The calcium requirement for functional vesicle development and nitrogen fixation by Frankia strains EAN1pec and CpI1

A calcium requirement was shown for both vesicle development and nitrogenase activity by Frankia strains EAN1_pec and CpI1. Washing cells with EGTA or EDTA inhibited both vesicle development and nitrogenase activity. The inhibition of both was reversed by the addition of calcium. A variety of agents known to affect calcium-dependent biological processes, such as a Ca-ATPase inhibitor, Ca-channel blockers, Ca-ionophores, calmodulin antagonists and the local anaesthetics, tetracaine and dibucaine, inhibited nitrogenase activity. Respiratory studies showed that a CN-insensitive respiration process occurred only under nitrogen derepressing conditions. Respiration by NH₄Cl-grown cells was completely inhibited by KCN while N₂-grown cells were inhibited by only 70%. Removal of calcium ions by EGTA or by the addition of dibucaine or tetracaine blocked the CN-insensitive respiration. This CN-insensitive respiration may be involved in protecting nitrogenase inside the vesicles from oxygen.Abbreviations EDTA ethylenediaminetetraacetic acid - EGTA ethyleneglycol-bis-( amino-ethyl ether) N,N¹-tetraacetic acid - GI germination inhibitor - MOPS 3-[N-morpholino] propane sulfonic acid - PCMBS p-chloromercuribenzene sulphonate - TMB 8,8-(diethylamino)-octyl-3,4,5-trimethoxybenzoate     

Utilization of amino acids by Frankia sp. strain CpI1

The utilization of some amino acids, added at 1 mM and 10 mM concentrations, as the sole combined nitrogen sources by Frankia sp. strain CpI1, has been investigated. Glutamine, like NH ₄ ⁺ , provided rapid growth without N₂ fixation. Histidine at 1 mM yielded poor N₂-fixing activity but better cell growth than N₂. Aspartate, glutamate, alanine, proline, each at 1 mM concentration, supported similar levels of N₂ fixation and growth. Growth on 10 mM glutamate, proline, or histidine resulted in poor N₂-fixing activity and poor cell growth. Cells grown on 10 mM alanine had about half the N₂-fixing activity of cells grown on N₂ but growth was good. Aspartate at 10 mM concentration, however, stimulated N₂-fixing activity dramatically and promoted faster growth. Enzyme analysis suggested that asparate is catabolized by glutamate-oxaloacetate transaminase (GOT), since GOT specific activity was induced, and aspartase activity was not detected, in cells grown on aspartate as the sole combined nitrogen source. Thinlayer chromatography (TLC) of metabolites extracted from N₂-grown cells fed with [¹⁴C]-aspartate showed that label was rapidly accumulated mainly on aspartate and/or glutamate, depending on the cells' physiological state, without detectable labeling on fumarate or oxaloacetate (OAA). These findings provide evidence that aspartate is catabolized by GOT to OAA which, in turn, is rapidly converted to -ketoglutarate through the TCA cycle and then to glutamate by GOT or by glutamate synthase (GOGAT). The stimulation of N₂ fixation and growth by aspartate is probably caused by an increased intracellular glutamate pool.     

Growth,acetylene reduction activity and localization of nitrogenase in relation to vesicle formation in Frankia strains Cc1.17 and Cp1.2

A comparative study was conducted on the effect of NH₄Cl on growth, vesicle formation and formation of nitrogenase of Frankia strains Cc1.17 and Cp1.2, derived from root nodules of Colletia cruciata and Comptonia peregrina, respectively. On a medium without combined nitrogen (P-N), both strains formed spherical cells, called vesicles, like many other Frankia strains. Data are presented on the number of vesicles per mg protein, after cultivation in media with sodium propionate as C-source without combined nitrogen (P-N) or with 0.2 g NH₄Cl/l (P+N). Strain Cp1.2 as may other Frankia strains, showed on P+N medium a very strong reduction of vesicle formation of 99% relative to the number of vesicles formed on P-N medium, after 11 days growth. However, in strain Cc11.17 this reduction was only 70%. The occurence of relatively large numbers of vesicles in P+N media has not yet been reported for other Frankia strains. No acetylene reduction activity was found in NH ₄ ⁺ -grown cells. The regulation of induction of nitrogenase in Frankia by NH₄Cl was tested by immuno-gelectrophoresis using antisera against nitrogenase of Rhizobium leguminosarum PRE. The component I of the enzyme showed crossreactivity while the component II had only a weak crossreaction. The experiments indicated that no nitrogenase was detectable in the NH ₄ ⁺ -grown cells. For the localization of nitrogenase, relative amounts of the enzyme were compared in whole cells and vesicle-enriched fractions. Western blots showed a significant enrichment of nitrogenase in the vesicle fractions, which indicated that most of the nitrogenase was localized in the vesicle.     

