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  • 1
    Electronic Resource
    Electronic Resource
    Springer
    Archives of microbiology 139 (1984), S. 162-166 
    ISSN: 1432-072X
    Keywords: Frankia ; Nitrogenase ; Oxygen protection ; Alnus rubra isolate
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract 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 1μM O2. In N2-fixing cultures a second K m O2 of about 215 μM 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.
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  • 2
    Electronic Resource
    Electronic Resource
    Springer
    Archives of microbiology 137 (1984), S. 194-199 
    ISSN: 1432-072X
    Keywords: Heterocysts ; Immunoferritin labelling ; Cyanobacteria ; Anabaena ; Nitrogenase
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract The question of whether the vegetative cells of Anabaena cylindrica synthesize nitrogenase under anaerobic conditions was studied by immunoferritin labelling of the Fe-Mo protein (Component I). Differentiating cultures, incubated under an argon atmosphere, were treated with DCMU 12 h following initiation of induction. DCMU inhibited photosynthetic O2 production, thus insuring strict anaerobic conditions, but had no effect on nitrogenase induction. Fe-Mo protein levels, as determined by rocket immunoelectrophoresis, increased 5-fold within 24h of DCMU treatment. Immunoferritin labelling of aldehyde fixed, ultrathin cryosections of anaerobically induced filaments showed that the Fe-Mo protein was restricted to the heterocyst. Ferritin labelling was shown to be specific by the following criteria: (a) substituting preimmune goat serum for the anti-Fe-Mo protein IgG prevented ferritin labelling; (b) ferritin-conjugated, non-homologous rabbit anti-goat IgG did not bind; (c) incubation of anti-Fe-Mo protein IgG treated sections with rabbit anti-goat IgG prior to the treatment with the ferritin label also prevented labelling. The results provide direct immunochemical evidence that nitrogenase is restricted to the heterocysts even under strictly anaerobic conditions.
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  • 3
    Electronic Resource
    Electronic Resource
    Springer
    Archives of microbiology 145 (1986), S. 209-214 
    ISSN: 1432-072X
    Keywords: Frankia ; Carbon metabolism ; Oxygen effects ; Nitrogen-fixation
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract 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 μM to more than 300 μM. 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.
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  • 4
    Electronic Resource
    Electronic Resource
    Springer
    Archives of microbiology 151 (1989), S. 469-474 
    ISSN: 1432-072X
    Keywords: Anabaena ; Heterocysts ; Nitrogenase ; Oxygen-protection ; Mutants
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Mutants of Anabaena sp. PCC 7120 with O2-sensitive acetylene-reducing activity were studied to identify envelope components that contribute to the barrier limiting diffusion of oxygen into the heterocyst. Mutant strain EF114, deficient in a heterocyst-specific glycolipid, reduced acetylene only under strictly anaerobic conditions. Analysis of in vivo O2 uptake as a function of dissolved pO2 showed that EF114 has lost the low affinity, diffusion-limited respiratory component associated with heterocysts in wild-type filaments. The low affinity respiratory activity was also lost in EF116, a mutant in which the cohesiveness of the outer polysaccharide layer was reduced. Restoration of aerobic nitrogen fixation in a spontaneous revertant of EF116 and in a strain complemented with cosmid 41E11 was associated with restoration of the low affinity component of respiratory activity. The results provide evidence that the barrier to diffusion of gas into heterocysts depends upon both the glycolipid layer and the polysaccharide layer of the heterocyst envelope.
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  • 5
    ISSN: 1574-6968
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology
    Notes: An 8551-bp plasmid, pFQ11, from Frankia alni strain CpI1 was sequenced. Its sequence was found to be very similar to that presented for pFQ31 from strain ArI3. Six potential protein-encoding open reading frames (ORFs) were identified, and transcriptional activity was shown within four of those regions of the plasmid by reverse transcriptase-polymerase chain reaction (RT-PCR) analysis. An earlier study reported that ORF EF of pFQ31, which is nearly identical to the 3′ 45% of ORF1 of pFQ11, is significantly similar to RepF. We found no such similarity. ORF2 and ORF3 predict products that are similar to a repressor protein and a partition protein, respectively. We found inverted repeats within and covering the start codon of ORF3; palindromic sequences and direct repeats between ORF3 and ORF4; and 3′ from ORF3, an AT-rich sequence that extensively overlaps the promoter region of a uvrB homolog in strain ArI3.
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  • 6
    ISSN: 1399-3054
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology
    Notes: Our understanding of the actinorhizal symbiosis, in particular of the Frankia-Ceanothus association, has been hampered by the failure to isolate infective strains in pure culture. Recently, the polymerase chain reaction (PCR) has been utilized to amplify regions of the Frankia genome, allowing analysis of the microsymbiont genome without first isolating the microbe in pure culture. Root nodules were collected from six Ceanothus spp. common to the coastal regions of the Santa Monica Mountains of southern California. Individual lobes were surface-sterilized, total DNA was extracted and amplified using prokaryotic-specific primers. To assess the genetic diversity of Frankia endophytes in the population studied, the BOX primer was used to generate genomic fingerprints of prokaryotic nodule inhabitants using rep-PCR. Fingerprint patterns fell into twelve distinct groups indicating the occurrence of genetic diversity of Frankia in the nodules sampled. DNA extracts of individual lobes that gave distinct BOX-PCR fingerprints were also amplified by PCR using primers directed against conserved regions of the 16S ribosomal RNA gene. The nucleotide sequences of the PCR products were determined and aligned with the corresponding region from other taxa for phylogenetic analysis. The sequences from Ceanothus nodules share a common ancestor to that of the Elaeagnus–infective strains.
