P. S. Bisen

Ca2+ uptake and its regulation in the cyanobacterium Nostoc MAC

Abstract. The uptake of Ca2+ and its regulation in the cyanobacterium Nostoc MAC were investigated. Cation uptake pattern was found to be biphasic, consisting of (a) rapid binding of cations to the negatively charged cell surface and (b) its metabolism dependent on intracellular import at least up to 60 min with the saturation at 2 mM Ca2+ (Km, 1.5 mM, Vmax 42.1 nmol Ca2+ mg−1 protein min−1 ). The cellular Ca2+ uptake was light and ATP dependent, and the addition of 3(3,4-dichlorophenyl)-1,1-dimethyl urea (DCMU) or exogenous ATP proved the vital role of PS II-generated energy to drive the process. The significant inhibition of Ca2+ uptake by different metabolic inhibitors and uncouplers like p-chloromercuribenzoate (pCMB), carbonylcyanide-p-nitrofluoromethoxylphenyl hydrazone (FCCP), N′N-dicyclohexylcarbodiimide (DCCD) and azide revealed that -SH group(s), proton gradient across the cell membrane, and ATP hydrolysis were involved in the transmembrane movement of Ca2+ in Nostoc MAC cells. Verapamil showed antagonism, abscisic acid (ABA) agonism, while trifluoroperazine (TFP) and N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7) exerted no effect on Ca2+ uptake.

Isolation and characterization of the thylakoid membranes from the NaCl-resistant (NaClr) mutant strain of the cyanobacterium Anabaena variabilis.

NaCl-induced changes in the thylakoid membrane of wild-type Anabaena variabilis and its NaClr mutant strain have been studied. Biochemical characterization of the thylakoid membrane was done by taking its absorption and fluorescence spectra at different wavelength. The thylakoid membranes of both strains were isolated by mechanical disruption of the freeze-dried and lysozyme-treated cells, followed by differential and density gradient centrifugation. The light absorption spectra of the thylakoid membrane showed three and two peaks in NaClr mutant strain and its wild-type counterpart respectively at wavelengths of 400–850 nm. These peaks revealed that the thylakoid membrane contains a large amount of carotenoid and chlorophyll a. Fluorescence emission spectra of thylakoid membrane of NaClr mutant and its wild-type strain at excitation wavelength of 335 nm showed two different peaks, one at 340 nm and the other at 663 nm respectively. The light absorption and fluorescence spectra of the thylakoid membrane also revealed that the membrane contained carotenoid pigment, chlorophyll (Chl) a, and a pigment with an emission peak at 335 nm. The HPLC analysis of the pigments of the thylakoid membrane indicates that the NaClr mutant strain under NaCl stress contained an additional peak for the carotenoid pigment, which was lacking in its wild-type counterpart. The major peak in thylakoid membrane was that of echinenone and β-carotene. Whereas the polypeptide composition of thylakoid membrane differed in the wild-type and its NaClr mutant strain, no difference in the cell wall protein pattern was observed in both strains. The thylakoid membrane of NaClr mutant strain contained two additional protein bands that were absent in its wild-type counterpart. The thylakoid membrane of the wild-type and its NaClr mutant strain also showed morphological variations under NaCl stress.

Regulation of assimilatory nitrate reductase in the cyanobacterium Anabaena doliolum

The assimilatory nitrate reductase (NR) from the cyanobacteriumAnabaena doliolum was membrane bound and solubilized by sonication. The Km value of the enzyme was 870 µM for nitrate with dithionite-reduced methyl viologen (MV) as electron donor. The pH optimum was 10.5 in the MV assay. Nitrate acted as an inducer and ammonium as repressor of the enzyme synthesis. In the presence ofl-methionine-d,l-sulfoximine (MSX) or azaserine, inhibitors of the glutamine synthetase-glutamate synthase (GS-GOGAT) pathway, ammonium did not exhibit any inhibitory effect on the enzyme. The photosynthetic nature of NR was shown with PS II inhibitor, 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU). This enzyme fromA. doliolum has been shown to be a light-driven process, requiring de novo protein synthesis. It was inhibited by chlorate, the structural analog of nitrate;p-chloromercuribenzoate, a thiol reagent; sodium tungstate; and certain cations.

Isolation and partial characterization of Het- Fix- mutant strain of the diazotrophic cyanobacterium Anabaena variabilis showing chromatic adaptation.

We propose a model to describe the changes taking place in biochemical processes/events to explain the development of heterocyst and nitrogenase in a diazotrophic cyanobacterium Anabaena variabilis. For this purpose, a mutant strain of A. variabilis lacking heterocyst differentiation and incapable of growth with dinitrogen as the sole source of nitrogen has been isolated after nitrosoguanidine (NTG) mutagenesis and selection by penicillin enrichment. The mutant strain (Het− Fix−) thus isolated has morphological variation and was incapable of reducing acetylene under anaerobic conditions, indicating its mutational loss of the process of nitrogen fixation. The Het− Fix− mutant strain had reduced glutamine synthetase (transferase) activity compared with its wild-type counterpart, suggesting a link between nif gene expression and the expression of gln A, the structural gene of GS. The Het− Fix− mutant strain compared with its wild-type strain also had an extremely high level of phycobiliprotein and a low level of carotenoids. Furthermore, the coiling of vegetative filaments in the Het− Fix− mutant strain, which reduced the surface area to be exposed to light, was a direct indication of the chromatic adaptation, because the mutant strain was found to be photosensitive, showing bleaching of the cells under high light intensity.

