Effect of ammonia and sulfide on rifampicin-induced heterocyst formation inNostoc linckia

The effect of ammonia and sulfide on rifampicin-induced heterocyst differentiation was studied in the nitrogen-fixing cyanobacteriumNostoc linckia. Aerobic growth with nitrogen gas of the cyanobacterium was greatly affected by rifampicin with formation of multiple heterocysts in chains in the filaments whereas ammonia in the medium reversed the rifampicin inhibition of growth and prevented the induction of heterocysts. In a sulfide medium the suppression exerted by rifampicin on aerobic growth with nitrogen gas and heterocyst induction was found to be considerably reduced. The results suggest two interesting points,viz. that (i) rifampicin interferes with the nitrogen-fixing function of heterocysts, and (ii) it checks the synthesis of an unknown heterocyst, inhibitor and thus permits the adjacent vegetative cells to differentiate into heterocysts in chains.     

THE HETEROCYSTS OF BLUE-GREEN ALGAE (MYXOPHYCEAE)

1. Heterocysts are found in many species of filamentous blue-green algae. They are cells of slightly larger size and with a more thickened wall than the vegetative cells. 2. Structural details of the heterocyst are: the presence of three additional wall layers, the absence of granules, sparse thylakoid network throughout, except at the poles where a dense coiling of membranes occurs. Other characters include the two pores at opposite poles ‘plugged’ with refractive material called the polar granule. 3. Peculiarities in the pigment composition of the heterocyst include an abundance of carotenoids and absence of phycobilins, and a short-wave form of chlorophyll a. 4. Unique glycolipids and an acyl lipid, not found in the vegetative cells of the algae or in other plant cells, are associated with the heterocyst. The glycolipids constitute the laminated layer of the wall and probably regulate diffusion of substances through it, whereas the acyl lipids are supposed to function as carriers and intermediates in the biosynthesis of the wall. 5. The heterocysts develop from vegetative cells, and the visible changes during differentiation include cell enlargement, synthesis of additional wall layers, disappearance of granules and reorientation and synthesis of the thylakoids. 6. Heterocysts are formed sequentially with characteristic cellular spacing during the growth of cultures in medium free from combined nitrogen. 7. Various sources of combined nitrogen inhibit heterocyst formation when supplied in the culture medium. Ammonium salts are among the most powerful inhibitors. Heterocysts are formed simultaneously and within a short period after transference of ammonia-grown non-heterocystous filaments to ammonia-free medium. 8. Incompletely differentiated heterocysts or proheterocysts are found in cultures grown in the presence of combined nitrogen. If two or more proheterocysts are close together generally a single one develops to maturity after a competitive interaction in medium free from combined nitrogen. This indicates that heterocyst formation is completed in two phases: phase I, synthesis and conservation of macromolecules, which takes place during growth in ammonia-containing medium: and phase 11, morphological differentiation of the heterocyst which is unaccompanied by growth in cell number. In the ammonia-free medium phase 11 quickly succeeds phase 1 and the whole process appears as a continuum. 9. Heterocyst formation shows a definite requirement for light. Red light favours heterocyst formation, whereas green and blue light do not. The effects of light seem to be mainly due to photosynthesis, although some effects may be morphogenetic. 10. Studies with metabolic inhibitors have revealed the involvement of photosynthesis, respiration and protein synthesis in heterocyst formation. Photosynthesis provides carbon skeletons, whereas ATP is most probably supplied by oxidative metabolism. 11. Various functions have been assigned to the heterocyst from time to time. Their role in akinete formation is suggested by (i) the formation of akinetes adjacent to the heterocysts and (ii) prevention of sporulation by detachment of the heterocysts from the vegetative cells (potential akinetes). Despite substantial evidence for such a role, it is not applicable to all akinete-forming genera. 12. Heterocysts are now widely believed to be the site of nitrogen fixation in blue-green algae. The main facts in favour of such a role are: (i) fixation of nitrogen by all heterocystous algae, (ii) inhibition of heterocyst formation by combined nitrogen and (iii) direct observations on acetylene reduction by isolated heterocysts. 13. Some non-heterocystous and unicellular algae, and vegetative cells of heterocystous algae fix nitrogen under microaerophilic conditions suggesting that absence of oxygen favours nitrogenase activity. Heterocysts lack the oxygen-evolving photo-system 11, possess oxidative enzymes, and reduce externally supplied tetrazolium salts - all indicating that they are the most suitable sites for harbouring nitrogenase in aerobic conditions. 14. Heterocysts probably originated in the Precambrian in response to the earth's changing environment and seem to be the first example of morphological differentiation in the plant kingdom.     

