Characterization and infectivity of a spontaneous variant isolated fromFrankia sp. WEY 0131391

Summary A spontaneous variant, obtained from aFrankia isolate fromAlnus rubra nodules, was compared with the parent strain with regard to infectivity, nitrogenase activity, and electrophoretic and immunological profiles. Both the parent and the variant strain were equally effective in inducing nodulation in seedlings ofA. rubra. All inoculated plants had an active nitrogenase system as measured by the acetylene reduction assay. Electrophoresis of whole cell homogenates on SDS-polyacrylamide slab gels showed similar electrophoretic profiles; however, the variant strain also exhibited striking differences in protein patterns that distinguish it from the parent strain. Immunological analysis of the originalFrankia strain and its variant revealed shared antigens as well as immunologically distinct antigenic determinants in the two strains. The variant strain exhibits a distinct morphology and growth patterns which remain stable after many passages through culture.     

Effects of nickel on Frankia and its symbiosis with Alnus glutinosa (L.) Gaertn

The tolerance of nickel by Frankia in culture and in symbiosis with Alnus was determined. Yield of three Frankia strains was not affected significantly by 2.25 mM nickel when cultured in propionate medium containing hydolysed casein as nitrogen source. Yield of two strains in medium without combined nitrogen, and thus reliant on fixed nitrogen, was stimulated markedly by the same nickel concentration. Utilisation of nickel for synthesis of uptake hydrogenases is presumed to be the cause of enhanced nitrogenase activity.Although growth was reduced, treatment of 2-month-old seedlings with 0.025 mM nickel for 4 weeks did not affect nodulation significantly while nitrogenase activity was doubled. Nodulation and nitrogenase activity of seedlings receiving 0.075 mM nickel were inhibited markedly, while 0.5 mM nickel was lethal to all seedlings after 4 weeks of treatment. A few small, ineffective nodules were initiated early on some of the latter seedlings, suggesting that effects of nickel on host plant processes rather than Frankia are the primary cause of inhibition of nodulation. This interpretation is supported by the retention of substantial nitrogenase activity in 10-month-old plants 1 day after the treatment with 0.59 mM nickel, when the nickel content of roots and nodules was already maximal. No nitrogenase activity was detected after 3 days, by which time the leaves were almost completely necrotic. Over a 4 day period, most nickel was retained in the roots and nodules. Supplying histidine simultaneously at concentrations equal to, or in excess of, nickel prevented wilting and leaf necrosis, but did not increase translocation of nickel to the shoot.     

The regulation of nodulation,nitrogen fixation and ammonium assimilation under a carbohydrate shortage stress in the Discaria trinervis-Frankia symbiosis

N₂-fixation is sensitive to limitation in the availability of newly synthesised carbohydrates for the nodules. We decided to explore the response of the D. trinervis - Frankia symbiosis to a transient decrease in carbohydrate supply to nodules. Feedback inhibition of nodulation as well as nodule growth was not released by a 6-day dark stress in D. trinervis nodulated plants. However, nitrogen fixation and assimilation were affected by the imposed stress. Nitrogenase activity was totally inhibited after 4 days of darkness although high levels of nitrogenase components were still detected at this time. Degradation of FeMo and Fe nitrogenase subunits – both at similar rates – was observed after 6 days of dark stress, revealing the need for inactivation to precede enhancement of protein turnover. Glutamine synthetase (GS), malate dehydrogenase (MDH) and asparagine synthetase (AS) polypeptides were also degraded during the dark stress, although at a lower rate than nitrogenase. ARA and nitrogenase were totally recovered 8 days after resuming normal illumination. It seems that current nitrogenase activity and ammonium assimilation are not, or are only weakly linked with the feedback control of nodulation in D. trinervis. These observations give support to the persistence of an autoregulatory signal in mature nodules that is not sensitive to transient shortages of carbon supply and sustains the inhibition of nodulation in the transient absence of N₂ fixation.     

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.     

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

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

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     

Immobilized Frankia spores remained viable on dry storage and on restoration to medium regenerated active colonies

Spores of Frankia strain ACN^1AG, immobilized in calcium alginate beads, germinated to produce colonies that increased in protein content and showed nitrogenase activity. Air dried immobilized spores remained viable for at least 15 days in dry condition, making the storage and transport of Frankia strains easy. This also opens the possibility of using beaded spores as inocula.     

