Nostoc PCC Research Articles

Characterization of a restriction barrier and electrotransformation of the cyanobacterium Nostoc PCC 7121.

We have investigated host restriction as a barrier to transformation and developed a method for gene transfer into the previously untransformable, heterotrophic cyanobacterium Nostoc PCC 7121. A restriction endonuclease, designated Nsp 7121I, has been partially purified by phosphocellulose chromatography of Nostoc cell extracts. Comparisons of Nsp 7121I digests of bacteriophage lambda and plasmid DNAs with computer-generated restriction fragment profiles showed that Nsp 7121I is an isoschizomer of restriction endonucleases, such as Asu I, Nsp 7524IV, Sau 96I, and Eco 47II, that recognize the sequence GGNCC. Cleavage by Nsp 7121I within this sequence was confirmed by sequence analysis of DNA fragments cleaved at a unique Nsp 7121I site. These data further suggested that cleavage occurs after the first G (5'-G/GNCC-3') in this site to generate a three base 5' overhang. Nsp 7121I degraded all plasmids used in previous transformation attempts but modification of these DNA molecules by Eco 47II methylase effectively prevented digestion by Nsp 7121I. Plasmids premethylated by passage through Escherichia coli carrying a plasmid encoded Eco 47II methylase have now been used in an electroporation procedure to transform Nostoc PCC 7121 to neomycin resistance at frequencies as high as one transformant per 10(3) viable cells. Transformation, and stable replication within Nostoc of one of the transforming plasmids (pRL25), was confirmed by recovery of pRL25, in its original form, from transformants. Conjugal transfer of pRL25 from E. coli into Nostoc was also possible but at much lower efficiency than by electroporation. These findings establish the basis for genetic analysis of Nostoc PCC 7121, from which genes for photosynthetic electron transport have been cloned.

Ornithine cycle in Nostoc PCC 73102. Arginase, OCT and arginine deiminase, and the effects of addition of external arginine, ornithine, or citrulline

Arginase, ornithine carbamoyl transferase (OCT) and arginine deiminase activities were found in cell-free extracts of Nostoc PCC 73102, a free-living cyanobacterium originally isolated from the cycad Macrozamia. Addition of either arginine, ornithine or citrulline to the growth medium induced significant changes in their in vitro activities. Moreover, growth in darkness, compared to in light, induced higher in vitro activities. The in vitro activities of arginase and arginine deiminase, two catabolic enzymes primarily involved in the breakdown of arginine, increased substantially by a combination of growth in darkness and addition of either arginine, or ornithine, to the growth medium. The most significant effects on the in vitro OCT activities where observed in cells grown with the addition of ornithine. Cells grown in darkness exhibited about 6% of the in vivo nitrogenase activity observed in cells grown in light. However, addition of external carbon (glucose and fructose) to cells grown in darkness resulted in in vivo nitrogenase activity levels similar to, or even higher than, cells grown in light. Growth with high in vivo nitrogenase activity or in darkness with the addition of external carbon, resulted in repressed levels of in vitro arginase and arginine deiminase activities. It is suggested that nitrogen starvation induces a mobilization of the stored nitrogen, internal release of the amino compound arginine, and an induction of two catabolic enzymes arginase and arginine deiminase. A similar and even more pronunced induction can be observed by addition of external arginine to the growth medium.

Cloning and sequencing of the petBD operon from the cyanobacterium Synechococcus sp. PCC 7002.

The genes encoding the photosynthetic cytochrome b6 (petB) and subunit 4 (petD) have been cloned and sequenced from the unicellular, photoheterotrophic, transformable cyanobacterium Synechococcus sp. PCC 7002, formerly designated Agmenellum quadruplicatum. The gene arrangement was found to be similar to that reported in the cyanobacterium Nostoc PCC 7906. The DNA and derived protein sequences were compared to chloroplast and the other cyanobacterial sequences. By pulsed-field electrophoresis, the petBD operon and the petCA operon, encoding the Rieske iron-sulfur protein and cytochrome f, were found to be located on separate, unlinked, Not I-digested DNA fragments. The petBD operon was found on the third largest Not I fragment (NC-325) while the petCA operon was found on the second largest Not I fragment (NB-370). These results suggest the two operons are not in proximity. The 1.35 kb transcript was shown to be light-regulated. Transcripts from cells grown under constant illumination showed a decrease in petB transcript levels to undetectable levels within 2 h after the cells were placed in the dark. Upon reillumination, transcript levels rose to three-fold over that seen initially under constant illumination.

