Frankia Sp Research Articles

The cyanobacterium Synechococcus sp. strain PCC 7942 contains a second alkaline phosphatase encoded by phoV

A gene (phoV) encoding an alkaline phosphatase from Synechococcus sp. strain PCC 7942 was isolated by screening a plasmid gene bank for expression of alkaline phosphatase activity in Escherichia coli JM103. Two independent clones carrying the same alkaline-phosphatase-encoding gene were isolated. One of these clones (pKW1) was further analysed and the nucleotide sequence of a contiguous 3234 bp DNA fragment was determined. Two complete open reading frames (ORF1 and phoV) and an incomplete ORF3 were identified reading in the same direction. The deduced phoV gene product showed 34% identity to the alkaline phosphatase PhoA from Zymomonas mobilis, and the N-terminal part of the putative ORF3 protein exhibited 57% identity to a protein of unknown function from Frankia sp. Insertional inactivation of the Synechococcus PCC 7942 phoV gene failed, indicating an essential role for either the phoV or the ORF3 gene product. PhoV consists of 550 amino acid residues, resulting in a molecular mass of 61.3 kDa. To overexpress the Synechococcus PCC 7942 phoV gene in E. coli, plasmid pKW1 was transformed into a phoA mutant of E. coli (CC118). In E. coli strain CC118(pKW1) PhoV was expressed constitutively with high rates of activity, and was shown to be membrane associated in the periplasmic space. After partial purification of the recombinant PhoV, it was shown that, like other alkaline phosphatases, the Synechococcus PhoV had a broad pH optimum in the alkaline region and a broad substrate specificity for phosphomonoesters, required Zn2+ for activity, and was inhibited by phosphate. In contrast to several other alkaline phosphatases, PhoV was inhibited by Mn2+. Due to the lack of a Synechococcus PCC 7942 phoV mutant strain, the function of PhoV remains uncertain. However, the present results show that Synechococcus PCC 7942 has a second, probably phosphate-irrepressible, alkaline phosphatase (PhoV, 61.3 kDa) in addition to the phosphate-repressible enzyme (PhoA, 145 kDa) already described.

Heterologous hybridization ofFrankiaDNA toRhizobium melilotiandKlebsiella pneumoniae nifgenes

A nif gene probe from Rhizobium meliloti was used to isolate a recombinant bacteriophage from a Frankia sp. ArI3 gene bank. There is a large homology between nifD and nifH genes of R. meliloti or Klebsiella pneumoniae and Frankia DNA sequences. Approximately 4.5 kb to the right of nifK, we have localized a DNA region hybridizing to a R. meliloti probe containing nifA and nifB genes. The extent of the homology was greater for nifB than for nifA.

Cytokinin Secretion by Frankia sp. HFP ArI3 in Defined Medium

Frankia sp. HFP ArI3 (host plant Alnus rubra Bong.) was grown in defined medium and the culture solution was analyzed for the presence of various cytokinins and related compounds. N(6)- (Delta(2)-isopentenyl) adenosine was the only cytokinin detected by both high performance liquid chromatography and gas chromatography-mass spectrometry, at levels of approximately 1 ng/ml culture medium.

Studies on Frankia sp. LLR 02022 from Casuarina cunninghanmiana and its mutant LLR 02023

Frankia sp. LLR 02022, isolated from nodules of Casuarina cunninghamiana Miq., and its spontaneous mutant LLR 02023, show similar morphology but slight differences in cell chemistry and major differences in carbohydrate utilization. The mutant has the same phospholipids, phospholipid fatty acids and menaqmnones as the parent, but shows quantitative differences in peptidoglycan composition, pigment production and triglyceride fatty acids. The parent utilizes only rhamnose; the mutant also utilizes arabinose, glucose and xylose under certain conditions of growth. The significance of these findings is discussed.

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.

Enzyme-linked immunosorbent assay (ELISA) as a means of taxonomic analysis of Streptomyces and related organisms.