INFLUENCE OF NITROGEN SOURCE ON MORPHOLOGY OF RIVULARIACEAE (CYANOPHYTA)1

Thirty-four heterocyst-producing strains of Rivulariaceae (29 Calothrix, 1 Dichothrix, 2 Gloeotrichia, 2 Rivularia), which produced tapered trichomes in medium minus combined nitrogen, were grown in the presence of nitrate. One strain was unchanged in morphology under this condition. The remaining 33 strains developed trichomes lacking heterocysts. In 19 strains almost all the trichomes became untapered and in the other 14, similar untapered trichomes were produced, but also many tapered trichomes resembling Homoeothrix or Hammatoidea. Similar results were obtained when representative strains were incubated with ammonia as the source of combined N. Only five strains formed colorless hairs in the control medium (minus combined N). The presence of combined N did not diminish hair development in the two strains which had only a few short hairs, but hair frequency and length were both reduced considerably in the three strains with many long hairs in the control medium. Two strains of the non-heterocystous genus Homoeothrix were incubated in medium without combined N. Neither strain showed any growth or heterocyst development, indicating that neither is simply a growth form of a heterocystous genus.     

The Anabaena sp. strain PCC 7120 asr1734 gene encodes a negative regulator of heterocyst development

The novel asr1734 gene of Anabaena (Nostoc) sp. strain PCC 7120 inhibited heterocyst development when present in extra copies. Overexpression of asr1734 inhibited heterocyst development in several strains including the wild type and two strains that form multiple contiguous heterocysts (Mch phenotype): a PatS null mutant and a hetR(R223W) mutant. Overexpression of asr1734 also caused increased nblA messenger RNA levels, and increased loss of autofluorescence in vegetative cells throughout filaments after nitrogen or sulphur depletion. Unlike the wild type, an asr1734 knockout mutant formed 5% heterocysts after a nitrogen shift from ammonium to nitrate, and formed 15% heterocysts and a weak Mch phenotype after step-down to medium lacking combined nitrogen. After nitrogen step-down, the asr1734 mutant had elevated levels of ntcA messenger RNA. A green fluorescent protein reporter driven by the asr1734 promoter, P(asr1734)-gfp, was expressed specifically in differentiating proheterocysts and heterocysts after nitrogen step-down. Strains overexpressing asr1734 and containing P(hetR)-gfp or P(patS)-gfp reporters failed to show normal patterned upregulation 24 h after nitrogen step-down even though hetR expression was upregulated at 6 h. Apparent orthologues of asr1734 are found only in two other filamentous nitrogen-fixing cyanobacteria, Anabaena variabilis and Nostoc punctiforme.     

Formation of one-dimensional patterns by stochastic processes and by filamentous blue-green algae

The distribution of distances between centers in a pattern arising on a line, based on the Johnson-Mehl and associated models, is investigated analytically and by computer simulation. The distribution of spacings between heterocysts arising in a filament initially free of heterocysts was found to differ from the distribution expected if cells differentiating into heterocysts inhibit nearby cells from so differentiating by means of an undamped domino effect, and to approximate the distribution expected if inhibition is by a diffusion phenomenon. The data presented support the premise that the inhibition, by heterocysts, of heterocyst formation is mediated by a diffusible substance produced by heterocysts. The inhibition can be impaired by rifampicin.     