Ornithine cycle in Nostoc PCC 73102. Occurrence and localization of ornithine carbamoyl transferase, and the effects of external carbon and ornithine on nitrogenase activity and citrulline synthesis

Cells of free‐living nitrogen‐fixing Nostoc PCC 73102, a filamentous heterocystous cyanobacterium originally isolated from coralloid roots of the cycad Macrozamia. were examined for the presence of ornithine carbamoyl transferase (OCT) by native‐PAGE/in situ activity stain, and SDS‐PAGE/Western immunoblots. Transmission electron microscopy and immunocytological labeling were used to study the cellular and subcellular distribution of OCT in the Nostoc cells. Moreover, the effects of photoautotrophic and dark heterotrophic growth metabolism on growth, nitrogenase activity and in vivo citrulline synthesis were investigated. PAGE in combination with in situ activity staining demonstrated an in vitro active OCT with a molecular weight of approximately 80 kDa. SDS‐PAGE/Western immunoblots revealed that a polypeptide with a molecular weight of approximately 38 kDa was immunologically related to OCT purified from pea (Pisum sativum L. cv. Alaska). Immunolocalization demonstrated that the OCT protein was located both in vegetative cells and heterocysts. Using the particle analysis of an image processor, the labeling associated with the photosynthetic vegetative cells was calculated to be 75.6 (± 5.5) gold particles μm−2 compared with 62.0 (± 7.5) in the nitrogen‐fixing heterocysts. Glucose and fructose stimulated both cyanobacterial growth and nitrogenase activity in light and darkness. Addition of exogenous ornithine decreased nitrogenase activity. In light grown cells, additions of glucose and fructose in combination with ornithine not only stimulated growth and nitrogenase activity but also in vivo citrulline synthesis, measured as 14CO2‐fixation into [14C]‐citrulline. In darkness no stimulation was observed on in vivo citrulline synthesis. The substantial stimulation of nitrogenase activity by additions of external glucose and fructose, both in the light and in darkness, was not followed by a simultaneous stimulation of in vivo citrulline synthesis.

Ornithine cycle in Nostoc PCC 73102. Occurrence and localization of ornithine carbamoyl transferase, and the effects of external carbon and ornithine on nitrogenase activity and citrulline synthesis

Ornithine cycle in Nostoc PCC 73102. Occurrence and localization of ornithine carbamoyl transferase, and the effects of external carbon and ornithine on nitrogenase activity and citrulline synthesis

The cloning and sequencing of Synechococcus sp. PCC 7002 petCA operon: Implications for the cytochrome c-553 binding domain of cytochrome f

The genes encoding the Rieske iron-sulfur protein and cytochrome f from a unicellular, naturally transformable, photoheterotrophic cyanobacterium, Synechococcus sp. PCC 7002, formerly Agmenellum quadruplicatum, have been isolated and sequenced. The two genes were found to be on a single operon, petCA.The Synechococcus sp. PCC 7002 iron-sulfur protein contains 181 amino acids, the conserved putative iron-binding domains CTHLGCV, residues 108-114, and CPCHGS, residues 128-133, no presequence and has a 73% sequence identity to the Nostoc PCC 7906 iron-sulfur protein. The 325 amino acid apocytochrome f sequence contains a 42 amino acid presequence, a CANCH heme binding domain, residues 20-24 from the presumed start of the mature protein, and a predicted hydrophobic membrane-spanning domain, residues 250-269. The mature cytochrome f sequence has a 71.5% sequence identity with Nostoc PCC 7906 cytochrome f and possesses a large (-14) negative charge and low calculated pI of 4.47 compared to higher plant chloroplast sequences. Nine separate domains showing differences in charged residues among cyanobacteria and plants have been identified and the possibility that these domains are involved in the ionic interactions with plastocyanin or cytochrome c-553 is discussed.

N2-Fixation by Freshly Isolated Nostoc from Coralloid Roots of the Cycad Macrozamia riedlei (Fisch. ex Gaud.) Gardn.