Isolation and nitrogenase activity of vesicles from Frankia sp. strain EAN1pec.

Vesicles, specialized cell structures thought to be the site of nitrogen fixation in the actinorhizal bacteria, were isolated from Frankia sp. strain EAN1pec by using French pressure disruption of mycelia followed by differential and isopycnic gradient centrifugation. The isolated vesicles reduced acetylene when incubated anaerobically with Mg2+ ions, ATP, and dithionite. No nitrogenase activity was detected in the disrupted mycelial fractions. Vesicles permeabilized by freeze-thaw or detergents showed increased rates of acetylene reduction due to increased permeability of dithionite. The effect on nitrogenase activity of different ATP concentrations was the same in normal and permeabilized vesicles. The endogenous respiratory rate of vesicles was significantly lower than that of mycelia, and the respiration rate of vesicles did not increase following the addition of succinate. The low respiratory activity of vesicles and their apparent dependence on externally supplied ATP for acetylene reduction suggest that the energy and reducing power for nitrogen fixation may be supplied from the mycelia to which they are attached.     

Lipid composition and nitrogenase activity of symbioticFrankia (Alnus incana) in response to different oxygen concentrations

Summary The role ofFrankia vesicle envelope lipids in regulating oxygen diffusion of symbiotic nitrogen fixation inAlnus incana was examined. Total lipids of symbioticFrankia (vesicle clusters) that had been adapted to oxygen tensions of 5,21, or 40 kPa were analyzed with a normal phase HPLC system. During the oxygen treatment, nitrogenase activity was measured as hydrogen evolution in an open flow-through system. When plants were transferred to low oxygen (5 kPa) or high oxygen (40 kPa), nitrogenase activity dropped initially. Activity recovered in both treatments with a rate comparable to the controls (21 kPa O₂). Both lipid content and lipid composition of vesicle clusters were affected by the oxygen treatments. With increasing oxygen tension, the vesicle cluster lipid content increased. This correlated with structural data (fluorescence microscopy and TEM) which showed a thicker vesicle envelope at higher oxygen tension. Three hopanoid lipids, bacteriohopanetetrol (bht) and two isomers of phenylacetyl monoester of bht, made up approximately 80% of the vesicle cluster lipids. With changing oxygen concentrations, the ratio of the two bht esters changed whereas the relative proportion of bht remained fairly constant. Therefore, in theFrankia-Alnus incana symbiosis, adaptation to different ambient oxygen tensions occurs at least partly by increasing the thickness of theFrankia vesicle envelope and by changing its lipid composition.Abbreviations dw dry weight - bht bacteriohopanetetrol - SE standard error - TEM transmission electron microscopy Dedicated to the memory of Professor John G. Torrey     

Effects of oxygen and chloramphenicol on Frankia nitrogenase activity

The kinetics of asymbiotic nitrogenase activity in three strains of the actinomycete Frankia were studied. Decay rates for enzyme activity were determined by adding chloramphenicol to active acetylene-reducing cells and measuring the time required for all activity to cease. Synthesis rates were measured by bubbling oxygen through actively-reducing cells (which totally destroyed all activity) and then measuring the time required for activity to return to normal. Decay rates (t _1/2) for these three strains were approximately 30 to 40 min. Synthesis rates were slower and initial nitrogenase activities were recorded about 110 min (DDB 011610) or 210 min (DDB 020210 and WgCc1.17) after return to air-equilibrated cultures. Frankia strain WgCc1.17 showed a greater sensitivity to oxygen and nitrogenase activity was totally lost when cells were bubbled only with atmospheric concentrations of oxygen. The results presented here indicate that nitrogenase activity turnover time is relatively rapid, on the order of minutes rather than hours or days. However, regulation of nitrogenase activity will differ from one strain to another and asmmbiotic characterization will be useful for understanding nitrogenase regulation in the bacterial-plant symbiosis.Contribution no. 879 from the Battelle-Kettering Laboratory     

