Gloeothece Sp Research Articles

LED light for microalgal carotenoids and antioxidant capacity increase - a profitable advance in blue biotechnology

LED light for microalgal carotenoids and antioxidant capacity increase - a profitable advance in blue biotechnology

ITRAQ-based quantitative proteomic analysis of Gloeothece sp. PCC 6909: Comparison with its sheathless mutant and adaptations to nitrate deficiency and sulfur limitation

Gloeothece sp. PCC 6909 is a unicellular N(2)-fixing cyanobacterium with a well defined and highly developed sheath surrounding its cells. A sheathless mutant of this strain was previously obtained by chemical mutagenesis and, although lacking the sheath, it releases large amounts of polysaccharides into the culture medium. To provide a global understanding on the metabolic differences between the two phenotypes, the proteomes of the wild type and mutant were analyzed using a cross-species proteomics approach coupled with iTRAQ isobaric tagging technology, since their genome sequences are not yet available. Effects arising from the presence/absence of nitrate and sulfur are presented as two metabolically directed follow-up iTRAQ studies. These nutrients are believed to play a major role in Gloeothece's metabolism, including the production of extracellular polymeric substances - EPS. 454, 124, and 53 proteins were identified and reliably quantified using homology anchoring approaches for iTRAQ previously described. The results obtained strongly suggest that the chemical mutagenesis affected the regulation of a number of key cellular processes, as revealed by the significant fold changes observed for proteins covering a large spectrum of functional groups. Moreover, they provide new insights on the adaptations of Gloeothece cells to nitrate-deficiency and sulfur-limitation.

English

The goal of this study was to determine the effect of bispyribac-sodium on the nitrogenase activities and growth of cyanobacteria isolated from paddy fields. Ten cyanobacterialspecies were used in this study. Cyanobacterial species were isolated from soil and water samples obtained from rice fields in Corum, Turkey. Among all Anabaena strains, the maximum activity was determined in Anabaena sp. O-22 (2.26 µl ethylene/mg.h) whereas; the lowest activity was shown in Gloeothece sp. O-Y (0.04 µl ethylene/mg.h).The maximum inhibition was seen in Anabaena sp. O-22, Synechocystis sp. O-X andAnabaena sp. O-16 in 100 µg/ml bispyribac-sodium concentration. Although low bispyribac-sodium concentrations somewhat stimulated growths of Anabaena sp. O-X2, O-C, O-4, O-16, O-8 and Synechocystis sp. O-X, the biomass of all cultures were severely inhibited at higher concentrations. The growths of Anabaena sp. O-22 andSynechocystis sp. O-X completely repressed at 100 µg/mL and at higher bispyribac-sodium concentrations, whereas, Anabaena O-X2, O-6, O-4 and O-16 completely suppressed at 500 µg/mL bispyribac-sodium concentration. The end of the studyAnabaena sp. O-22 has been proposed as biofertilizer. The results obtained may be useful for the production of rice. Key words: Cyanobacteria, nitrogenase activity, herbicide.

Using extracellular polymeric substances (EPS)-producing cyanobacteria for the bioremediation of heavy metals: do cations compete for the EPS functional groups and also accumulate inside the cell?

Many cyanobacteria produce extracellular polymeric substances (EPS) mainly of polysaccharidic nature. These EPS can remain associated to the cell surface as sheaths, capsules and/or slimes, or be liberated into the surrounding environment as released polysaccharides (RPS). The ability of EPS-producing cyanobacteria to remove heavy metals from aqueous solutions has been widely reported in the literature, focusing mainly on the biotechnological potential. However, the knowledge of the effects of the metals in the cell's survival/growth is still scarce, particularly when they are simultaneously exposed to more than one metal. This work evaluated the effects of different concentrations of Cu(2+) and/or Pb(2+) in the growth/survival of Gloeothece sp. PCC 6909 and its sheathless mutant Gloeothece sp. CCY 9612. The results obtained clearly showed that both phenotypes are more severely affected by Cu(2+) than Pb(2+), and that the mutant is more sensitive to the former metal than the wild-type. Evident ultrastructural changes were also observed in the wild-type and mutant cells exposed to high levels (10 mg l(-1)) of Cu(2+). Moreover, in bi-metal systems, Pb(2+) was preferentially removed compared with Cu(2+), being the RPS of the mutant that is the most efficient polysaccharide fraction in metal removal. In these systems, the simultaneous presence of Cu(2+) and Pb(2+) caused a mutual inhibition in the adsorption of each metal.

