Heterocyst Frequency Research Articles

Growth Characteristics of an Estuarine Heterocystous Cyanobacterium

A new estuarine filamentous heterocystous cyanobacterium was isolated from intertidal sediment of the Lagoa dos Patos estuary (Brazil). The isolate may represent a new genus related to Cylindrospermopsis. While the latter is planktonic, contains gas vesicles, and is toxic, the newly isolated strain is benthic and does not contain gas vesicles. It is not known whether the new strain is toxic. It grows equally well in freshwater, brackish and full salinity growth media, in the absence of inorganic or organic combined nitrogen, with a growth rate 0.6 d-1. Nitrogenase, the enzyme complex responsible for fixing dinitrogen, was most active during the initial growth phase and its activity was not different between the different salinities tested (freshwater, brackish, and full salinity seawater). Salinity shock also did not affect nitrogenase activity. The frequency of heterocysts was high, coinciding with high nitrogenase activity during the initial growth phase, but decreased subsequently. However, the frequency of heterocysts decreased considerably more at higher salinity, while no change in nitrogenase activity occurred, indicating a higher efficiency of dinitrogen fixation. Akinete frequency was low in the initial growth phase and higher in the late growth phase. Akinete frequency was much lower at high salinity, which might indicate better growth conditions or that akinete differentiation was under the same control as heterocyst differentiation. These trends have hitherto not been reported for heterocystous cyanobacteria but they seem to be well fitted for an estuarine life style.

In silico characterization and transcriptomic analysis of nif family genes from Anabaena sp. PCC7120.

In silico approaches in conjunction with morphology, nitrogenase activity, and qRT-PCR explore the impact of selected abiotic stressor such as arsenic, salt, cadmium, copper, and butachlor on nitrogen fixing (nif family) genes of diazotrophic cyanobacterium Anabaena sp. PCC7120. A total of 19 nif genes are present within the Anabaena genome that is involved in the process of nitrogen fixation. Docking studies revealed the interaction between these nif gene-encoded proteins and the selected abiotic stressors which were further validated through decreased heterocyst frequency, fragmentation of filaments, and downregulation of nitrogenase activity under these stresses indicating towards their toxic impact on nitrogen fixation potential of filamentous cyanobacterium Anabaena sp. PCC7120. Another appealing finding of this study is even though having similar binding energy and similar interacting residues between arsenic/salt and copper/cadmium to nif-encoded proteins, arsenic and cadmium are more toxic than salt and copper for nitrogenase activity of Anabaena which is crucial for growth and yield of rice paddy and soil reclamation.

Increased heterocyst frequency by patN disruption in Anabaena leads to enhanced photobiological hydrogen production at high light intensity and high cell density.

The effects of increasing the heterocyst-to-vegetative cell ratio on the nitrogenase-based photobiological hydrogen production by the filamentous heterocyst-forming cyanobacterium Anabaena sp. PCC 7120 were studied. Using the uptake hydrogenase-disrupted mutant (ΔHup) as the parent, a deletion-insertion mutant (PN1) was created in patN, known to be involved in heterocyst pattern formation and leading to multiple singular heterocysts (MSH) in Nostoc punctiforme strain ATCC 29133. The PN1 strain showed heterocyst differentiation but failed to grow in medium free of combined-nitrogen; however, a spontaneous mutant (PN22) was obtained on prolonged incubation of PN1 liquid cultures and was able to grow robustly on N2. The disruption of patN was confirmed in both PN1 and PN22 by PCR and whole genome resequencing. Under combined-nitrogen limitation, the percentage of heterocysts to total cells in the PN22 filaments was 13-15 and 16-18% under air and 1% CO2-enriched air, respectively, in contrast to the parent ΔHup which formed 6.5-11 and 9.7-13% heterocysts in these conditions. The PN22 strain exhibited a MSH phenotype, normal diazotrophic growth, and higher H2 productivity at high cell concentrations, and was less susceptible to photoinhibition by strong light than the parent ΔHup strain, resulting in greater light energy utilization efficiency in H2 production on a per unit area basis under high light conditions. The increase in MSH frequency shown here appears to be a viable strategy for enhancing H2 productivity by outdoor cultures of cyanobacteria in high-light environments.