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     

Respiratory capacity,nitrogenase activity and structural changes ofFrankia,in symbiosis withAlnus incana,in response to prolonged darkness

Plants ofAlnus incana (L.) Moench in symbiosis with a local source ofFrankia were exposed to prolonged darkness under controlled climate conditions.Frankia vesicle clusters were prepared from the root nodules, and the condition ofFrankia was measured as respiratory capacity by supplying the preparation with saturating amounts of four different substrates. During darkness, nitrogenase (EC 1.7.99.2) activity decreased in intact plants and in the vesicle-cluster preparations. The respiratory capacity ofFrankia also decreased. After 4 d in darkness most respiration was lost, though all nitrogenase activity was already lost after 3 d. When the dark treatment was ended after 2 d and normal light/dark conditions restored, nitrogenase activity immediately started to recover. The respiratory capacity continued to decrease and no recovery was observed until the third day after the end of the dark treatment. Whole-plant nitrogenase activity slowly increased at a rate similar to the rate of increase observed in untreated plants. Transmission electron micrographs of the root nodules showed that the cytoplasm of infected host cells and the cells ofFrankia were structurally degraded in response to dark treatment, while young vesicles were frequent during recovery. Growth and differentiation ofFrankia cells were apparently important for recovery of the enzyme activities studied.     

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.     

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.     

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.     

Nodulation of actinorhizal plants byFrankia strains capable of both root hair infection and intercellular penetration

Summary A morphological analysis of the initiation and development of root nodules ofElaeagnus angustifolia andMyrica cerifera inoculated with pure-culturedFrankia strains DDB 011610 or DDB 020110 was undertaken. From ultrastructural observations it was determined that both of theseFrankia strains can infectElaeagnus by an intercellular penetration mechanism andMyrica by the root hair infection mechanism. This indicates that both of these strains have the ability to infect host plant roots by either of two mechanisms. The reverse, thatElaeagnus orMyrica could be infected by both mechanisms, was not observed. The infection and nodule development processes of these two plants in combination with these strains were similar to observations made in previous studies (Miller andBaker 1985,Torrey andCallaham 1979). However, one exception was identified in the development of the prenodule ofMyrica when infected with strain 011610, in that endophytic hyphae developed vesicles within the cells of the prenodule. This event has not been described before for any of the actinorhizal genera and may be an indication of less than optimal compatibility between the host plant and the symbiont.Contribution no. 876 of the Battelle-Kettering Laboratory.     

Effect of oxygen on substrate utilization for nitrogen fixation and growth in Frankia spp.

Frankia, the actinomycete partner in the nitrogenfixing symbiosis of certain woody non-legumes, has been shown to fix nitrogen in pure culture under aerobic conditions. The sensitivity of in vivo nitrogen-fixation (acetylene reduction) to oxygen tension in the gas phase was measured in short-term assays with two Frankia isolates designated ARI3 and CcI3. The carbon source utilized had an effect on the optimum O₂ concentration for acetylene reduction. Cells utilizing an organic acid, e.g., propionate or pyruvate had maximum nitrogenase activity at an oxygen concentration of 15 to 20%. In contrast, cells respiring a sugar, e.g., trehalose or glucose, or endogenous reserves (glycogen or trehalose) had maximum acetylene reduction activity at 5 to 10% in the gas phase. Oxygen uptake kinetics showed that respiration in vesicle-containing cells utilizing trehalose had a biphasic response to oxygen concentration with a diffusion limited component at oxygen concentrations of 20 M to more than 300 M. These results suggested that trehalose was oxidized in the vesicles as well as in the vegetative hyphae. Oxygen concentration also had an effect on the trehalose-supported growth of cells (non nitrogenfixing, [⁺NH₄Cl]). Cells grown with 5–10% O₂ in the gas phase had a doubling time approximately half those grown with 20% O₂ (atmospheric). Propionate-grown cells showed similar growth rates at the two oxygen tensions, and grew faster (almost 2x) than the trehalose cells at 5–10% O₂. Trehalose also supported approximately 40% lower rates of oxygen uptake than propionate in vesicle-containing cells.     