Oxygen and the light–dark cycle of nitrogenase activity in two unicellular cyanobacteria

Cyanobacteria capable of fixing dinitrogen exhibit various strategies to protect nitrogenase from inactivation by oxygen. The marine Crocosphaera watsonii WH8501 and the terrestrial Gloeothece sp. PCC6909 are unicellular diazotrophic cyanobacteria that are capable of aerobic nitrogen fixation. These cyanobacteria separate the incompatible processes of oxygenic photosynthesis and nitrogen fixation temporally, confining the latter to the dark. Although these cyanobacteria thrive in fully aerobic environments and can be cultivated diazotrophically under aerobic conditions, the effect of oxygen is not precisely known due to methodological limitations. Here we report the characteristics of nitrogenase activity with respect to well-defined levels of oxygen to which the organisms are exposed, using an online and near real-time acetylene reduction assay combined with sensitive laser-based photoacoustic ethylene detection. The cultures were grown under an alternating 12-12 h light-dark cycle and acetylene reduction was recorded continuously. Acetylene reduction was assayed at 20%, 15%, 10%, 7.5%, 5% and 0% oxygen and at photon flux densities of 30 and 76 mumol m(-2) s(-1) provided at the same light-dark cycle as during cultivation. Nitrogenase activity was predominantly but not exclusively confined to the dark. At 0% oxygen nitrogenase activity in Gloeothece sp. was not detected during the dark and was shifted completely to the light period, while C. watsonii did not exhibit nitrogenase activity at all. Oxygen concentrations of 15% and higher did not support nitrogenase activity in either of the two cyanobacteria. The highest nitrogenase activities were at 5-7.5% oxygen. The highest nitrogenase activities in C. watsonii and Gloeothece sp. were observed at 29 degrees C. At 31 degrees C and above, nitrogenase activity was not detected in C. watsonii while the same was the case at 41 degrees C and above in Gloeothece sp. The differences in the behaviour of nitrogenase activity in these cyanobacteria are discussed with respect to their presumed physiological strategies to protect nitrogenase from oxygen inactivation and to the environment in which they thrive.

Characterization and transcriptional analysis of hupSLW in Gloeothece sp. ATCC 27152: an uptake hydrogenase from a unicellular cyanobacterium

The structural genes (hupSL) encoding an uptake hydrogenase in the unicellular cyanobacterium Gloeothece sp. ATCC 27152, a strain capable of aerobic N(2) fixation, were identified and sequenced. 3'-RACE experiments uncovered the presence of an additional ORF 184 bp downstream of hupL, showing a high degree of sequence identity with a gene encoding an uptake-hydrogenase-specific endopeptidase (hupW) in other cyanobacteria. In addition, the transcription start point was identified 238 bp upstream of the hupS translational start. RT-PCR experiments revealed that hupW is co-transcribed with the uptake hydrogenase structural genes in Gloeothece sp. ATCC 27152. In addition, Northern hybridizations clearly showed that hupSLW are transcribed under nitrogen fixing conditions, but not in the presence of combined nitrogen. A putative NtcA binding site was identified in the promoter region upstream of hupS, centred at -41.5 bp with respect to the transcription start point. Electrophoretic retardation of a labelled DNA fragment (harbouring the putative NtcA-binding motif) was significantly affected by an Escherichia coli cell-free extract containing overexpressed NtcA, suggesting that NtcA is involved in the transcriptional regulation of hupSLW.

Synthesis and proteolytic degradation of nitrogenase in cultures of the unicellular cyanobacterium Gloeothece strain ATCC 27152.