Physiological characteristics of glutamine synthetases I and II of Frankia sp. strain CpI1

Frankia sp. strain CpI1 has two glutamine synthetases designated GSI and GSII. Biosynthetic activities of both GSI and GSII were strongly inhibited by ADP and AMP. Alanine, aspartate, glycine and serine inhibited both GSI and GSII activities, whereas asparagine and lysine inhibited only slightly. Glutamine inhibited GSII but did not affect GSI. Since GSII is more heat labile than GSI, their relative heat stabilities can be used to determine their contribution to total GS activity. In cells grown on ammonia and on glutamine as sole combined-nitrogen sources most GS activity detected in crude extracts was due to GSI. In cells transferred to glutamate, GSI accounted for all GS activity in the first 15 h and then heat labile GSII was induced and increased to account for 40% of total GS activity within 50 h. Transfer of N₂-fixing cells to ammonia-containing medium led to a rapid decrease of GSII and a slow increase of GSI activity within 24 h. Conversely, when ammonia-grown cells were transferred to combined nitrogen-free medium, GSI activity gradually decreased and GSII increased before total activity leveled off in 50 h. GSII appears to be an ammonia-assimilating enzyme specifically synthesized during perceived N-starvation of Frankia cells.     

Oxygen protection of nitrogenase in Frankia sp. HFPArI3

O₂ protection of nitrogenase in a cultured Frankia isolate from Alnus rubra (HFPArI3) was studied in vivo. Evidence for a passive gas diffusion barrier in the vesicles was obtained by kinetic analysis of in vivo O₂ uptake and acetylene reduction rates in response to substrate concentration. O₂ of NH ₄ ⁺ -grown cells showed an apparent K _m O₂ of approximately 1M O₂. In N₂-fixing cultures a second K _m O₂ of about 215 M O₂ was observed. Thus, respiration remained unsaturated by O₂ at air-saturation levels. In vivo, the apparent K _m for acetylene was more than 10-fold greater than reported in vitro values. These data were inter oreted as evidence for a gas diffusion barrier in the vesicles but not vegetative filaments of Frankia sp. HFPArI3.     

Infectivity and effectivity of Frankia strains from the Rhamnaceae family on different actinorhizal plants

Ten strains of Frankia isolated from root nodules of plant species from five genera of the host family Rhamnaceae were assayed in cross inoculation assays. They were tested on host plants belonging to four actinorhizal families: Trevoa trinervis (Rhamnaceae), Elaeagnus angustifolia (Elaeagnaceae), Alnus glutinosa (Betulaceae) and Casuarina cunninghamiana (Casuarinaceae). All Frankia strains from the Rhamnaceae were able to infect and nodulate both T. trinervis and E. angustifolia. Strain ChI4 isolated from Colletia hystrix was also infective on Alnus glutinosa. All nodules showed a positive acetylene reduction indicating that the microsymbionts used as inoculants were effective in nitrogen fixation. The results suggest that Frankia strains from Rhamnaceae belong to the Elaeagnus-infective subdivision of the genus Frankia.     

Conservation of nif sequences in Frankia

Summary Southern blots of Frankia total DNAs were hybridized with nifHDK probes from Rhizobium meliloti, Klebsiella pneumoniae and Frankia strain Arl3. Differences between strains were noted in the size of the hybridizing restriction fragments. These differences were more pronounced among Elaeagnus-compatible strains than among Alnus- or Casuarina-compatible strains. Gene banks constructed for Frankia strains EUN1f, HRN18a, CeD and ACoN24d were used to isolate nif-hybridizing restriction fragments for subsequent mapping and comparisons. The nifH zone had the highest sequence conservation and the nifH and nifD genes were found to be contiguous. The complete nucleotide sequence of the nifH open reading frame (ORF) from Frankia strain Arl3 is 861 bp in length and encodes a polypeptide of 287 amino acids. Comparisons of these nucleic acid and amino acid sequences with other published nifH sequences suggest that Frankia is most similar to Anabaena and Azotobacter spp. and K. pneunoniae and least similar to the Gram-positive Clostridium pasteurianum and to the archaebacterium Methanococcus voltae.     

