Effect of 7-azatryptophan on heterocyst differentiation inAnabaena doliolum Bharadwaja

The effect of DL-7-azatryptophan, an analogue of tryptophan, has been studied on the heterocyst spacing pattern and the probability of proheterocyst regression inAnabaena doliolum. 7-azatryptophan suppressed growth and induced heterocyst differentiation in nitrogen-free medium. In ammonium (1 mM), nitrite (2 mM) and nitrate (2 mM) supplemented media, it caused proheterocyst regression with a frequency of 100%, 35% and 10% respectively. The role of azatryptophan in nitrogen metabolism has been discussed in relation to ammonia-uptake study.     

Canavanine-lnduced inhibition of growth and heterocyst differentiation inAnabaena doliolum and isolation of a canavanine-resistant mutant

The effect of the arginine analogue, canavanine on growth and heterocyst differentiation in the nitrogen-fixing algaAnabaena doliolum has been studied. The analogue inhibited growth and heterocyst differentiation at a concentration as low as 1 μM. The treated algal cells lacked conspicuous granular inclusions, whereas treatment with chloramphenicol led to increased synthesis of granules (probably cyanophycin granules). Exogenously added arginine completely reversed the effect of the analogue but lysine could only partially relieve the effect. A time course study with canavanine indicated inhibition of fresh protein(s) synthesis at all steps where a new class of proteins is synthesized so that the action of the analogue does not seem to be specific for a particular kind of protein. A mutant resistant to this analogue has been successfully isolated indicating that this alga does not show mutational immunity at least to the amino acid analogues unlike in the observation with different antibiotics. Our observations indicate that canavanine either directly inhibits protein synthesis or forms defective protein(s) which produces all the observed effects.     

Response of a wild type and a non-nitrogen-fixing mutant of Anabaena doliolum towards different amino acids

The effects of various amino acids on growth and heterocyst differentiation have been studied on wild type and a heterocystous, non-nitrogen-fixing (het+ nif-) mutant of Anabaena doliolum. Glutamine, arginine and asparagine showed maximum stimulation of growth. Serine, proline and alanine elicited slight stimulation of growth of wild type but failed to show any stimulatory effect on mutant strain. Valine, glutamic acid, iso-leucine and leucine at a concentration of as low as 0.1 mM were inhibitory to growth of parent type. Methionine, aspartic acid, threonine, cysteine, and tryptophan did not affect growth at concentrations lower than 0.5 mM. But at 1 mM, these amino acids were inhibitory. In addition to the stimulatory effects of glutamine, arginine and asparagine, the heterocyst frequency was also repressed by these amino acids. Glutamine and arginine at 2 mM completely repressed heterocyst differentiation in the mutant strain; however, other amino acids failed to repress the differentiation of heterocysts. Our results suggest that glutamine and arginine are utilized as nitrogen sources. This is strongly supported from the data of growth and heterocyst differentiation of mutant strain, where at least with glutamine there is good growth without heterocyst formation. Studies with glutamine and arginine on other N2-fixing blue-green algae may reveal the regulation of the heterocyst-nitrogenase sub-system.     

Genetic control of heterocyst formation in the blue-Green algae Nostoc muscorum and nostoc linckia

Non-heterocystous, non-nitrogenfixing (het ^- nif^-), heterocystous, non-nitrogenfixing (het ⁺ nif^-) and multiple heterocystous, nitrogen-fixing (M-het ⁺ nif⁺) mutants of heterocystous, nitrogen-fixing (het ⁺ nif⁺) wild-type Nostoc muscorum and Nostoc linckia were isolated and characterized with respect to (a) nitrogenfixing activity, (b) reversion frequency, (c) ammonium repressibility of heterocyst formation, (d) heterocyst spacing pattern, and (e) action of L-methionine-DL-sulphoximine (MSO), an inhibitor of glutamine synthetase (GS), on heterocyst regulation. The mutant and revertant results suggest: (i) either involvement of a common genetic determinant in the formation of heterocyst and nitrogenase or the organization of het genes and nif genes in a single operon prone to complete inactivation by a single polar mutation, (ii) non-participation of active nitrogenase in regulation of heterocyst spacing; (iii) involvement of genetic factor(s) in the control of heterocyst spacing pattern in N. linckia, and (iv) apparently different nature of the mechanism of heterocyst inhibition by proheterocyst from that of heterocyst inhibition by NO ₃ ^- or NH ₄ ⁺ . L-Methionine-DL-sulphoximine inhibits growth and causes heterocyst formation in chains in N. linckia growing in nitrogen-free, NO ₃ ^- , NO ₂ ^- or NH ₄ ⁺ medium, thus indicating a close physiological linkage between heterocyst and inorganic nitrogen metabolism regulation.     