Regulation of CO on heterocyst differentiation and nitrate uptake in the cyanobacterium Anabaena sp. PCC 7120

AIMS: The aim of the present investigation was to study the effects of different inorganic carbon and nitrogen sources on nitrate uptake and heterocyst differentiation in the culture of cyanobacterium Anabaena sp. PCC 7120. METHODS AND RESULTS: Anabaena was cultivated in media BG11 containing combined nitrogen and supplementary NaHCO3 or CO2. Cell growth, heterocyst differentiation, nitrate reductase (NR, EC 1.7.7.2), glucose-6-phosphate dehydrogenase (G6PDH, EC 1.1.1.49) and NO uptake were analysed. The cells cultivated in BG11(0) medium with aeration were taken as reference. Experimental results showed that the differentiation frequency of heterocysts when the cells were cultivated with elevated CO2 was higher than that of the cells grown with air or bicarbonate. Heterocysts appeared unexpectedly when CO2 was introduced into the medium containing nitrate. However, no heterocysts emerged when CO2 was added to medium containing NH or urea, or when NaHCO3 was supplied to the medium with nitrate. Both nitrate uptake rate and nitrate reduction enzyme activity were depressed by the supplement of CO2 to the culture. The activity of G6PDH was enhanced with the increase in heterocyst differentiation frequency. CONCLUSION: CO2 might compete with NO for energy and electrons in the uptake process and CO2 appears favoured. This led to a high intracellular C/N ratio and a relative N limitation. So the process of heterocyst differentiation was activated to supplement nitrogen uptake. SIGNIFICANCE AND IMPACT OF THE STUDY: This study provided an attractive possibility to form more heterocysts by rapid growth of Anabaena cells cultivated in the medium containing nitrate in order to increase nitrogen fixation and hydrogen production.     

The Accumulation and Degradation Dynamics of Cyanophycin in Cyanobacterial Cells Grown in Symbiotic Associations with Plant Tissues and Cells

Five different artificial associations of cyanobacterial cells with the cells or tissues of nightshade and rauwolfia were studied. The associations grown on nitrogen-containing media produced heterocysts. Cyanobacterial cells in the associations retained their ability to take up combined nitrogen from the medium, to store it in the form of cyanophycin granules, and to use them in the process of symbiotic growth. The synthesis and degradation of cyanophycin granules in cyanobacterial cells were more active in the associations than in monocultures. In the symbiotic associations of Chlorogloeopsis fritschii ATCC 27193 with Solanum laciniatum cells and of Nostoc muscorum CALU 304 with the Rauwolfia serpentina callus, heterocysts were produced with a 3- to 30-fold higher cyanophycin content than in pure cyanobacterial cultures. In contrast, in the association of N. muscorum CALU 304 with the Solanum dulcamara callus, heterocysts were produced with a lower cyanophycin content than in the N. muscorum CALU 304 pure culture. The degradation of cyanophycin granules in N. muscorum CALU 304 cells grown in associations with plant tissues or cells was subjected to mathematical analysis. The activation of cyanophycin degradation and heterocyst differentiation in the associations N. muscorum CALU 304–R. serpentinaand C.fritschii–S. laciniatum was accompanied by an enhanced synthesis of the nitrogen-containing alkaloids in plant cells. The data obtained suggest that an integrated system of nitrogen homeostasis can be formed in symbiotic associations. Depending on the growth stage of an association, its plant member can either stimulate the accumulation of combined nitrogen in vegetative cyanobacterial cells in the form of cyanophycin granules, activate their degradation, or initiate the formation of heterocysts independently of the cyanobacterial combined nitrogen deprivation sensing-signaling pathway.     