Isolation of infective and effective Frankia strains from root nodules of Alnus acuminata (Betulaceae)

Two Frankia strains were isolated from root nodules of Alnus acuminata collected in the Tucumano-oranense forest, Argentina. Monosporal cultures were obtained by plating a spore suspension of each strain and isolating a single colony. The strains (named AacI and AacIII) showed branched mycelia with polymorphic sporangia and NIR-vesicles. They differed in their ability to use carbon sources: the AacI strain grew well on pyruvate, while the AacIII strain grew on mineral medium supplemented with glucose or, alternatively, with sucrose. The two strains were sensitive to oleandomycin, erythromycin, kanamycin, penicillin G, streptomycin and chloramphenicol at 5 μg/ml. The AcIII strain exhibited a moderate resistance to rifampicin, ampicillin and vancomycin. The nitrogenase activity in vitro of the strains was significantly higher in basal medium without nitrogen than that determined in the presence of ammonium chloride. Both strains were infective on seedlings of Alnus glutinosa, inducing an approximately similar percentage of nodulated plants (80%), although strain AacIII produced a higher number of nodules per plant (≤15) than strain AacI (≤6). They were also effective for nitrogen fixation in planta, determined by the acetylene reduction assay. This revised version was published online in July 2006 with corrections to the Cover Date.     

Patterns of [13N]ammonium uptake and assimilation by Frankia HFPArl3

Nitrogen-starved cells of Frankia strain HFPArl3 incorporated [¹³N]-labeled ammonium into glutamine serine (glutamate, alanine, aspartate), after five-minute radioisotope exposures. High initial endogenous pools of glutamate were reduced, while total glutamine increased, during short term NH _inf4 ^sup+ incubation. Preincubation of cells in methionine sulfoximine (MSX) resulted in [¹³N]glutamine reduced by more than 80%, while [¹³N]glutamate and [¹³N]alanine levels increased. The results suggest that glutamine synthetase is the primary enzyme of ammonium assimilation, and that glutamate dehydrogenase and alanine dehydrogenase may also function in ammonium assimilation at low levels. Efflux of [¹³N]serine and lesser amounts of [¹³N]glutamine was detected from the Frankia cells. The identity of both Ser and Gln in the extracellular compartment was confirmed with gas chromatography/mass spectrometry. Serine efflux may be of significance in nitrogen transfer in Frankia.Abbreviations Pthr phosphothreonine - Aad -amino-adipate - MSX methionine sulfoximine     

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.     

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.     

Effect of carbon source on growth,nitrogenase and uptake hydrogenase activities of Frankia isolates from Casuarina sp.

The effect of different carbon sources on the growth of Frankia isolates for Casuarina sp. was studied. In addition, regulation of nitrogenase and uptake hydrogenase activity by carbon sources was investigated. For each of the three isolates, JCT287, KB5 and HFPCcI3, growth was greatest on the carbon sources pyruvate and propionate. In general the carbon sources which gave the greatest growth gave the highest levels of nitrogenase activity, but repressed the activity of uptake hydrogenase. The regulation of growth, uptake hydrogenase activity and nitrogenase activity is discussed.     

Studies of two Frankia strains isolated from Trevoa trinervis Miers

The Frankia strains TtI 11 and TtI 12 isolated from T. trinervis Miers were characterized regarding their carbon source utilization, intrinsic antibiotic resistance, infectivity, and effectivity on the original host. Both strains grew on BAP medium supplemented with glucose, maltose, and sucrose, but differed in their ability to use other carbon sources such as propionate, pyruvate, acetate, succinate, citrate, and mannitol. The isolates were sensitive to five of the twelve antibiotics tested at 1 μg mL⁻¹ concentration: chloramphenicol, tobramycin, eritromycin, streptomycin, and rifampicin. They exhibited a variable degree of resistance at 1 μg mL⁻¹ concentraction to penicillin G, 4-fluorouracil, oleandomycin, and lincomycin. Both isolates were able to infect and nodulate the original host plant, and thus represent the first reported infective and effective microsymbionts for T. trinervis Miers, a rhamnaceous actinorhizal host. R O D Dixon Section editor     