Nitrogenase (EC 1.7.99.2) activity (acetylene reduction) and nitrogen fixation ((15)N(2) fixation) were measured in cyanobacteria freshly isolated from the coralloid roots of Macrozamia riedlei (Fisch. ex Gaud.) Gardn. Light and gas phase oxygen concentration had marked interactive effects on activity, with higher (up to 100-fold) rates of acetylene reduction and (15)N(2) fixation in light. The relationship between ethylene formation and N(2)-fixation varied in the freshly isolated cyanobacteria from 4 to 7 nanomoles of C(2)H(4) per nanomole (15)N(2). Intact coralloid roots, incubated in darkness and ambient air, showed a value of 4.3. Maximum rates of nitrogenase activity occurred at about 0.6% O(2) in light, while in darkness there was a broad optimum around 5 to 8% O(2). Inhibition of nitrogenase, in light, by pO(2) above 0.6% was irreversible. Measurements of light-dependent O(2) evolution and (14)CO(2) fixation indicated negligible photosynthetic electron transport involving photosystem II and, on the basis of inhibitor studies, the stimulatory effect of light was attributed to cyclic photophos-phorylation. Nitrogenase activity of free-living culture of an isolate from Macrozamia (Nostoc PCC 73102) was only slightly inhibited by O(2) levels above 6% O(2) and the inhibition was reversible. These cells showed rates of light-dependent O(2) evolution and (14)CO(2) fixation which were 100- to 200-fold higher than those by the freshly isolated symbiont. Furthermore, nitrogenase activity was dependent on both photosynthetic electron transport and photophosphorylation. These data indicate that cyanobacteria within cycad coralloid roots are differentiated specifically for symbiotic functioning in a microaerobic environment. Specializations include a high heterocyst frequency, enhanced permeability to O(2), and a direct dependence on the cycad for substrates to support nitrogenase activity.

Occurrence of azoxyglycosides in Macrozamia riedlei and their effects on the free-living isolated Nostoc PCC 73102

The presence of azoxyglycosides in the Australian cycad Macrozamia reidlei was examined using high performance liquid chromatography. Cycasin and macrozamia were present in all tissues examined, cycasin being three to 17 times more abundant than macrozamin. The symbiotic organ, the coralloid root, contained 0.16% [g/g (fresh weight)] cycasin and 0.01 % (same unit) macrozamin. Addition of these azoxyglycoside concentrations to nitrogen-fixing Nostoc PCC 73102 cultures, a filamentous heterocystous cyanobacterium originally isolated from Macrozia, inhibited light and dark nitrogenase (acetylene reduction) activity. No effects were observed on in vitro glutamine synthetase activity or net in vivo CO 2 fixation. Cycasin (1.6%) and macrozamin (0.1%), i.e. 10 times the concentrations observed in the coralloid root, decreased phycobiliprotein content by 25 and 45%, 1 and 4 hr after the addition, respectively. The relative distribution of individual phycobiliproteins was not affected.

Characterization of two operons encoding the cytochrome b6-f complex of the cyanobacterium Nostoc PCC 7906. Highly conserved sequences but different gene organization than in chloroplasts.

We have isolated and determined the nucleotide and derived protein sequences for the four genes, petCA and BD, which encode the cytochrome b6-f, electron-transfer complex of the filamentous cyanobacterium, Nostoc PCC 7906. The primary structure and cotranscription of the petCA genes encoding the Rieske-FeS (nuclear encoded in plants) and apocytochrome f proteins has been described previously (Kallas, T., Spiller, S., and Malkin, R. (1988) Proc. Natl. Acad. Sci. U.S.A., in press). The petBD genes (645 and 480 protein-coding nucleotides, respectively) for the apocytochrome b6 (24.3 kDa) and subunit-IV (17.5 kDa) proteins comprise a second operon located at least 12 kilobases (kb) from petCA. The Nostoc petBD genes are not closely linked to the psbB gene (encoding the 51-kDa photosystem II polypeptide) and do not contain introns as do the closely related chloroplast genes. DNA probes specific for each of the Nostoc cytochrome-complex genes hybridized to single bands in genomic DNA blots at intensities expected for single copy genes. These data suggest that a single set of cytochrome b6-f proteins function in the different types of membranes found in Nostoc vegetative and heterocyst cells. RNA blot hybridizations identified an 1.8-kb mRNA common to cytochrome b6 and subunit IV, and an intensely hybridizing 0.8-kb mRNA specific to the subunit IV gene probe. The role of the latter RNA is not clear but it may represent a transcript from the opposite strand. The deduced Rieske, apocytochrome f, apocytochrome b6, and subunit IV proteins exhibit 59, 58-63, 84-85, and 79-83% sequence identity with the proteins from chloroplast cytochrome b6-f complexes. The Nostoc proteins show lower but still significant sequences identity with the corresponding proteins of the mitochondrial-type b-c1 complexes. The four probable heme-liganding His residues, and the approximate spacings between them, have been conserved in all of the available cytochrome b6 and b sequences from divergent sources. The Nostoc apocytochrome b6 and subunit IV proteins, as well as the Rieske, appear to be translated and thus inserted into the membrane as mature forms without cleavable presequences. Hydropathy analyses revealed five potential membrane spans in cytochrome b6 and three in the subunit IV protein, consistent with the profiles observed for the chloroplast proteins and the related cytochrome b proteins of cytochrome b-c1 complexes.