Overexpression of AhpC enhances stress tolerance and N2–fixation in Anabaena by upregulating stress responsive genes

The study explores the significance of peroxides in regulating the CO2- and N2-fixation capacities in Anabaena sp. PCC7120. To this end Anabaena strains were generated carrying an extra copy of ahpC (An+ahpC) or by deleting from their endogenous functional ahpC (AnΔahpC). AhpC levels were 2.2- to 6.0-fold higher in An+ahpC than in wild type. An+ahpC revealed 1.4- to 2-fold upregulation of photosystems I and II, nitrogenase, superoxide dismutase and catalase activities while same activities were 1.3- to 2.5-fold downregulated in the insertional mutant (AnΔahpC) compared to the wild type. Peroxide, superoxide and malondialdehyde contents were low in An+ahpC and high in AnΔahpC. Growth was inhibited in AnΔahpC by approximately 40–60% compared to a 33–40% enhanced growth in An+ahpC under selected stresses. Most interestingly, heterocyst frequency was increased in An+ahpC. In order to address transcriptional and posttranscriptional effects, transcripts of genes including groEL, fld, kat, gor, gst, dps, bfr, tf, sodA, dnaK, prx, uspA, pcs and apx were quantified and found to be increased 1.33- to 7.70-fold in unstressed and 1.76- to 13.80-fold in stressed An+ahpC. In a converse manner, they were downregulated by 1.20- to 7.50-fold in unstressed and 1.23 to 10.20-fold in stressed AnΔahpC. It is concluded that the level of AhpC controls a major set of metabolic and developmental genes in normal and stress conditions and thus likely is in the core of the redox regulatory system of Anabaena.

English

Cyanobacteria are a cheap source of nitrogen and quite suitable for farmers of developing countries. Although, they live in a diverse range of environments, different environmental variables influence their nitrogen fixing ability. Thus, this study evaluated the effect of pond depth and lining plastic colors on nitrogen fixing capacity of Anabaena species strain E3. Factorial combinations of four pond lining plastic colors and two depths were laid out in a complete randomized design with three replications. The ANOVA results revealed that the 20 cm depth pond had a higher mean growth rate (0.063 OD day -1 ), dissolved oxygen (17.63 mg L -1 ), dry biomass (0.58 g L -1 ) and total nitrogen (27.77 mg L -1 ) than the 40 cm depth. The highest mean growth rate (0.089 OD day -1 ), dissolved oxygen (19.07 mg L -1 ), dry biomass (0.66 g L -1 ) and total nitrogen (41.60 mg L -1 ) were registered in the treatment with a transparent lining plastic color. Moreover, it was noted that, the test strain under the treatment combinations of 20 cm depth and transparent plastic lining expressed the highest mean optical density (1.227), heterocyst frequency (2.92%) and pH (10.28). Therefore, for mass-production of E3-strain-based biofertilizer, the strain should be grown in ponds of 20 cm depth with transparent plastic. Keywords: Cyanobacteria, dry biomass, growth rate, shallow pond, total nitrogen

Salinity induced physiological and biochemical changes in the freshly separated cyanobionts of Azolla microphylla and Azolla caroliniana