In cultures of the unicellular cyanobacterium Gloeothece sp. ATCC 27152 growing under alternating 12 h light and 12 h darkness, nitrogenase activity appears as cultures enter the dark phase. Synthesis of both component proteins of nitrogenase commences immediately prior to the appearance of activity and continues until about 8 h into the period of darkness. The two components (Fe-protein and MoFe-protein) are synthesized in a molar ratio of about 3:1. Degradation of the nitrogenase proteins starts as early as 4 h into the dark period and increases markedly as cultures enter the light phase. As a result, both nitrogenase proteins are completely absent from cultures during most of the light phase. In contrast, all of the other proteins investigated appeared to be present throughout the cycle of alternating light and darkness. Degradation of nitrogenase depends upon protein synthesis during the last 6 h of darkness and is prevented by addition of protease inhibitors. Two proteins, of M(r) 47,000 and 29,000, are specifically synthesized during this period and it is possible that they have a role in nitrogenase degradation. Proteolytic activity of extracts of Gloeothece, measured as the ability to degrade azocasein, increased markedly during the early part of the light period, but this increase did not depend on protein synthesis. This activity does not therefore correspond to that specifically involved in nitrogenase catabolism, though it may act on initial breakdown products generated by a nitrogenase-specific degradative system. A phycobiliprotein appears to act as a temporary store of the degradation products of nitrogenase.

Effect of sulfur starvation on the morphology and ultrastructure of the cyanobacterium Gloeothece sp. PCC 6909

Gloeothece sp. PCC 6909 is a unicellular, nitrogen-fixing cyanobacterium that accumulates sulfate in its sheath. An ultrastructural study of sulfate-deficient and normal Gloeothece sp. PCC 6909 cells was carried out. The physiological alterations, caused by sulfur starvation, were related to important morphological alterations in the cell: a structureless sheath, accumulation of cyanophycin, polyhydroxybutyrate and glycogen granules, and disintegration of thylakoidal membranes. Most of these changes were reversed by the addition of sulfate to the culture medium. The important role of sulfate in the sheath structure was demonstrated.

Modification of the Fe protein of the nitrogenase of Gloeothece (Nägeli) sp. ATCC 27152 during growth under alternating light and darkness.

The Fe protein of the nitrogenase of the unicellular Cyanobacterium Gloeothece (Nägeli) sp. ATCC 27152 can be resolved by SDS-PAGE into two antigenically detectable components of approximate Mr 38500 and 40000 respectively. The larger form of this protein may be produced by modification of the smaller form. Modification of the Fe protein is promoted under conditions where O2 (but not O2 - or H2 O2 ) has increased access to the enzyme, but does not allow nitrogenase to function under conditions of O2 stress. During growth of Gloeothece under alternating light and darkness, antigenically detectable Fe protein is absent throughout most of the light period. The restriction of nitrogenase activity to the period of darkness is better explained in terms of regulation of nitrogenase synthesis and degradation than by reversible modification of a constant intracellular concentration of Fe protein. However, newly synthesized Fe protein always appeared initially as its larger form, which may be catalytically inactive in aerobic cultures of Gloeothece. Conversion of this form to the smaller, assumed active, form of the Fe protein may be an additional factor in explaining the increase in nitrogenase activity that occurs during the first few hours of each dark phase.

Poly(hydroxyalkanoate) in cyanobacteria: an overview

In this paper an overview is given on the occurrence of poly(hydroxyalkanoate) (PHA) in cyanobacteria and its possible role as a putative reserve compound. Comparisons are made with the function of other storage compounds that occur in cyanobacteria. For the cyanobacteria Oscillatoria limosa and Gloeothece sp. PCC 6909, some experimental data on the accumulation and mobilization of PHA are presented. O. limosa presumably contains poly(hydroxyvalerate) (PHV), whereas in Gloeothece poly(hydroxybutyrate) (PHB) was detected. Both species accumulated PHA to 6–9% of the dry weight. In Gloeothece PHB accumulation was stimulated by the addition of acetate but in O. limosa this was not the case. PHA was not involved in dark metabolism in either of the strains.

Poly(hydroxyalkanoate) in cyanobacteria: an overview

In this paper an overview is given on the occurrence of poly(hydroxyalkanoate) (PHA) in cyanobacteria and its possible role as a putative reserve compound. Comparisons are made with the function of other storage compounds that occur in cyanobacteria. For the cyanobacteria Oscillatoria limosa and Gloeothece sp. PCC 6909, some experimental data on the accumulation and mobilization of PHA are presented. O. limosa presumably contains poly(hydroxyvalerate) (PHV), whereas in Gloeothece poly(hydroxybutyrate) (PHB) was detected. Both species accumulated PHA to 6–9% of the dry weight. In Gloeothece PHB accumulation was stimulated by the addition of acetate but in O. limosa this was not the case. PHA was not involved in dark metabolism in either of the strains.