Primary structure of cotranscribed genes encoding the Rieske Fe-S and cytochrome f proteins of the cyanobacterium Nostoc PCC 7906.

The thylakoid membrane cytochrome b6-f complex (plastoquinol:oxidized-plastocyanin oxidoreductase, EC 1.10.99.1) catalyzes electron-transfer and proton-translocation reactions essential for oxygenic photosynthesis. We have isolated and determined the nucleotide sequences of the petC and petA genes encoding the Rieske Fe-S and cytochrome f polypeptides from the filamentous cyanobacterium Nostoc PCC 7906. These genes occur as single genomic copies, are tightly linked, and, as indicated by hybridization of gene-specific probes to Nostoc RNA, are cotranscribed as a 2.0-kilobase message. The Rieske Fe-S/cytochrome f gene pair thus represents an example of clustering and cotranscription in cyanobacteria of functionally related genes that, in photosynthetic eukaryotes, reside on separate nuclear and plastid genomes. These data are consistent with the progressive degeneration of the modern chloroplast genome from the ancestral, cyanobacterial-like genome of an endosymbiont. The Rieske Fe-S and the mature cytochrome f apoproteins are encoded by 537 and 867 nucleotides and have molecular masses of 19.2 and 31.2 kDa, respectively. They show 59% and 60% protein sequence identity, respectively, relative to spinach. Forty-four amino acids (4.7 kDa) resembling a prokaryotic signal sequence precede apocytochrome f. In contrast, the Rieske Fe-S protein appears to be translated without a presequence. The 183 bases separating the Rieske Fe-S and preapocytochrome f genes contain two families of 7- to 9-base tandem repeats, and some part of this sequence is highly reiterated in the genome. The C terminus of the Rieske Fe-S protein contains cysteine and histidine residues (probable ligands for the Fe2S2 center) in two peptides, Cys-Thr-His-Leu-Gly-Cys-Val and Cys-Pro-Cys-His-Gly-Ser, which have been conserved in spinach and in the five available Rieske Fe-S sequences from the mitochondrial-type cytochrome b-c1 complexes. Cytochrome f shows the heme binding residues Cys-Xaa-Xaa-Cys-His near its N terminus. Single, long hydrophobic stretches occur near the N and C termini, respectively, of the Rieske Fe-S and cytochrome f proteins and may form membrane-spanning helices.

86] Isolation and characterization of genes for cytochrome [formula omitted] complex

This chapter describes procedures used for the isolation and preliminary characterization of genes encoding the cytochrome b 6 /f complex from the filamentous cyanobacterium Nostoc PCC 7906. The proton-translocating thylakoid membrane cytochrome b 6 /f complex catalyzes electron transfer between the two photosystems (PS) and is required for cyclic electron flow around photosystem I (PSI). In cyanobacteria as in plant chloroplasts, the complex consists of four essential polypeptides: cytochromes f and b 6 , the Rieske Fe-S protein, and a subunit IV. In the cyanobacterium Anabaena these have apparent molecular weights of 31,000, 22,500, 22,000 and 16,000, respectively. Cloned genes can now be introduced by transformation or conjugation into several strains of cyanobacteria enabling, among other applications, a molecular genetic analysis of the structure and function of thylakoid membrane protein complexes. An additional requirement for this approach is the availability of cloned genes for these proteins and the determination of their nucleotide and derived amino acid sequences. Heterologous hybridization (the use of a gene from one source as a probe for isolating a gene from another source) was the approach first used to isolate cyanobacterial genes and perhaps remains the most widely used one. Its success ultimately depends on the level of nucleotide sequence similarity between the probe and the target DNA.