Freshly separated cyanobionts of Azolla microphylla and Azolla caroliniana plants exposed to salinity showed decline in the cellular constituents such as chlorophyll (23.1 and 38.9%) and protein (12.9 and 19.3%). However, an increase in the carotenoid and sugar content was observed. Exposure to salinity stress reduced the heterocyst frequency (35.4 and 57.2%) and nitrogenase activity (37.7 and 46.3%) of the cyanobionts. Increase in the activity of antioxidant enzymes such as super oxide dismutase (50.6 and 11.5%), ascorbate peroxidase (63.7 and 57.9%), catalase (94.2 and 22.5%) as well as non-enzymatic antioxidant proline (18.8 and 13.3%) was also observed in response to salinity. The cyanobionts exhibited significant increase in the intracellular Na+ level and reduced intracellular K+/Na+ and Ca2+/Na+ ratio in response to salinity. The results demonstrate the adverse impact of salinity on the freshly separated cyanobionts as similar to free living cyanobacteria. These results may be helpful in the critical evaluation of salinity tolerance mechanism of the cyanobiont and its interaction with the host.

Effect of gibberellic acid on the cyanobacterium Nostoc linckia

We investigated the influence of gibberellic acid (GA3; 0, 1, 10, and 100 μM) on Nostoc linckia culture at 7, 14, and 21 days. The fresh and dry weight of N. linckia was increased considerably by the 10 and 100 μM GA3 treatments. A reduction in heterocyst frequency was observed in cultures treated with 1 and 10 μM GA3. Adding GA3 to N. linckia culture had a little effect on cell size. The amount of chlorophyll a and carotenoids decreased at all concentrations of GA3. The amount of phycocyanin increased up to twofold in 7-day-old culture treated with 1 μM GA3, and similar changes were observed for allophycocyanin and phycoerythrin content after 7 days. The effect of GA3 on reducing sugar content was different and was dependent on the growth period. A reduction in soluble sugar content was detected after GA3 application in 7- and 14-day-old cyanobacteria. Cultures treated with GA3 had a higher protein content after 14 days and a lower protein content after 7 and 21 days, and reduced nitrogenase activity after 7, 14, and 21 days. Our data show that GA3 application can be a suitable and inexpensive way to increase N. linckia biomass and phycobiliprotein production.

Investigation of the links between heterocyst and biohydrogen production by diazotrophic cyanobacterium A. variabilis ATCC 29413.

This work investigates the effect of heterocyst toward biohydrogen production by A. variabilis. The heterocyst frequency was artificially promoted by adding an amino acid analog, in this case DL-7-azatryptophan into the growth medium. The frequency of heterocyst differentiation was found to be proportional to the concentration of azatryptophan (0-25 µM) in the medium. Conversely, the growth and nitrogenase activity were gradually suppressed. In addition, there was also a distinct shortening of the cells filaments and detachment of heterocyst from the vegetative cells. Analysis on the hydrogen production performance revealed that both the frequency and distribution of heterocyst in the filaments affected the rate of hydrogen production. The highest hydrogen production rate and yield (41 µmol H2 mg chl a(-1) h(-1) and 97 mL H2 mg chl a(-1), respectively) were achieved by cells previously grown in 15 µM of azatryptophan with 14.5 % of heterocyst frequency. The existence of more isolated heterocyst has been shown to cause a relative loss in nitrogenase activity thus lowering the hydrogen production rate.

Facile and novel synthetic method to prepare nano molybdenum and its catalytic activity.

This study reports on a facile and economical synthetic method to prepare nano molybdenum by solid-state reaction technique. Metallic nano molybdenum was synthesised from molybdenum trioxide, molybdenum IV oxide and molybdenum VI oxide through thermal decomposition technique. Metallic nano molybdenum prepared from molybdenum IV oxide was used to study the catalytic effect of molybdenum nanoparticles on the growth of Anabaena sp. The increase in concentration of nano molybdenum from 0.1 to 100% in BG11 (N⁻ Mo⁻ + nano Mo) medium increases heterocyst frequency. The chlorophyll and protein content in Anabaena sp. was found to improve when compared with bulk molybdenum particles and showed a positive influence to be used as a nano nutrient for Anabaena sp.