Fourteen Streptomyces strains from various numerical taxonomic classes and representatives of three other genera of actinomycetes were studied using an indirect enzyme-linked immunosorbent assay (IND-ELISA) to determine their serological relationships. The IND-ELISA results agreed with those from previous numerical taxonomic analyses and Ouchterlony double-diffusion studies. The IND-ELISA method is quicker, more quantitative and less subjective than Ouchterlony assays and thus should be useful in Streptomyces taxonomy. The results indicated that Frankia sp. CpI1 was related to Streptomyces.

Effect of O2 on vesicle formation, acetylene reduction, and O2-uptake kinetics in Frankia sp. HFPCcI3 isolated from Casuarina cunninghamiana.

The effect of the partial pressure of oxygen (PO2) on the formation of vesicles, which are thought to be the site of N2 fixation in Frankia, was studied in HFPCcI3, an effective isolate from Casuarina cunninghamiana. Unlike other actinorhizal root nodules, vesicles are not produced by the endophyte in Casuarina nodules. However, in culture under aerobic conditions, large, phase-bright vesicles are formed in HFPCcI3 within 20 h following removal of NH+4 from the culture medium and reach peak numbers within 72 to 96 h. In vivo acetylene reduction activity parallels vesicle formation. Optimum rates of acetylene reduction in short-term assays occurred at 20% O2 (0.2 atm (1 atm = 101.325 kPa] in the gas phase. O2 uptake (respiration) determined polarographically showed diffusion-limited kinetics and remained unsaturated by O2 until 300 microM O2. In contrast, respiration in NH+4-grown cells was saturated by O2 between 8 and 10 microM O2. These results indicate the presence of a diffusion barrier associated with the vesicles. Vesicle development was repressed in cells incubated in N-free media sparged with gas mixtures with PO2 between 0.001 and 0.003 atm. Nitrogenase was induced under these conditions, but acetylene reduction was extremely O2 sensitive. The kinetics of O2 uptake as a function of dissolved O2 concentration in avesicular cells were similar to those in NH+4-grown cells indicating the lack of a diffusion barrier. These results demonstrate that vesicle formation and the development of the O2 protection mechanisms of nitrogenase are regulated by ambient PO2 in HFPCcI3.

Enzymes of glucose metabolism in Frankia sp.

Enzymes of glucose metabolism were assayed in crude cell extracts of Frankia strains HFPArI3 and HFPCcI2 as well as in isolated vesicle clusters from Alnus rubra root nodules. Activities of the Embden-Meyerhof-Parnas pathway enzymes glucokinase, phosphofructokinase, and pyruvate kinase were found in Frankia strain HFPArI3 and glucokinase and pyruvate kinase were found in Frankia strain HFPCcI2 and in the vesicle clusters. An NADP+-linked glucose 6-phosphate dehydrogenase and an NAD-linked 6-phosphogluconate dehydrogenase were found in all of the extracts, although the role of these enzymes is unclear. No NADP+-linked 6-phosphogluconate dehydrogenase was found. Both dehydrogenases were inhibited by adenosine 5-triphosphate, and the apparent Km's for glucose 6-phosphate and 6-phosphogluconate were 6.86 X 10(-4) and 7.0 X 10(-5) M, respectively. In addition to the enzymes mentioned above, an NADP+-linked malic enzyme was detected in the pure cultures but not in the vesicle clusters. In contrast, however, the vesicle clusters had activity of an NAD-linked malic enzyme. The possibility that this enzyme resulted from contamination from plant mitochondria trapped in the vesicle clusters could not be discounted. None of the extracts showed activities of the Entner-Doudoroff enzymes or the gluconate metabolism enzymes gluconate dehydrogenase or gluconokinase. Propionate- versus trehalose-grown cultures of strain HFPArI3 showed similar activities of most enzymes except malic enzyme, which was higher in the cultures grown on the organic acid. Nitrogen-fixing cultures of strain HFPArI3 showed higher specific activities of glucose 6-phosphate and 6-phosphogluconate dehydrogenases and phosphofructokinase than ammonia-grown cultures.