Heterocyst-specific expression of patB,a gene required for nitrogen fixation in Anabaena sp. strain PCC 7120

The patB gene product is required for growth and survival of the filamentous cyanobacterium Anabaena sp. strain PCC 7120 in the absence of combined nitrogen. A patB::gfp fusion demonstrated that this gene is expressed exclusively in heterocysts. patB mutants have a normal initial pattern of heterocyst spacing along the filament but differentiate excess heterocysts after several days in the absence of combined nitrogen. Expression of hetR and patS, two critical regulators of the heterocyst development cascade, are normal for patB mutants, indicating that patB acts downstream of them in the differentiation pathway. A patB deletion mutant suffers an almost complete cessation of growth and nitrogen fixation within 24 h of combined nitrogen removal. In contrast, a new PatB mutant that is defective in its N-terminal ferredoxin domain, or a previously described mutant that has a frameshift removing its C-terminal helix-turn-helix domain, grows very slowly and differentiates multiple contiguous heterocysts under nitrogen-deficient conditions.     

FREQUENT HETEROCYST GERMINATION IN THE BLUE-GREEN ALGA GLOEOTRICHIA GHOSEI SINGH1

A unique feature, frequent heterocyst germination, has been observed in a nonsporulating mutant clone (of spontaneous origin) of the blue-green alga Gloeotrichia ghosei Singh. The controlling factor seems to be the presence of ammoniacal nitrogen in the medium. In addition, such a medium supports differentiation of successive crops of new heterocysts and their germination in the name medium and in the same algal culture. Contrary to previous observations with oilier blue-green algae, ammoniacal nitrogen does not seem to inhibit heterocyst differentiation in this alga. Both the parent alga and its mutant clone grow poorly in a nitrogen-free medium, which, although they are not completely free from bacteria, may indicate that they tire poor fixers or nonfixers. However, they form a large number of heterocysts under these conditions. The general conclusion is that the heterocysts of blue-green algae show a multiplicity of structure and function. In the present case they have reproductive function leading to direct propagation of the alga. The bearing of these findings on the interrelationships of the genera Gloeotrichia and Rivularia has been discussed. It has been concluded that the distinction between them is purely artificial.     

Morphological and ultrastructural changes in vegetative cells and heterocysts of Anabaena variabilis grown with fructose.

The morphology and ultrastructure of Anabaena variabilis grown in medium with and without 40 mM fructose were compared. Vegetative cells and young heterocysts in fructose-supplemented medium were significantly larger, were filled with glycogen granules, and had fewer thylakoids. Developing heterocysts contained large numbers of glycogen granules well into mature stages, and envelope formation was precocious. As heterocysts enlarged in fructose medium, their shape became more broadly oblong compared with the more rectangular heterocysts in fructose-free medium.     

Cellular differentiation and nitrogenase activity in the cyanobacteriumAnabaena

Nitrogenase activity at periods of differentiation of heterocysts and akinetes was assayed by the acetylene reduction technique. There was no nitrogenase activity in ammoniumgrown, non-heterocystousAnabaena sp.; the activity appeared only after a lag-phase of about 17 – 21 h after the ammonium-grown culture had been transferred to medium free of combined nitrogen. This activity started appearing as the proheterocysts were developing to mature heterocysts. Maximum nitrogenase activity was attained with exponential phase of culture and mature heterocysts. This activity gradually decreased with the differentiation of akinetes. Only insignificant nitrogenase activity was observed in old cultures in which most cells had matured into akinetes.     