DNA topoisomerases in Gloeothece (Nägeli) sp. ATCC 27152: a role in N2 fixation?

summaryExtracts from aerobically grown cultures of the unicellular cyanobacterium Gloeothece (Nägeli) sp. ATCC 27152 converted relaxed ColE1 plasmid cccDNA into a supercoiled form. They therefore contained a DNA gyrase. However, when DNA gyrase was inhibited, these same extracts catalyzed a net relaxation of ColE1 DNA, implying that they also contained an enzyme similar to DNA topoisomerase I. During the first 2 h after transfer of aerobically grown cultures of Gloeothece to an atmosphere of O2, the ability of extracts to generate supercoiled DNA was temporarily lost. Instead, extracts catalyzed net relaxation of ColE1 DNA during this period, which coincided with a transient inhibition of nitrogenase synthesis. O2 and some related compounds also inhibited DNA supercoiling by extracts of Gloeothece in vitro. As in other diazotrophs therefore, O2 may inhibit nitrogenase synthesis in Gloeothece by altering the relative activities of DNA gyrase and DNA topoisomerase I in such a way that relaxation of one or more of the nif genes may occur, with consequent inhibition of transcription. However, this effect is only transient. In contrast, addition of NH4+ to cultures of Gloeothece permanently inhibited nitrogenase synthesis and did not abolish the ability of extracts to catalyze net supercoiling of DNA. Moreover, NH4+ did not abolish DNA supercoiling in vitro. NH4+ therefore appears to inhibit nitrogenase synthesis in Gloeothece by a mechanism different from that of O2.

Diazotrophic growth of the unicellular cyanobacterium Gloeothece sp. PCC 6909 in continuous culture

The unicellular cyanobacterium Gloeothece sp. PCC 6909 was grown diazotrophically in continuous culture. Various light-dark regimes and dilution rates were applied. Nitrogenase activity (acetylene reduction), oxygen-production and -consumption, and glycogen were measured over 24 h periods covering one complete light-dark cycle. Gloeothece fixed nitrogen predominantly in the light, concomitant with photosynthetic oxygen evolution. This result was found irrespective of the imposed light-dark regime or dilution rate. It is concluded that temporal separation of photosynthesis and nitrogen fixation is not obligatory in Gloeothece.

Fine-structural and chemical analyses on inner and outer sheath of the cyanobacteriumGloeothece sp. PCC 6909

Cells of the unicellular cyanobacterium Gloeothece sp. PCC 6909 are surrounded by an inner (enclosing 1-2 cells) and an outer (enclosing cell groups) sheath. Using conventional Epon-embedding in combination with ruthenium-red staining, the inner and outer sheaths appeared similar and displayed multiple bands of electron-dense subunits. However, embedding in Nanoplast resin to avoid shrinkage led to the detection of two distinct zones (inner and outer zone) each with several distinct layers. The zone delimited by the electron-dense thick inner sheath layer, and the zone enclosed by the thin electron-dense outer sheath layer, are composed of a homogeneous material of little electron-contrast. Whereas the outer zone appears to be of even contrast, the inner zone is characterized by a distinct electron-transparent layer. Element distribution analysis revealed that the electron-transparent layer contained relatively large amounts of sulfur, carbon, and oxygen but only little nitrogen. Inner and outer sheath fractions were isolated by differential mechanical cell breakage and centrifugation. The outer sheath fraction was less hydrated than the inner one. The two fractions differed little in their contents of uronic acids, carbohydrate and protein, although the outer sheath fraction contained less sulfate. A soluble polysaccharide with a chemical composition similar to that of inner and outer sheath fractions was also obtained from the culture supernatant.