Synchronous Akinete Germination and Heterocyst Differentiation in Anabaena PCC 7937 and Nostoc PCC 6720

Isolates of the filamentous cyanobacteria Anabaena PCC 7937 and Nostoc PCC 6720 were selected in which the germling extruded from the akinete coat at the single cell stage and in which heterocyst differentiation occurred at the two or three cell stage after germination in the absence of combined nitrogen. The process of heterocyst differentiation of these germinating akinetes was synchronous and highly reproducible, and it resulted in transient yields of mature heterocysts exceeding 35% of the total cell population.

Akinetes of the Cyanobacterium Nostoc PCC 7524: Macromolecular and Biochemical Changes during Synchronous Germination

Summary: Akinetes of Nostoc PCC 7524 germinated synchronously when diluted into fresh medium in the light in the absence of a source of combined nitrogen. The akinetes and their daughter cells were unable to fix N2 until the first heterocysts differentiated in the young three-celled filaments after 19 h. The large glycogen reserves of the mature akinetes were not necessary for germination, since fixation of CO2 commenced immediately and 70% of the fixed carbon accumulated as glycogen. Although cyanophycin was degraded after 6 h, this reserve material was subsequently resynthesized with concomitant breakdown of phycocyanin. These nitrogen reserves were not required for protein synthesis, which was initiated immediately after the induction of germination, apparently at the expense of another, non-proteinaceous, reserve material. Although RNA synthesis occurred without lag, DNA synthesis commenced only after 80 min. Quantitatively similar changes were observed during germination in the presence of combined nitrogen except that neither cyanophycin nor phycocyanin were degraded and balanced growth was achieved more rapidly.

Resistance of DNA from filamentous and unicellular cyanobacteria to restriction endonuclease cleavage

Chromosomal DNA from nine species of filamentous cyanobacteria as diverse as Nostoc, Gloeotrichia and Plectonema is suggested to be extensively modified (methylated) by its resistance to cleavage by a number of restriction endonucleases. A remarkably similar pattern of DNA modification in these species contrasts with the known heterogeneity of their type II restriction endonuclease content. In particular, Nostoc PCC 73102, which lacks detectable sequence-specific endonucleases, is shown to possess extensive DNA modification. The use of isoschizomers demonstrates the presence of a methylase in the filamentous strains analogous to the dam enzyme of Escherichia coli. As a preliminary to assessing the significance of the DNA modification, a study of susceptibility to restriction endonuclease cleavage of the genomes of five unicellular cyanobacteria revealed considerable variation between the different strains. The significance of the DNA modification patterns elucidated is discussed in terms of the restriction endonuclease content and cellular differentiation of the relevant cyanobacterial strains.

A freezing method for purification and storage of akinete suspensions of the cyanobacterium Nostoc PCC 7524

A freezing method for purification and storage of akinete suspensions of the cyanobacterium Nostoc PCC 7524

Comparison of plasmids from the cyanobacterium Nostoc PCC 7524 with two mutant strains unable to form heterocysts

Comparison of plasmids from the cyanobacterium Nostoc PCC 7524 with two mutant strains unable to form heterocysts

Akinetes of the Cyanobacterium Nostoc PCC 7524: Macromolecular Composition, Structure and Control of Differentiation

Synchronized akinete differentiation occurred following the transition from exponential to non-exponential (linear) growth, the major trigger being energy limitation. Young akinetes first accumulated cyanophycin, then developed a multilayered extracellular envelope and a thickened wall. The dry weight, chlorophyll a, glycogen and carbon contents of mature akinetes were greater than those of vegetative cells, while their contents of DNA, RNA, protein, phycocyanin and nitrogen were similar to those of vegetative cells. Akinetes were resistant to desiccation and low temperatures, but not to temperatures above the maximum for vegetative cell growth. In N2-grown cultures heterocyst differentiation ceased at the end of exponential growth, while cell division continued, and akinetes first appeared in a regular pattern at a fixed distance (9 cells) from the nearest heterocyst. Exogenous NH4 + inhibited the differentiation of heterocysts and, in their absence, akinetes developed in irregular positions. The regular spatial pattern imposed on akinete differentiation by heterocysts was, like the heterocyst spatial pattern itself, independent of N2 fixation. Similar changes in both patterns induced by 7-azatryptophan suggested that they share a common mechanism of control.