Effects of L-methionine-DL-sulphoximine on the assimilation of newly fixed NH3, acetylene reduction and heterocyst production in Anabaena cylindrica.

The addition of exogenous L-methionine-DL-sulphoximine (MSO) to N₂-fixing cultures of the blue-green alga Anabaena cylindrica results in over half of the newly fixed NH₃ being released into the medium. MSO also inhibits glutamine synthetase (GS) activity, has negligible effect on alanine dehydrogenase activity, and glutamate dehydrogenase activity under N₂-fixing conditions is negligible. In the presence of MSO, intracellular pools of glutamate and glutamine decrease, those of aspartate and alanine + glycine show little change, and the NH₃ pool increases. MSO alleviates the inhibitory effect of exogenous NH₄⁺ on nitrogenase synthesis and heterocyst production. The results suggest that in N₂-fixing cultures of A. cylindrica the primary NH₃ assimilating pathway involves GS, and probably glutamate synthase (GOGAT), and that the repressor of nitrogenase synthesis and heterocyst production is not NH₄⁺ but is GS, GOGAT, or a product of their reactions.     

Heterocyst patterns without patterning proteins in cyanobacterial filaments

We have quantitatively modeled heterocyst differentiation after fixed nitrogen step-down in the filamentous cyanobacterium Anabaena sp. PCC 7120 without lateral inhibition due to the patterning proteins PatS or HetN. We use cell growth and division together with fixed-nitrogen dynamics and allow heterocysts to differentiate upon the local exhaustion of available fixed nitrogen. Slow transport of fixed nitrogen along a shared periplasmic space allows for fast growing cells to differentiate ahead of their neighbors. Cell-to-cell variability in growth rate determines the initial heterocyst pattern. Early release of fixed nitrogen from committed heterocysts allows a significant fraction of vegetative cells to be retained at later times. We recover the experimental heterocyst spacing distributions and cluster size distributions of Khudyakov and Golden [Khudyakov, I.Y., Golden, J.W., 2004. Different functions of HetR, a master regulator of heterocyst differentiation in Anabaena sp PCC 7120, can be separated by mutation. Proc. Natl. Acad. Sci. U. S. A. 101, 16040-16045].     

Effect of norleucine on growth,protein and catechol oxidase synthesis in tobacco suspension cultures

Tobacco cells were grown in artificial media with defined amino acid composition. In such media, the addition of methionine or norleucine caused increases in the specific activity of the catechol oxidase, while in the normal medium norleucine depressed it. The differences of the effect of norleucine on synthesis of catechol oxidase and on cell growth is demonstrated, as is the reversibility of the norleucine effect by methionine. The incorporation of norleucine into a purified enzyme fraction is shown. The change in the electrophoretic patterns of the enzyme during growth in the absence and presence of norleucine was followed. [¹⁴C]-Leucine incorporation by control and norleucine treated cells was examined and it was shown that protein synthesis in the norleucine treated cells was markedly changed and total incorporation reduced. Incorporation into soluble protein was reduced, but increased in the 20 000 g precipitate fraction. Nevertheless use of autoradiography indicates that some catechol oxidase is apparently synthesised in the presence of norleucine.     