Polysaccharide-hydrolyzing enzymes ofFrankia (Actinomycetales)

Studies were made of the polysaccharide-hydrolyzing activity inFrankia (Actinomycetales) grown in synthetic media using modifications of three standard assay procedures. In screening five different strains ofFrankia for cellulase activity, based on the method of utilization of cellulose in liquid culture, only one strain, CcI3, degraded filter paper cellulose to complete disintegration and only under very specific conditions of pH and primary carbon source. When carboxymethylcellulose (CMC) at 1% was used as substrate, all five strains showed the capacity to produce reducing sugars as hydrolytic products. Microcystalline cellulose, xylans and gum arabic were hydrolyzed to a lesser extent. Optimum activity depended upon pH and primary carbon source with pH 5.0 and pyruvate or propionate producing highest activities. In fractionation studies of culturedFrankia, assays for hydrolysis of 1% CMC in liquid medium showed that highest activity was in the enzyme preparation supernatant with lesser activity in the cell-free extract and cell wall fractions.Frankia strain CpI1 showed the greatest total hydrolytic activity against CMC after 2 weeks of culture. Strains ArI3 and CcI3 also showed good activity. The agar plate method for direct dye-polysaccharide interaction proved to be the least sensitive assay method with only ArI3 showing significant activity using CMC as substrate. It appears that theFranka strains grown in synthetic media all showed hydrolytic activity but the degree of hydrolysis of polysaccharides to reducing sugars depends upon strain of bacteria and very specific cultural conditions.     

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

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

Nitrate and nitrite reductase negative mutants of N2-fixingAzospirillum spp.

Chlorate resistant spontaneous mutants ofAzospirillum spp. (syn.Spirillum lipoferum) were selected in oxygen limited, deep agar tubes with chlorate. Among 20 mutants fromA. brasilense and 13 fromA. lipoferum all retained their functional nitrogenase and 11 from each species were nitrate reductase negative (nr^–). Most of the mutants were also nitrite reductase negative (nir^–), only 3 remaining nir⁺. Two mutants from nr⁺ nir⁺ parent strains lost only nir and became like the nr⁺ nir^– parent strain ofA. brasilense. No parent strain or nr⁺ mutant showed any nitrogenase activity with 10 mM NO ₃ ^– . In all nr^– mutants, nitrogenase was unaffected by 10 mM NO ₃ ^– . Nitrite inhibited nitrogenase activity of all parent strains and mutants including those which were nir^–. It seems therefore, that inhibition of nitrogenase by nitrate is dependent on nitrate reduction. Under aerobic conditions, where nitrogenase activity is inhibited by oxygen, nitrate could be used as sole nitrogen source for growth of the parent strains and one mutant (nr^– nir^–) and nitritite of the parent strains and 10 mutants (all types). This indicates the loss of both assimilatory and dissimilatory nitrate reduction but only dissimilatory nitrite reduction in the mutants selected with chlorate.     

Localization of nitrogenase in vesicles of Frankia sp. Cc1.17 by immunogoldlabelling on ultrathin cryosections

Immunogoldlabelling on ultrathin cryosections of Frankia sp. Cc1.17 showed specific labelling of nitrogenase in the spherical cells called vesicles. No label was found in the hyphae in any cells grown on a medium with combined nitrogen, nor in those to which no specific antiserum was added. Similar results were obtained with cultures grown under high (20%) and low (2%) oxygen tension in the gas phase.Abbreviations BSA Bovine serum albumin fraction V (Sigma) - PBS phosphate buffered saline. Phosphate buffer 0.1 M, 8 g NaCl/1, 0.2 g KCl/1 - PIPES Piperazine-1,4-bis(ethane sulphonic acid)     

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.     

Nitrogen fixation by hydrogen-utilizing bacteria

Seventeen strains of nitrogen-fixing bacteria, isolated from different habitats on hydrogen and carbon dioxide as well as on other substrates, morphologically resembled each other. All strains, including Mycobacterium flavum 301, grew autotrophically with hydrogen. The isolate strain 6 was sensitive to oxygen when dependent on N₂ as nitrogen source, a consequence of the sensitivity of its nitrogenase towards oxygen. At the same time, strain 6 was sensitive to hydrogen when growing autotrophically on N₂ as nitrogen source, but hydrogen did not affect acetylene reduction by these cells.Abbreviations MPN Most probable number - BS medium basal salts medium