Novel benzo[a]naphthacene quinones from an actinomycete, Frankia G-2 (ORS 020604)

NANCY N. GERBER and MARY P. LECHEVALIER. Can. J. Chem. 62, 2818 (1984). Frankia sp. G2 (ORS 020604), grown for one month without shaking, produced a mixture of 2 quinonoid pigments. The structure of one, G-2N, was shown by chemical and physical methods to be 2-methyl-4,7,9,12-tetrahydroxy-5,6-dihydro benzo[a]naphthacene-8,13-dione. The other, G-2A, has the same structure with the addition of a carboxyl group at position 10 or 11. Natural quinones with this ring system have not been reported previously.

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 NH4+-grown cells showed an apparent KmO2 of approximately 1 microM O2. In N2-fixing cultures a second Km O2 of about 215 microM O2 was observed. Thus, respiration remained unsaturated by O2 at air-saturation levels. In vivo, the apparent Km for acetylene was more than 10-fold greater than reported in vitro values. These data were interpreted as evidence for a gas diffusion barrier in the vesicles but not vegetative filaments of Frankia sp. HFPArI3.

Levels of trehalose and glycogen in Frankia sp. HFPArI3 (Actinomycetales)

The levels of soluble trehalose were compared in Frankia sp. ArI3 under various conditions in batch culture. Trehalose appears to be a specialized carbon reserve which is synthesized upon transfer of cultures to fresh medium; thereafter it is metabolized and broken down. Levels reach a peak of 10–20% cell dry weight depending on cultural conditions, then drop to a minimum of 1–2% cell dry weight. A similar pattern is observed whether cells are grown in a medium containing NH4Cl or in a medium lacking nitrogen substrates; however, the total levels of trehalose are higher in the latter. The levels of endogenous trehalose showed an inverse correlation with a sharp increase in nitrogenase activity. When cells were inoculated into a medium lacking nitrogen to induce fixation of atmospheric nitrogen, trehalose levels reached their peak in 4 days. The nitrogenase activity (measured with the acetylene reduction technique) increased sharply on the 4th to 5th day. Trehalose levels fell sharply from day 4 to day 5; when they fell to half their maximum, nitrogenase activity decreased sharply. Thereafter, the decline in trehalose concentration paralleled that of nitrogenase activity. When cells were washed free of exogenous carbon, the endogenous trehalose levels dropped to half the initial value in 1 h. The cells continued to respire for 2 days on endogenous reserves. In addition to trehalose, glycogen reserves were extracted from Frankia. At its maximum, the glycogen totalled approximately 10% of the dry weight of cultures grown on a medium supplemented with NH4Cl as exogenous nitrogen source, and decreased to ca. 1.0% dry weight in starved cultures. Glycogen did not accumulate significantly in cultures that were induced to fix nitrogen.

Plasmids in Frankia sp.

A method to achieve cell lysis and isolate Frankia sp. plasmid DNA was developed. A screening of Frankia sp. strains belonging to different host compatibility groups (Alnus sp., Elaeagnus sp., Ceanothus sp.) showed that, of 39 strains tested, 4 (strains Cp11, ARgN22d, ArI3, and EUN1f) possessed plasmids ranging in size from 7.1 to 32.2 kilobase pairs as estimated from agarose gel electrophoresis and electron microscopy. A total of 11 plasmids were detected.

Structural features of the vesicle of Frankia sp. CpI1 in culture

The filamentous bacterium Frankia sp. CpI1 of the Actinomycetales, responsible for symbiotic nitrogen fixation in the nodules of certain woody dicots, also fixes dinitrogen when grown independently of the host in a nitrogen-free synthetic nutrient medium under aerobic conditions. In structural studies of Frankia grown in culture it has been shown that the bacterial filaments form vesicles, enlarged terminal endings in which the enzyme nitrogenase is formed. Microscopic examination of cultures shows that the vesicles possess a specialized envelope consisting of a number of thin layers or laminae which in polarized light show birefringence and in freeze-etch electron microscopy are resolved as multiple (12–15) laminae approximately 35–40 Å (1 Å = 0.1 nm) in thickness. Comparisons are made between the structure of the vesicle envelope in cultured Frankia and the strikingly similar innermost laminated layer in the dinitrogen-fixing heterocysts of the cyanobacterium Anabaena. Comparable protective functions in limiting oxygen to the dinitrogen-fixing sites are suggested for these similar structures in two quite unrelated microorganisms.