Phylogeny of nitrogenase sequences inFrankia and other nitrogen-fixing microorganisms

Summary The complete nucleotide sequence of a nitrogenase (nifH) gene was determined from a second strain (HRN18a) ofFrankia, an aerobic soil bacterium. The open reading frame is 870 bp long and encodes a polypeptide of 290 amino acids. The amino acid and nucleotide sequences were compared with 21 other published sequences. The twoFrankia strains were 96% similar at the amino acid level and 93% similar at the nucleotide level. A number of methods were used to infer phylogenies of these nitrogen fixers, based onnifH amino acid and nucleotide sequences. The results obtained do not agree completely with other phylogenies for these bacteria and thus make probable occurrences of lateral transfer of thenif genes. The time of divergence of the twoFrankia strains could be estimated at about 100 million years. The vanadium-dependent (Type 2) nitrogenase present inAzotobacter spp. appears to be a recent derivation from the conventional molybdenum-dependent (Type 1) enzyme, whereas the iron-dependent (Type 3) alternative nitrogenase would have a much older origin.     

Effect of spring flooding on endophyte differentiation,nitrogenase activity,root growth and shoot growth inMyrica gale

Summary Spring flooding was investigated as a possible limiting factor in the development of nitrogenase activity, root growth, and shoot growth inMyrica gale. Dormant, one year oldMyrica gale plants were placed in a greenhouse in early April and given three treatments: control (not flooded), flooded-water (flooded with water to 2.5 cm above the soil level) and flooded-peat (flooded with water-saturated peat to 4.0 cm above the soil level). Nitrogenase activity was absent at budbreak but appeared concurrently with the differentiation of vesicles by theFrankia sp. endophyte. Flooding delayed the onset of nitrogenase activity, substantially reduced the specific nitrogenase activity of the nodules, and also severely limited the production of the new nodule biomass. Consequently by 67 days past budbreak nitrogenase activity was much greater in the control plants (5.55±0.42 mol C₂H₄/plant.h; ± SE; N=9) than in the flooded-water (1.18±0.29) and flooded-peat (0.15±0.05) plants. Production of new secondary roots was substantially reduced in the flooded plants but adventitious roots were rapidly produced along the flooded portion of the stem in the better aerated zone near the surface. New nodules formed on several adventitious roots by 67 days indicating that the plants are able to replace their largely nonfunctional deeply flooded nodules with new nodules in the aerobic zone. Initially shoot growth was unaffected by flooding but by 67 days the flooded plants had substantially less leaf biomass, lower leaf and stem nitrogen concentrations, and less total shoot nitrogen content than the control plants.     

Nitrogen fixation in strains ofAzotobacter chroococcum bearing deletions of a cluster of genes coding for nitrogenase

Strains of the obligately aerobic nitrogen fixing organismAzotobacter chroococcum were constructed which contained defined chromosomal deletions in which the nitrogenase structural genenifHDK cluster (nifH for the polypeptide of the Fe-protein component of nitrogenase andnifD andnifK for the alpha and beta subunits respectively of the MoFe-protein component of the enzyme) was replaced by a kanamycin resistance gene. N₂ fixation was nevertheless observed in deletion strains though only in a molybdenum-deficient medium or in spontaneously arising tungstate-resistant derivatives. In comparison with the parent strain growing in molybdenum-sufficient medium, diazotrophic growth was slow and the nitrogenase activity in vivo was characterised by disproportionately low rates of C₂H₂-reduction compared to H₂-evolution and relative insensitivity of H₂-evolution to inhibition by C₂H₂. The findings show reiteration of functional structural genes for nitrogenase inA. chroococcum consistent with our previous observation of twonifH genes in this organism and detection in this work of a secondnifK-like sequence in the genomes of both parent and deletion strains whenA. chroococcum nifK DNA was used as a probe.