Cadmium removal by Anabaena doliolum Ind1 isolated from a coal mining area in Meghalaya, India: associated structural and physiological alterations

ABSTRACT The cyanobacterium Anabaena doliolum Ind1 isolated from a coal mining site was tested for removal of cadmium at optimum pH 7.0 and temperature 25˚C. The organism recorded high percentage of metal removal (92-69%) within seven days of exposure to 0.5-2.0 ppm cadmium. Biosorption onto the cell surface was the primary mode of metal removal. Fourier transform infrared spectroscopy (FTIR) established hydroxyl, amides, carboxyl, sulphate and carbonyl groups to be the major functional groups on the cell surface involved in cadmium binding. Cellular ultrastructure and a range of vital physiological processes (i.e., photosynthetic pigments, respiration, photosynthesis, heterocyst frequency and nitrogenase activity) remained unaffected upon 0.5 ppm treatment; higher concentrations of cadmium exerted visible adverse effects. Amongst the five photosynthetic pigments tested, phycocyanin was the most targeted pigment (inhibition was 15-89%). Both respiration and photosynthetic activities were inhibited by cadmium with more severe effect seen on respiration. 2.0 ppm cadmium exposure also had drastic negative effect on nitrogenase activity (87% decreased).

The Potential of Cyanobacteria Growth in Different Water Sources

Many studies have addressed the effect of different environmental factors on growth, heterocyst frequency and N fixation of cyanobacteria. One of the factors is water quality. Different cyanobacteria strains different efficiency to adapt the quality of water. Therefore, this study was conducted to select the best combination of N-fixing cyan bacterial strains under different water sources. A factorial combination of the three Anabaena species strains (E10, E3 and E8) and two water sources (tap water and distilled water) were laid out in a complete randomized design in the laboratory set up. The ANOVA revealed that cyanobacteria strain E3 performed best on distilled than in tap water in terms of optical density, heterocyst frequency and total nitrogen than the other strains but no significant differences appeared between the two water sources. Thus, indicated that cyaobabacteria strains are growing in different water sources and the ability to grow more than one water sources.

Intercellular transfer along the trichomes of the invasive terminal heterocyst forming cyanobacterium Cylindrospermopsis raciborskii CS-505.

Cylindrospermopsis raciborskii CS-505 is an invasive freshwater filamentous cyanobacterium that when grown diazotrophically may develop trichomes of up to 100 vegetative cells while differentiating only two end heterocysts, the sole sites for their N2-fixation process. We examined the diazotrophic growth and intercellular transfer mechanisms in C. raciborskii CS-505. Subjecting cultures to a combined-nitrogen-free medium to elicit N2 fixation, the trichome length remained unaffected while growth rates decreased. The structures and proteins for intercellular communication showed that while a continuous periplasmic space was apparent along the trichomes, the putative septal junction sepJ gene is divided into two open reading frames and lacks several transmembrane domains unlike the situation in Anabaena, differentiating a 5-fold higher frequency of heterocysts. FRAP analyses also showed that the dyes calcein and 5-CFDA were taken up by heterocysts and vegetative cells, and that the transfer from heterocysts and 'terminal' vegetative cells showed considerably higher transfer rates than that from vegetative cells located in the middle of the trichomes. The data suggest that C. raciborskii CS-505 compensates its low-frequency heterocyst phenotype by a highly efficient transfer of the fixed nitrogen towards cells in distal parts of the trichomes (growing rapidly) while cells in central parts suffers (slow growth).

Structural and functional stability of regenerated cyanobacteria following immobilization

Cyanobacterial preservation is important to ensure availability of pure strains for research without repeating the extensive process of isolation and purification. Preservation techniques should be able to conserve various wild-type characteristics of a strain and deter any loss of phenotypic characters that were originally present in the organism. In the present study, immobilization of four different cyanobacterial strains in calcium alginate beads was studied extensively to evaluate the merits of this technique in conserving various characteristics of these strains over a long period of time. Since the immobilized cultures were stored in dehydrated conditions and in dark for a period of 3 years, their viability and retention of assorted characteristics were of interest to credit this method for cyanobacterial preservation. Morphological investigations carried out using SEM provided excellent proof in favor of the technique. In addition, various biochemical analyses showed retention of growth rate, heterocyst frequency, and photosynthetic and respiratory abilities as well as performances of various enzymes of nitrogen metabolism such as nitrogenase, glutamine synthetase, nitrate reductase, and nitrite reductase. An investigation into molecular fingerprints of the organisms using PCR also presented proof of genetic stability in the organisms stored immobilized in calcium alginate beads for 3 years in dark under dehydrated conditions.