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  • 7
    ISSN: 1573-5036
    Keywords: actinorhizae ; Casuarina cunninghamiana ; Frankia ; nitrogen fixation ; oxygen
    Source: Springer Online Journal Archives 1860-2000
    Topics: Agriculture, Forestry, Horticulture, Fishery, Domestic Science, Nutrition
    Notes: Abstract The effects of time after exposure to acetylene and of nodule excision were examined using a flow-through system. After a transient depression in the rate of acetylene reduction that began about 1.5 min after exposure to acetylene, the rate recovered to 98% of the initial maximum value after 40 min. After nodule excision the rate stabilized to 90% of the initial maximum value observed in the intact plant. Excised nodules, measured at 6-min intervals in a closed system, with frequent changes of the gas mixture, were used for the remaining experiments. Acetylene reduction by the nodules increased rapidly as temperature was increased between 6 and 26°C. Between 26 and 36°C there was relatively little effect of temperature on acetylene reduction. Nodules and cultures ofFrankia were compared with respect to the effect of temperature and pO2 (partial pressure of oxygen) on oxygen uptake. Cultures ofFrankia were grown on a nitrogen-free medium at either 0.3 kPa O2 (vesicles absent) or 20 kPa O2 (vesicles present). Oxygen uptake by nodules (vesicles absent) and by vesicle-containing cultures was strongly dependent on pO2 at values below 20 kPa. This suggests the presence of a barrier to oxygen diffusion. Oxygen uptake was dependent on temperature as well as on pO2, but the Q10 was much larger for the cultures than for the nodules. This suggests that vesicles or related structures are not the source of the diffusion barrier in Casuarina nodules. Respiration by cultures ofFrankia lacking vesicles became O2-saturated at low pO2 values. Thus these cultures did not have a significant diffusion barrier. From these results it is concluded that nodules ofCasuarina cunninghamiana have a barrier to oxygen diffusion supplied by the host tissue and not byFrankia.
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  • 8
    ISSN: 1573-5036
    Keywords: Batch culture ; Frankia ; HFPAr13 ; Nitrogenase ; Vesicles
    Source: Springer Online Journal Archives 1860-2000
    Topics: Agriculture, Forestry, Horticulture, Fishery, Domestic Science, Nutrition
    Notes: Summary Kinetics of growth and nitrogenase induction inFrankia sp. Ar13 were studied in batch culture. Growth on defined medium with NH 4 + as the N source displayed typical batch culture kinetics; however, a short stationary phase was followed by autolysis. Removal of NH 4 + arrested growth and initiated vesicle differentiation. Vesicle numbers increased linearly and were paralleled by a rise in nitrogenase (acetylene reduction) activity. Nitrogenase activity (10 nM C2H4·mg protein−1·min−1) was sufficient to support growth on N2 and protein levels rose in parallel with nitrogenase induction. Optimal conditions for vesicle and nitrogenase induction were investigated. Maximum rates of acetylene reduction were obtained with 5 to 10 mM K2 HPO4/KH2PO4, 0.1 mM CaCl2 and MgSO4. The optimum pH for acetylene reduction and respiration was around 6.7. The amount (5 to 10 μg protein/ml) and stage (exponential) of growth of the ammonium-grown inoculum strongly influenced the subsequent development of nitrogenase activity. Propionate was the most effective carbon source tested for nitrogenase induction. Respiration in propionate-grown cells was stimulated by CO2 and biotin, suggesting that propionate is metabolized via the propionyl CoA pathway.
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  • 9
    ISSN: 1573-5036
    Keywords: Casuarina ; Frankia ; Nitrogenase
    Source: Springer Online Journal Archives 1860-2000
    Topics: Agriculture, Forestry, Horticulture, Fishery, Domestic Science, Nutrition
    Notes: Summary The optimum conditions for growth ofFrankia sp. HFPCcI3 isolated fromCasuarina cunninghamiana, were studied in batch culture using defined media. Maximum growth (doubling time was 24 h)_was achieved at 33°C and at pH 6.3 with pyruvate and NH 4 + as the sole C and N sources, respectively. Removal of NH 4 + from the culture medium resulted in vesicle differentiation which was paralleled by induction of acetylene reduction activity. Growth on atmospheric N2 was optimal with combined pyruvate and glucose as the carbon sources and displayed a doubling time of about 48 h. Comparisons in growth and N2-fixing activity ofFrankia strains grown in a variety of cultural conditions demonstrate the range of behavior among the strains.
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  • 10
    ISSN: 1617-4623
    Keywords: Cyanobacteria ; Anabaena variabilis ; Plasmids ; Shuttle vectors ; Conjugation
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary Anabaena variabilis ATCC 29413 contains two cryptic plasmids. Clones of the smaller (41 kb) plasmid, designated pRDS1, in cosmid vectors were used to construct a physical map. A clone bank of pRDS1 constructed by ligating fragments from aXhoII digest of a pRDS1 cosmid clone into a mobilizable plasmid was used to locate an origin of replication of pRDS1. Because we were unable to cureA. variabilis of pRDS1, the clone bank was transferred by conjugation to another strain ofAnabaena sp., strain M-131. A 5.3 kb fragment of pRDS1 contained all of the sequences necessary for replication inAnabaena sp. strain M-131 as judged by the ability to rescue the hybrid vector from exconjugants in unchanged form after many generations. Hybrid plasmids derived from pRDS1, one bearing genes for luciferase, were also transferred by conjugation toA. variabilis, where they appeared to recombine with pRDS1.
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