Erratum: Factors affecting vesicle formation and acetylene reduction (nitrogenase activity) in Frankia sp. CpIl

Erratum: Factors affecting vesicle formation and acetylene reduction (nitrogenase activity) in <i>Frankia</i> sp. CpIl

Dinitrogen Fixation by Cultures of Frankia sp CpIl Demonstrated by 15N2 Incorporation

The filamentous bacterium Frankia of the Actinomycetales, isolated from the nitrogen-fixing root nodules of certain woody plants, has shown nitrogenase activity in culture, using the acetylene reduction method. In the present work, nitrogenase activity in pure cultures of Frankia sp. CpIl is confirmed using mass spectrometric measurements of (15)N(2) incorporation. After addition of carrier NH(4) (+) to digested cultures, those exposed to (15)N(2) (25 atom%) had a (15)N content of 3.16 atom% compared to 0.354 atom% (15)N in the controls.

Factors affecting vesicle formation and acetylene reduction (nitrogenase activity) in Frankia sp. CpI1.

Vesicle formation and acetylene reduction (nitrogenase activity) were observed when washed hyphae from cultures of Frankia sp. CpI1 were transferred to a nitrogen-free medium containing ethylenediaminetetraacetic acid and succinate. Succinate could be replaced by malate or fumarate, but not other carbon sources. Maximum acetylene reduction and vesicle numbers were observed at a pH of 6.0-6.5, at 25-30 degrees Centigrade, and at atmos pheric Po2 or somewhat less (5-20 kPa). Addition of 1 mM NH4Cl almost completely inhibited vesicle formation and acetylene-reducing activity, but did not immediately inhibit such reducing activity by cultures with preexisting vesicles. Acetylene-reducing activity was never observed in the absence of vesicle formation.

Vesicle formation and acetylene reduction activity in Frankia sp. CPI1 cultured in defined nutrient media

It has been reported that certain species of free-living Rhizobium were capable of producing nitrogenase1–5, thereby demonstrating conclusively that the nif genes reside in the bacterial component of the dinitrogen-fixing symbiosis between Rhizobium and leguminous host plants. In the comparable dinitrogen-fixing symbiosis between the actinomycete Frankia and the actinorhizal woody dicotyledons that serve as nodulated host plants, the endophytic filamentous bacteria have only recently been brought into culture6–8. We report here evidence that free-living Frankia sp. grown in appropriate nutrient conditions show nitrogenase activity (acetylene reduction) associated with a distinctive morphological structure formed in effective nodules and also developed in culture.

Characterization of an effective actinorhizal microsymbiont, Frankia sp. AvcI1 (Actinomycetales).

The actinomycete, Frankia sp. AvcI1, isolated from root nodules of Alnus viridis ssp. crispa was grown in axenic culture and used to inoculate host seedlings. This bacterium has been shown to be an infective and effective nitrogen-fixing microsymbiont which can be distinguished from other frankiae, in vitro, on the basis of size, distinctive morphology, and growth characteristics. Cross-inoculation studies indicated that the host range of this symbiont encompasses all of the members of the genera Alnus, Myrica, and Comptonia tested. In all cases, the symbioses developed were effective in fixing atmospheric dinitrogen.

Characterization of an ineffective actinorhizal microsymbiont, Frankia sp. EuI1 (Actinomycetales).

The actinomycete, Frankia sp. EuI1, isolated from root nodules of Elaeagnus umbellata is an infective endophyte but which lacks the ability to form an effective nitrogen-fixing symbiosis with its host. This ineffective organism can be distinguished easily from other frankiae, in vitro, on the basis of size, morphology, and the elaboration of a diffusible pigment. Cross-inoculation studies indicated that the host range of this symbiont is narrow and probably restricted to the Elaeagnaceae. In all cases of nodulation the symbiosis never developed nitrogenase activity and the microsymbiont never produced endophytic vesicles within the infected host cells. Sporangia were produced in vivo and in vitro so the morphogenetic block is apparently restricted to vesicle formation.