Regulation of heterocyst frequency in Baltic Sea Aphanizomenon sp.

Heterocyst frequencies in Baltic Sea Aphanizomenon sp. were similar along a strong nutrient gradient from the discharge point of a sewage treatment plant at the head of the Himmerfjarden bay to the ...

Regulation of calcium ion and its effect on growth and developmental behavior in wild type and ntcA mutant of Anabaena sp. PCC 7120 under varied levels of CaCl2

A study of calcium ion regulation in Anabaena 7120 and its derivative mutant (CSE2) strain impaired in ntcA gene were investigated in terms of altered morphological and physiological responses against various levels of calcium stress (0–100 mM). Calcium concentration of 10 mM was found to be inhibitory while 100 mM proved lethal for both wild type and mutant strain. The involvement of Ca2+ in the regulation of cellular processes has been described in terms of an influx or efflux of Ca2+ from the cytosol. A biphasic calcium uptake with difference in calcium influx and efflux rate was responsible for differential amount of remaining calcium which followed a decreasing trend both for wild type and mutant. Low K s 0.5 and high V max in mutant suggest heavy and less restricted influx of calcium ion. Further, the interactive effect of calcium influx/efflux rate, remaining Ca2+ and intracellular levels of Na+ and K+ may be attributed for the degree of membrane damage and growth sustenance during exogenous supply of calcium salt. Widening in heterocyst spacing pattern, decreased heterocyst frequency and formation of abnormal cell structures at higher concentration (100 mM CaCl2) suggest that calcium mediated regulatory process modulate heterocyst frequency and maintenance of cell structure. Further, poor regulation of calcium ion homeostasis in ntcA suggests that the calcium level and ntcA gene expression are inter-related.

Enhanced Resistance to UV-B Radiation in Anabaena sp. PCC 7120 (Cyanophyceae) by Repeated Exposure

In natural habitats, organisms especially phytoplankton are not always continuously subjected to ultraviolet-B radiation (UVBR). By simulation of the natural situation, the N2-fixing cyanobacterium Anabaena sp. PCC 7120 was subjected to UV-B exposure and recovery cycles. A series of morphological and physiological changes were observed in Anabaena sp. PCC 7120 under repeated UVBR when compared with controls. Such as the breakage of filaments, intervals between heterocysts, heterocyst frequency, total carbohydrate, and carotenoids were increased, while the nitrogenase activity and photosynthetic activity were inhibited by repeated UVBR; however, these activities could recover when UV-B stress was removed. Unexpectedly, the over-compensatory growth was observed at the end of the second round of exposure and recovery cycle. Our results showed that discontinuous UVBR could increase the growth rate and the tolerance as well as repair capacity of Anabaena sp. PCC 7120. These results indicate that moderate UVBR may increase the growth of cyanobacteria in natural habitats.