Induction of increase in the heterocyst frequency of Anabaena sp. strain ATCC33047. Effect on ammonium photoproduction

Abstract Several approaches have been followed to increase the nitrogenase level in filaments of Anabaena ATCC33047. In a nitrogen-free medium lacking added molybdate and supplemented with 10 mM tungstate, growth was impaired as a result of decreased nitrogenase activity level. Under these conditions, the filaments exhibited nitrogen starvation symptoms and a high heterocyst frequency, with heterocysts being up to 28% of the total number of cells in the filaments, while a regular pattern of heterocyst distribution was maintained. Normal nitrogenase level and nitrogen status were recovered upon molybdate addition, with resumption of growth and decrease of the heterocyst frequency with time until reaching a value of about 10%. The yield of ammonium photoproduction from N₂ by filaments displaying different heterocyst frequencies and treated with l -methionine- d,l -sulfoximine (MSX) was determined. Maximal rates were obtained with filaments containing 16% of the cells differentiated as heterocysts. Results indicate that appropriate manipulation of the heterocyst frequency leads to an improvement in the efficiency of conversion of light energy into chemical energy through photoreduction of N₂ to ammonium.     

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.     

Polyglutamylfolate synthesis in Neurospora crassa: changes in pool size following growth in glycine- and methionine-supplemented media

The effect of media supplements on total and polyglutamylfolate concentrations has been examined in Neurospora crassa wild type (FGSC 853), an ethionine-resistant mutant (FGSC 1212), and a methionine auxotroph (FGSC 1330) which lacks folylpolyglutamate synthetase. When the culture medium contained 1 mm glycine, folate concentrations in the wild type were increased by over 90% and more p-[³H]aminobenzoate was incorporated into folates. Growth in l-methionine-supplemented media (1–5 mm) decreased folate levels and labeling in all three strains. In the wild type, this effect of l-methionine was reversed on transfer to unsupplemented media but p-[³H]aminobenzoate pulse-chase experiments suggested that exogenous methionine did not increase the turnover of labeled folates. At 1 mm, d-methionine did not affect polyglutamylfolate labeling but l-methionine reduced ³H incorporation by 65% in the wild type. Ion-exchange chromatography showed that p-[³H]aminobenzoate was incorporated in formyl- and methyltetrahydrofolates which in the wild type, were principally hexaglutamyl derivatives. Glycine-supplemented growth yielded labeled folates that were 24% heptaglutamates but these and pentaglutamates were lacking when l-methionine was supplied. The specific activity of GTP cyclohydrolase was not significantly affected by culture in l-methionine-containing media. Dialysis and gel filtration both lowered enzyme activities and product formation was not changed when up to 10 μmol of l-methionine was added to the reaction system. The data suggest that methionine or its metabolic products exerts some control over folate production which is distinct from the established inhibition of methylenetetrahydrofolate reductase by AdoMet.     

Some characteristics of two morphological mutants of Nostoc linckia induced by nitrosoguanidine.

The blue-green alga Nostoc linckia was treated with nitrosoguanidine and two classes of morphological mutant clones were isolated. One class shows certain abnormal phenotypic features of vegetative cells, spores, and heterocysts. It has increased heterocyst frequency and impaired growth rate. The other class exhibits an altered heterocyst spacing pattern. Both classes of mutants have reduced nitrogenase activity.     

The role of HetN in maintenance of the heterocyst pattern in Anabaena sp. PCC 7120

The gene hetN encodes a putative oxidoreductase that is known to suppress heterocyst differentiation when present on a multicopy plasmid in Anabaena sp. PCC 7120. To mimic the hetN null phenotype and to examine where HetN acts in the regulatory cascade that controls heterocyst differentiation, we replaced the native chromosomal hetN promoter with the copper-inducible petE promoter. In the presence of copper, heterocyst formation was suppressed in undifferentiated filaments. When hetN expression was turned off by transferring cells to media lacking copper, the filaments initially displayed the wild-type pattern of single heterocysts but, 48 h after the induction of heterocyst formation, a pattern of multiple contiguous heterocysts predominated. Suppression of heterocyst formation by HetN appears to occur both upstream and downstream of the positive regulator HetR: overexpression of hetN in undifferentiated filaments prevents the wild-type pattern of hetR expression as well as the multiheterocyst phenotype normally observed when hetR is expressed from an inducible promoter. Green fluorescent protein fusions show that the expression of hetN in wild-type filaments normally occurs primarily in heterocysts. We propose that HetN is normally involved in the maintenance of heterocyst spacing after the initial heterocyst pattern has been established, but ectopic expression of hetN can also block the initial establishment of the pattern.     