Ascorbic Acid and Heterocyst Development in the Blue–Green Alga Amabaema ambigua

The nitrogen–fixing blue–green alga Anabaena ambigua was grown in a medium which contained either ammonium chloride as nitrogen source or molecular nitrogen. In the latter case the alga produced heterocysts. The material was analysed for ascorbic acid, dehydro-ascorbic acid and diketogulonic acid. The amount of a,scorbic acid was found always to he higher in the alga grown with molecular nitrogen. When the alga grown with combined nitrogen was transferred to the medium lacking it, there was an increase in the ascorbic acid content. Conversely, when material cultured on the nitrogen–free medium was suspended in the medium containing ammonium chloride, there was a decrease in the cellular ascorbic acid. Esogenously added ascorbic acid, up to 0.5 mg per ml, increased the heterocyst frequency to nearly three times that of the control. D–isoascorbic acid, an analogue of ascorbic acid, also showed an enhancement of heterocyst production. Algal extracts were fractionated by poiyacrylamide electro–phoresis, and the presence of ascorbic acid oxidase was detected on the gels. Two bands, with Rf values 0.34 and 1.0, were found to give positive test: for the enzyme. The total enzyme activity was 16.7 % higher in cells grown with molecular nitrogen than in those grown with combined nitrogen. The exact location of the enzyme in the alga ist not known although the heterocysts were earlier shown to contain ascorbic acid. Cytochemical tests, however, indicated strong per–oxidase activity in the heterocysts.     

The accumulation and degradation dynamics of cyanophycin in cyanobacteria grown in symbiotic associations with plant tissues and cells

Five different artificial associations of cyanobacterial cells with the cells or tissues of nightshade and rauwolfia were studied. The associations grown on nitrogen-containing media produced heterocysts. Cyanobacterial cells in the associations retained their ability to take up bound nitrogen from the medium, to store it in the form of cyanophycin granules, and to use them in the process of symbiotic growth. The synthesis and degradation of cyanophycin granules in cyanobacterial cells were more active in the associations than in monocultures. In the symbiotic associations of Chlorogloeopsis fritschii ATCC 27193 with Solanum laciniatum cells and of Nostoc muscorum CALU 304 with the Rauwolfia serpentina callus, heterocysts were produced at 3- to 30-fold higher cyanophycin contents than in cyanobacterial monocultures. In contrast, in the association of N. muscorum CALU 304 with the Solanum dulcamara callus, heterocysts were produced at lower cyanophycin contents than in the N. muscorum CALU 304 monoculture. The degradation of cyanophycin granules in N. muscorum CALU 304 cells grown in associations with plant tissues or cells was subjected to mathematical analysis. The activation of cyanophycin degradation and heterocyst production in the associations N. muscorum CALU 304-R. serpentina and C. fritschii-S. laciniatum was accompanied by an enhanced synthesis of the nitrogen-containing alkaloids in plant cells. The data obtained suggest that an integrated system of nitrogen homeostasis can be formed in symbiotic associations. Depending on the growth stage of an association, its plant member can either stimulate the accumulation of bound nitrogen in vegetative cyanobacterial cells in the form of cyanophycin granules, or activate their degradation, or initiate the formation of heterocysts independently of the cyanobacterial sensory-signalling system.     

A novel solid dosage form of rifampicin and isoniazid with improved functionality

The aim of the present investigation was to develop a novel dosage form of rifampicin and isoniazid to minimize degradation of rifampicin in acidic medium and to modulate the release of rifampicin in the stomach and isoniazid in the intestine. Gastroretentive tablets of rifampicin (150 mg) were prepared by the wet granulation method using hydroxypropyl methylcellulose, calcium carbonate, and polyethylene glycol 4000. The granules and tablets of rifampicin were characterized. Hard gelatin capsules (size 4) containing a compacted mass of isoniazid (150 mg) and dicalcium phosphate (75 mg) were enteric coated. Two tablets of rifampicin and 1 capsule (size 4) of isoniazid were put into a hard gelatin capsule (size 00). The in vitro drug release and in vitro drug degradation studies were performed. Rifampicin was released over 4 hours by zero-order kinetics from the novel dosage form. More than 90% of isoniazid was released in alkaline medium in 30 minutes. The results of dissolution studies with the US Pharmacopeia XXIII method revealed that a substantial amount of rifampicin was degraded from the immediate release capsule containing rifampicin and isoniazid powder owing to drug accumulation in the dissolution vessel and also to the presence of isoniazid. The degradation of rifampicin to 3-formyl rifampicin SV (3FRSV) was arrested (3.6%–4.8% degradation of rifampicin at 4 hours) because of the minimization of physical contact between the 2 drugs and controlled release of rifampicin in acidic medium in the modified Rossett-Rice apparatus. This study concludes that the problem of rifampicin degradation can be alleviated to a certain extent by this novel dosage form. Published: August 24, 2007     