Effects of lead accumulation on the Azolla caroliniana–Anabaena association

The effect of lead accumulation on photopigment production, mineral nutrition, and Anabaena vegetative cell size and heterocyst formation in Azolla caroliniana was investigated. Plants were exposed to 0, 1, 5, 10, and 20mgL−1 lead acetate for ten days. Lead accumulation increased when plants were treated with higher lead concentrations. Results revealed a statistically significant decline in total chlorophyll, chlorophyll a, chlorophyll b, and carotenoids in 5, 10, and 20mgPbL−1 treatment groups as compared to plants with 0 or 1mgPbL−1 treatments. No statistically significant change in anthocyanin production was observed. Calcium, magnesium, and zinc concentrations in plants decreased in increasing treatment groups, whereas sodium and potassium concentrations increased. Nitrogen and carbon were also found to decrease in plant tissue. Anabaena vegetative cells decreased in size and heterocyst frequency declined rapidly in a Pb dose-dependent manner. These results indicate that, while A. caroliniana removes lead from aqueous solution, the heavy metal causes physiological and biochemical changes by impairing photosynthesis, changing mineral nutrition, and impeding the growth and formation of heterocysts of the symbiotic cyanobacteria that live within leaf cavities of the fronds.

Effects of Copper Ion (Cu<sup>2+</sup>) on the Physiological and Biochemical Activities of the Cyanobacterium <i>Nostoc</i> ANTH

The effects of copper sulphate on the cyanobacterium Nostoc ANTH were studied. The copper concentrations chosen for the study were 0.5, 1.0, and 5.0 mg/L. From the results, it was observed that removal of Cu2+ ions by the organism was dependent on the external concentrations of Cu 2+ ions. Exposure to high concentrations of copper led to severe morphological changes in the cells, which were visible under transmission electron microscopy. Yet, complete elimination of copper from the growth medium led to poor growth, and ultimately the death, of the Nostoc ANTH cells. Cells exposed to low copper concentration (0.5 mg/L) in the medium showed comparable physiological and biochemical activities (such as growth, heterocyst frequency, nitrogenase activity, photosynthesis, and respiration) to that of the controlled cultures. However, these features were compromised as the copper concentration increased in the surrounding.

Expression of nifH, hetR, and ndaF genes in Nodularia spumigena under nitrogen sources

Acetylene reduction assays (ARAs) and real-time reverse transcriptase-polymerase chain reaction (RT-PCR) analyses were performed to determine the effect of exogenous nitrogen sources on nitrogen fixation activity in three Nodularia spumigena isolates (KAC11, KAC71, and KAC66). It was observed that nitrogen fixation activity decreased in all three isolates in the presence of relatively high levels of ammonium (0.5 mM and 1.0 mM NH4Cl). Concomitantly, real-time RT-PCR analysis showed that expression of nifH, which encodes the dinitrogenase reductase component of the nitrogenase enzyme, decreased in response to ammonium treatment. On the other hand, nitrate (NaNO3) does not seem to affect the nitrogen fixation process. All isolates continuously expressed hetR, encoding the master gene of heterocyst differentiation, and maintained heterocyst frequency along the filaments both in the presence of ammonium and nitrate, even though nitrogen fixation had ceased. Furthermore, expression of ndaF, a marker gene for nodularin production, dropped in the presence of ammonium, indicating a possible correlation between nitrogen fixation and toxin production in these N. spumigena isolates.

Morphological characterization and molecular fingerprinting of Nostoc strains by multiplex RAPD

Morphological parameters studied for the twenty selected Nostoc strains were mostly found to be consistent with the earlier reports. But the shape of akinetes observed in this study was a little deviation from the existing descriptions and heterocyst frequency was also found to be different in different strains in spite of growing in the same nitrogen free media. Multiplex RAPD produced reproducible and completely polymorphic amplification profiles for all the strains including some strain specific unique bands which are intended to be useful for identification of those strains. At least one to a maximum of two unique bands was produced by different dual primer combinations. For ten strains out of twenty, strain specific bands were found to be generated. Cluster analysis revealed a vast heterogeneity among these Nostoc strains and no specific clustering based on geographical origin was found except a few strains. It was also observed that morphological data may not necessarily correspond to the genetic data in most of the cases. CCC92 (Nostoc muscorum) and CCC48 (Nostoc punctiforme) showed a high degree of similarity which was well supported by high bootstrap value. The level of similarity of the strains ranged from 0.15 to 0.94. Cluster analysis based on multiplex RAPD showed a good fit revealing the discriminatory power of this technique.