Action of 2,4-Dichlorophenoxyacetic Acid onNostoc linckia: Impact of Glucose and Tryptophan

2,4-Dichlorophenoxyacetic acid (2,4-D) stimulated growth and heterocyst differen- tiation ofNostoc linckia in nitrogen-free medium at lower concentrations (100 μ.g/mL) while its higher concentrations inhibited both processes and 1500 μg/mL proved to be lethal. Dry mass and specific growth rate of the alga declined with increasing concentration of 2,4-D in the range of 100–1500 μg/mL. Glucose slightly increased the heterocyst frequency without any lag in their differentiation. Tryptophan promoted growth of the alga, and formation of heterocysts (nearly three-fold). Tryptophan (50 μg/mL) complex medium with 1 mg 2,4-D per mL did not produce mature heterocysts. The filaments were fragmented at the point of hererocyst development and detached heterocysts germinatedin situ. Glucose and tryptophan protected the alga, its growth and heterocyst differentiation even at the lethal concentration of the herbicide. We are grateful to the Head, Department of Botany,Banaras Hindu University, Varanasi, for providing the necessary facilities. The first author is also grateful to the Principal,K.D. College, Kutir-Chakkey, Jaunpur, for his interest in this study.     

Extracellular polypeptides ofAnabaena L-31: Evidence for their role in regulation of heterocyst formation

Extracellular polypeptides released by both N₂-grown [peptide I] and NO₃-grown [peptide II]Anabaena L-31 have molecular weight of approximately 3,500 but have distinctly different amino acid composition. Acid hydrolysis of the peptide I fraction (obtained by separation on Sephadex G-25) yielded ten amino acids whereas that from peptide II fraction yielded only 3 amino acids. On addition to a freshly inoculated N₂-grown culture, the peptide I fraction stimulated pro-heterocyst and to a lesser extent heterocyst differentiation, whereas the peptide II fraction strongly inhibited differentiation. The inhibitory effect of polypeptide II fraction could not be relieved by methionine sulphoximine, which by itself enhances differentiation, but was greatly relieved by addition of the peptide I fraction. The data suggest but does not prove, thatAnabaena L-31 synthesises “inducer” or “inhibitor” peptides which could possibly control pattern formation.     

Free Amino Acid and Storage Protein Composition of Soybean Fruit Explants and Isolated Cotyledons Cultured with and without Methionine

Thein vitroculture of immature soybean cotyledons (in direct contact with the medium) and immature fruit explants (stem dipping into the medium) on a defined medium containing glutamine and sulphate as sole sources of N and S for 7 d led to rates of growth and reserve protein accumulation close to, or greater than, those occurringin situ. Supplementation of the medium with 8.4 mMmethionine had little effect on growth and protein accumulation of the cotyledons in the explant system, but did result in significant increases in the isolated cotyledon system. Methionine suppressed the synthesis of the 7S β-subunit in both systems. The free amino pool of the cotyledons increased more than three-fold when methionine was present in the explant medium. In the isolated cotyledon system, the basal medium alone caused a large increase (over 30-fold) in the free amino acid fraction, but methionine resulted in an even greater increase (over 50-fold). In both systems the expansion involved a very large increase in the methionine pool, but many other amino acids also showed large increases. Specific effects of methionine on individual amino acids were more clear in the explant system, where its presence resulted in marked increases in serine, alanine and asparagine. The data show that an abnormal situation arises on feeding with methionine, a fact to be considered before attributing effects on growth and protein synthesis directly to methionine.     

