologically investigated for occurrence and loca... more Enzymes involved in hydrogen metabolism were immunologically investigated for occurrence and localization. Western immunoblots on free-living and symbiotic Frankia KB revealed that a 36 kDa protein found both in symbiotic and free-living Frunkia was immunologically related to the Fe-protein of nitrogenase purified from Rhodospiriilum rubrum. Also, a 63 kDa protein immunologically related to the large subunit of a dimeric hydrogenase from Akaligenes lam was found in free-living Frankia KB. Reversible hydrogenase could not be detected.
A single clone of Alnus incana (L.) Moench was grown in a controlled-environment chamber. The pla... more A single clone of Alnus incana (L.) Moench was grown in a controlled-environment chamber. The plants were either inoculated with Frankia and fixed atmospheric nitrogen or were left uninoculated but received ammonium at the same rate as the first group fixed their nitrogen. Nitrogen fixation was calculated from frequent measurements of acetylene reduction and hydrogen evolution. The diurnal variation of acetylene reduction was also taken into account. The relative efficiency of nitrogenase could be used in the calculations of fixed nitrogen since the Frankia used did not show any detectable hydrogenase activity. Alders fixing nitrogen developed more biomass, longer shoots, larger leaf areas and contained more nitrogen than alders receiving ammonium. In one experiment, almost all ammonium given to the non-nodulated alders was taken up and 15% of the nitrogen taken up was excreted. In the other experiment, 34% of the ammonium was left in the nutrient solution and 8% of the nitrogen taken up was excreted. Alders inoculated with Frankia did not excrete any detectable amount of nitrogen. It seems that the energy demand for nitrogen fixation is not so high that biomass production in alders is retarded. The symbiotic system of A. incana and Frankia seems to be more efficient in utilizing its nitrogen than non-symbiotic A. incana receiving ammonium.
Acetylene reduction, 15N 2 reduction and H2 evolution were measured in root systems of intact pla... more Acetylene reduction, 15N 2 reduction and H2 evolution were measured in root systems of intact plants of grey alder (Alnus ineana (L.) Moench) in symbiosis with Frankia. The ratios of C2H2 : 15N 2 were compared with CzHz:N 2 ratios calculated from C2H2 reduction and H 2 evolution, and with C2H2: N2 ratios calculated from accumulated C2H4 production and nitrogen content. It was possible to calculate C2H2:N2 ratios from C2H2 reduction and H 2 evolution because this source of Frankia did not show any hydrogenase activity. The ratios obtained using the different methods ranged from 2.72 to 4.42, but these values were not significantly different. It was also shown that enriched tSN could be detected in the shoot after a 1-h incubation of the root-system. It is concluded that the measurement of H2 evolution in combination with C2H2 reduction represents a nondestructive assay for nitrogen fixation in a Frankia symbiosis which shows no detectable hydrogenase activity.
Acetylene reduction assays were shown to inactivate uptake hydrogenase activity to different exte... more Acetylene reduction assays were shown to inactivate uptake hydrogenase activity to different extents in one Casuarina and two Alnus symbioses. Inactivation was found to be caused by C2H2 and not by C2H4. Acetylene completely inactivated the hydrogenase activity of intact root systems of Alnus incana inoculated with Frankia strain Avcll in 90 minutes, as shown by a drop in the relative efficiency of nitrogenase from 1.0 to 0.73. The hydrogenase of Frankia preparations (containing vesicles) and of cell-free extracts (not containing vesicles) from the same symbiosis was much more susceptible to acetylene inactivation. Cellfree extracts lost all hydrogenase activity after 5 minutes of exposure to acetylene. The hydrogenase activity of intact root systems of Casuarina obesa was less sensitive to acetylene than that of root systems of A. incana, since the relative efficiency of nitrogenase changed only from 1.0 to 0.95 over 90 minutes. Frankia preparations and cell-free extracts of C. obesa still retained hydrogenase activity after a 10 minute-exposure to acetylene.
Symbiotic and free-living Frankia were investigated for correlation between hydrogenase activitie... more 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 809 www.plant.org on March 1, 2016 -Published by www.plantphysiol.org Downloaded from
The effect of different carbon sources on the growth of Frankia isolates for Casuarina sp. was st... more The effect of different carbon sources on the growth of Frankia isolates for Casuarina sp. was studied.
Nitrogenase activity, hydrogen evolution, biomass production and nodulation were studied in three... more Nitrogenase activity, hydrogen evolution, biomass production and nodulation were studied in three Casuarina species, C. equisetifolia Forst., C. glauca Sieber ex Spreng and C. obesa Miq., either inoculated with a crushed nodule inoculum prepared from C. glauca nodules or inoculated with the pure culture HFP Cci3. Nodulation was also studied in C. cr&tata Miq. inoculated with the above mentioned Frankia sources.
... Microbiol. 85: 193-201. - 1987. Relative efficiency of nitrogen fixation and the occur-rence ... more ... Microbiol. 85: 193-201. - 1987. Relative efficiency of nitrogen fixation and the occur-rence ofhydrogenase in pea root nodules. - Plant Soil. 100: 149-156. - , Blunden, EAG & Searle, JW 1981. Intercellular space and hydrogen diffusion in pea lupin root nodules. - Plant Sci. Lett. ...
The plant responses to infection by pathogenic bacteria have been extensively reviewed in recent ... more The plant responses to infection by pathogenic bacteria have been extensively reviewed in recent years, including the spatial and temporal production of reactive oxygen species (ROS). The immediate and localized release of ROS upon infection, known as the oxidative burst, was shown not only to be part of the hypersensitive response but also likely responsible for mediating, directly or via signal transduction pathways, other plant defence strategies. This paradigm inspired studies in nitrogen-fixing root nodule symbioses, and a parallelism is unavoidable. In rhizobia-legume symbioses, histochemical data revealed the presence of ROS in the host infection threads and in the root nodules primordia. On the other hand, in actinorhizal infections, it has been shown that Alnus glutinosa root exudates induce several oxidative stress response-related proteins in compatible Frankia. These data suggest that the nitrogen-fixing microsymbionts must have had to evolve adaptations to overcome and possibly regulate an unfriendly environment. In this review, particular emphasis will be given to the bacteria antioxidant mechanisms at different developmental stages of the nitrogen-fixing root nodule symbioses.
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