The relation of heterocysts and hormogonia to nitrogen fixation and reproduction in blue-green algae

Indications are presented that the heterocysts of a member of theRivulariaceae germinate in NO₃ free medium containing low concentrations of NH₄Cl and glucose. Growth occurred regularly in the absence of combined nitrogen sources (NH₄, NO₃), suggesting N₂ fixation. Phosphate regulates hormogonia release, perhaps indirectly via an influence on differentiation of heterocysts.     

Pattern of akinete differentiation in the cyanobacteriumScytonema fritschii

The differentiation of akinetes inScytonema fritschii occurred adjacent to the newly developed heterocysts in late exponential phase. The filaments exhibited cell division leading to the formation of heterocysts, interspersed by the potential akinetes which could be identified by the accumulation of a large number of granules. Upon maturity, the akinetes acquired thick envelopes and were seen in elongated series interrupted by dead necridia which resulted from crumpling of the newly developed heterocysts. The formation of akinetes was accompanied by a change in color of cultures from blue-green to brown. Of the inorganic nitrogen sources tested, ammonium nitrate supported the formation of maximum percentage of akinetes. The incorporation of 7-azatryptophan and rifampicin in nitrate-free and nitrogen sources resulted in the production of heterocysts at a very high frequency in the late-exponential phase coinciding with akinete formation but the frequency of the latter was reduced. The activity of nitrogenase, nitrate reductase and glutamate-ammonia ligase was absent in mature akinetes. The absorption spectra of chlorophylla and phycobiliproteins revealed the presence of negligible amounts of the former white the latter were absent. The dry mass steadily increased during akinete differentiation with a concomitant decrease in C/N ratios.     

Feasibility of Fe Autoradiography as Performed on N(2)-Fixing Anabaena spp. Populations and Associated Bacteria

Fe emits low-energy X rays and Auger electrons by electron capture decay. Auger electrons are useful for autoradiographic examination of Fe incorporation among microbial communities. Attainable resolution, in terms of silver grain deposition, is excellent and comparable to H. Two known Fe-demanding processes, photosynthetic CO(2) fixation and N(2) fixation, were examined by autoradiography of Anabaena populations. During photosynthetically active (illuminated) N(2)-fixing periods, biological incorporation of FeCl(3) by vegetative cells and heterocysts was evident. When N(2) fixation was suppressed by NH(4) additions, heterocysts revealed no incorporation of Fe. Conversely, when N(2)-fixing Anabaena filaments were placed in darkness, Fe incorporation decreased in vegetative cells, whereas heterocysts showed sustained rates of Fe incorporation. Bacteria actively incorporated Fe under both light and dark conditions. The chelated (by Na(2)-ethylenediaminetetraacetate) form of FeCl(3) was more readily incorporated than the nonchelated form. Furthermore, abiotic adsorption of Fe to filters and nonliving particles proved lower when chelated Fe was used in experiments. Fe autoradiography is useful for observing the fate and cellular distribution of various forms of Fe among aquatic microbial communities.     

Zn(2+) site engineering at the oligomeric interface of the dopamine transporter

The hetN gene plays an important role in heterocyst differentiation and pattern formation. An immunoblotting study showed that the hetN gene in Anabaena sp. PCC 7120 was expressed in vegetative cells grown with combined nitrogen. After a switch to a medium without combined nitrogen, hetN expression first declined and was then followed by a rapid increase in its product, HetN, which was only present in mature heterocysts. HetN is located on both thylakoid membranes and plasma membranes as determined by immunoblotting using purified membranes. Overexpression of hetN completely prevented hetR up-regulation under nitrogen-deprivation conditions, suggesting that its role in pattern control may depend on its inhibition of hetR expression.