Amino acids and growth substances in barley root excretions (Hordeum distichon L.) and their biological effect

The biological effect of barley root excretions was studied during a 4 to 10 days cultivation period. The effect of root excretions changes according to the cultivation period of barley. It was ascertained by means of a bioassay that growth was either conclusively stimulated or the root excretions did not affect growth of the roots and of the upper part ofNasturtium. No significant inhibitory effect was observed. The effect of the single amino acids and of their mixtures found in the root excretions of barley was quite different. The following amino acids were determined by paper chromatography in root excretions: alanine, asparagine, phenylalanine, glycine, leucine, lysine, serine, tyrosine, glutamic acid and valine plus methionine. During cultivation their total quantity increased from 5·48. 10^?5 up to 1·13. 10^?3 mg per plant. Most of the 20 amino acids observed, displayed in theNasturtium test at a concentration from 0·1 to 1,000 mg/l an inhibitory effect onNasturtium growth. The effect of amino acid mixtures, corresponding qualitatively and quantitatively to the free amino acids in barley root excretions was dependent on their concentration. Growth regulators of the auxin type were found in culture solutions by chromatographic separation and with bioassay. As it may be seen from the results obtained, there are besides amino acids and indole derivatives other non-identified compounds involved in the effect of barley root excretions.     

Mutants of Anabaena cylindrica altered in heterocyst spacing

Nitrosoguanidine induced mutants of Anabaena cylindrica have been obtained, which are altered in heterocyst spacing. In the wild type organism the pattern is composed of single intercalary heterocysts. The mutant patterns fall into several classes: those with only terminal heterocysts, with both terminal and intercalary heterocysts, with groups of heterocysts and those totally lacking heterocysts. The mutants are described in detail, and the various pattern modifications are interpreted in terms of a model we have proposed.     

An azide-resistant mutant of the blue-green algaNostoc muscorum producing heterocyst and nitrogenase in the presence of fixed inorganic nitrogen source

The N₂, NO ₃ ^– , NO ₂ ^– , NH ₄ ⁺ and glutamine growing cultures of parentNostoc muscorum are found more or less equally sensitive to azide inhibition of growth. A mutant strain resistant to sodium azide was isolated from the parent strain in NO ₃ ^– medium and the two strains were compared with regard to their heterocyst formation and nitrogenase activity in NO ₃ ^– , NO ₂ ^– , NH ₄ ⁺ and glutamine media. While the parent strain stops production of both heterocyst and nitrogenase in all the fixed nitrogen media, the azide resistant strain forms both in the fixed inorganic nitrogen media but only heterocyst and no nitrogenase in the glutamine medium. Clearly a single genetic determinant of regulatory nature appears to mediate azide-resistance as well as relief of heterocyst and nitrogenase formation from inhibition by the fixed inorganic nitrogen source. The results of glutamine effect on the heterocyst and nitrogenase formation of the two strains indicate the operation of two levels of glutamine-sensitive regulation, one which operates through the common genetic determinant of heterocyst and nitrogenase regulation and the other exclusive to nitrogenase regulation. The in vivo functional nitrogenase does not appear to be the reason for azide-resistance and neither ammonia nor glutamine or its close metabolic product seems to function in the control of heterocyst spacing.     

The 2A resolution crystal structure of HetL, a pentapeptide repeat protein involved in regulation of heterocyst differentiation in the cyanobacterium Nostoc sp. strain PCC 7120

The hetL gene from the cyanobacterium Nostoc sp. PCC 7120 encodes a 237 amino acid protein (25.6kDa) containing 40 predicted tandem pentapeptide repeats. Nostoc sp. PCC 7120 is a filamentous cyanobacterium that forms heterocysts, specialized cells capable of fixing atmospheric N(2) during nitrogen starvation in its aqueous environment. Under these conditions, heterocysts occur in a regular pattern of approximately one out of every 10-15 vegetative cells. Heterocyst differentiation is highly regulated involving hundreds of genes, one of which encodes PatS, thought to be an intercellular peptide signal made by developing heterocysts to inhibit heterocyst differentiation in neighboring vegetative cells, thus contributing to pattern formation and spacing of heterocysts along the filament. While overexpression of PatS suppresses heterocyst differentiation in Nostoc sp. PCC 7120, overexpression of HetL produces a multiple contiguous heterocyst phenotype with loss of the wild type heterocyst pattern, and strains containing extra copies of hetL allow heterocyst formation even in cells overexpressing PatS. Thus, HetL appears to interfere with heterocyst differentiation inhibition by PatS, however, the mechanism for HetL function remains unknown. As a first step towards exploring the mechanism for its biochemical function, the crystal structure of HetL has been solved at 2.0A resolution using sulfur anomalous scattering.