2-O-methyl-d-xylose containing sheath in the cyanobacterium Gloeothece sp. PCC 6501

The sheath of the unicellular cyanobacterium Gloeothece sp. PCC 6501 was isolated from cell homogenates by differential and sucrose gradient centrifugation, followed by lysozyme treatment and hot sodium dodecyl sulfate extraction. The sheath contains a major fraction of carbohydrate consisting of galactose, glucose, mannose, rhamnose, 2-O-methyl-d-xylose, xylose, glucuronic and galacturonic acids, but only traces of fatty acids and phosphate. A protein content of about 2% (of fraction dry weight) could not be removed by the detergent treatment.

Nitrogen Fixation by Gloeothece sp. PCC 6909: Respiration and Not Photosynthesis Supports Nitrogenase Activity in the Light

SUMMARY: Nitrogenase activity of suspensions of the unicellular cyanobacterium Gloeothece sp. PCC 6909 plotted against the concentration of dissolved O2 (dO2) resulted in a bell-shaped curve, both in the light and in the dark, with optima of 25 or 80 μm-O2 depending on the age of the culture. At the optimum dO2, nitrogenase activity [typically 4 to 6 nmol C2H4 (mg protein)−1 min−1] was similar in the light or in the dark. Alteration of light intensity from zero to 2 klx, or addition of 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU), had no effect on nitrogenase activity. At 1 klx the ADP/ATP ratio was 0∙2 and showed only a marginal increase as the dO2 was increased. However, a high level of illumination (30 klx) stimulated or inhibited nitrogenase activity, depending on the external dO2, presumably as a consequence of changes in the intracellular O2 concentration; in the presence of DCMU, activity increased twofold, independent of dO2. In the dark, the dependence of the rate of respiration on O2 concentration suggested the presence of three O2-uptake systems with apparent K m values of 1 μm, 5 μm and 25 μm. The ADP/ATP ratio under anaerobic conditions was 0∙47 and showed a marked decrease as dO2 was increased to 25 μm. A CN-insensitive respiratory activity, which neither supported nitrogenase activity nor was coupled to ATP synthesis, was associated with the system with the apparent K m of 5 μm. The dependence of the specific activity of nitrogenase on dO2 indicated that both the high affinity (K m 1 μm) and low affinity (K m 25 μm) O2-uptake systems contributed ATP or reductant for N2-fixation. KCN (2∙5 mm) completely inhibited nitrogenase activity in the dark and at moderate levels of illumination and dO2. We conclude that respiration is the major source of reductant and ATP for nitrogenase activity both in the dark and in the light, but that photosystem I can contribute ATP at very high levels of illumination.

Studies on the Effects of Oxygen on Acetylene Reduction (Nitrogen Fixation) in Gloeothece sp. ATCC 27152

Cultures of Gloeothece sp. ATCC 27152 did not reduce acetylene when exposed to O2 concentrations greater than 0·7 atm. However, following exposure to 1 atm O2 for up to 12 h or to 0·8 atm O2 for up to 14 d, the ability to reduce acetylene recovered rapidly when cultures were returned to air. Complete recovery required active protein synthesis, probably for de novo synthesis of nitrogenase. Respiratory O2 consumption by cultures of Gloeothece was stimulated under elevated concentrations of O2. This respiration may contribute to the protection of nitrogenase from inactivation by O2 at concentrations up to 0·4 atm. The role of Ca2+ in nitrogen fixation may be related to respiratory protection.

Synthesis of Nitrogenase in the Cyanobacterium Gloeothece (Gloeocapsa) sp. CCAP 1430/3

55FeCI3 labelling and non-denaturing gel electrophoresis were used to study nitrogenase synthesis in Gloeothece sp. CCAP 1430/3. Nitrogenase synthesis was inhibited by addition of NHX but was unaffected by elevated concentrations of O2. Upon transfer of cultures of Gloeothece from light to darkness, there was initially a slight decrease in the rate of synthesis of nitrogenase but after 4–5 h there was an almost complete cessation of synthesis. This delayed effect of darkness on nitrogenase synthesis could not be related to any change in RNA synthesis, in protein synthesis or in the rate of breakdown of storage glucan. In cultures of Gloeothece, mRNA, including nif mRNA, was unstable, having a half-life of about 5 min. The synthesis of nif mRNA did not stop immediately upon transfer of cultures to the dark. If darkness exerts its effect on nitrogenase synthesis by inhibiting the synthesis of nif mRNA, it does so only after a lag of about 4 h.