Isolation and partial characterization of NaCl‐tolerant mutant strain of Anabaena variabilis with impaired glutamine synthetase activity

NaCl‐tolerant mutant of diazotrophic cyanobacterium Anabaena variabilis has been isolated by N‐methyl‐N‐nitro‐N‐nitrosoguanidine (NTG) mutagenesis and selection for NaCl resistance. The effect of NaCl (300 mM) on growth, heterocyst differentiation, nitrogenase, GS, NR, NO3— and NO2— uptake was studied by maintaining wild‐type A. variabilis as reference. The NaCl‐tolerant mutant grew reasonably well both in presence and absence of NaCl. It also produced more heterocysts and exhibited greater nitrogenase than its wild‐type counterpart in N2‐medium without NaCl. Exposure of wild‐type to NaCl (300 mM) for 7 days resulted a significant inhibition of growth and heterocyst differentiation with reduced nitrogenase activity. These activities were, however, significantly stimulated by NaCl in NaCl‐tolerant mutant. GS activity in NaCl‐tolerant mutant was reduced to about 50% causing liberation of ammonia in the external medium. No ammonia was however, liberated by wild‐type having normal GS activity, suggesting a metabolic linkage between GS (ammonia assimilation or release) and nitrogenase in NaCl‐tolerant mutant. GS activity in wild‐type was inhibited by NaCl, however, no such inhibition by NaCl was recorded in GS activity of NaCl‐tolerant mutant. Untreated cells of NaCl‐tolerant mutant showed greater levels of NO3— and NO2— uptake as compared to its wild‐type, which were further increased after NaCl‐treatment. In contrast, NaCl‐treatment to wild‐type cells resulted in stimulation of NO3— and NO2— uptake systems, however, it also led to almost complete inhibition of NR activity. This suggests that although wild‐type take up NO3— and NO2— more rapidly in response to NaCl stress, it could not metabolize these due to NaCl‐caused inhibition in NR and GS systems. Thus, NaCl‐caused impairment in nitrogen metabolism may be one of the reasons for reduced growth of wild‐type under NaCl stress.

Differential response in damage and repair of wild-type Anacystis nidulans and its UV-B plus heat shock tolerant (UV-HSt) strain under UV-B and heat shock stress

Abstract Both ultraviolet-B (UV-B) and heat shock (HS) tolerant (UV-HS t ) strain of the unicellular cyanobacterium Anacystis nidulans has been developed which showed remarkable growth under UV-B and heat shock (high temperature) compared to negligible growth of wild type cells under the conditions. In terms of oxygen production and Hill activity UV-HS t strain and wild type cells exhibited opposite responses at higher exposures to UV-B, heat shock or UV-HS beyond the normal condition. Photo-heat-shock-inactivation of the electron transport process occurred in both the strains beyond their respective photosynthesis-saturated normal light intensity. In wild type cells, both the oxidizing and reducing sides of the photosynthetic electron transport chain were found to be damaged by UV-HS exposure, unlike the UV-HS t strain in which only the oxidizing side of photosystem II (PS II) was inactivated. With the help of exogenous electron donors hydroxylamine (NH 2 OH) and diphenyl carbazide (DPC), photo-heat-shock-inactivated sites were delineated. The wild type cells showed quick and fast recovery within 24 h after imposition of the normal light condition compared to the UV-HS t strain. The significant recovery in NH 2 OH added wild type cells compared to DPC incubated cells suggested that only the reducing side of PS II could be repaired. The addition of L -methionine-DL-sufoximine (MSX), a glutamate analogue and irreversible inhibitor of glutamine synthetase (GS) inhibitor and protein synthesis inhibitors such as chloramphenicol (CAP) and rifampicin (RFM), suggested the vital role of nitrogen source and de novo protein synthesis in the regulation of the recovery process.

Regulation of Sodium Influx in the NaCl-Resistant (NaCl r ) Mutant Strain of the Cyanobacterium Anabaena variabilis

A NaClr mutant of the diazotrophic cyanobacterium Anabaena variabilis has been isolated by NTG mutagenesis and selection for NaCl resistance. The NaClr strain has been characterized with respect to its mechanism of NaCl tolerance and regulation of Na+ influx. NaClr strain exhibits low Na+ influx, accumulated high level of glycine betaine as a compatible solute, and persistent synthesis of SSPs at a higher rate than its wild-type counterpart. DCMU, an inhibitor of PS-II, inhibited Na+ influx, suggesting that Na+ influx is an energy-dependent process and that the energy is derived from photophosphorylation. This contention is further supported by the inhibition of Na+ influx under dark conditions. The inhibition of Na+ influx by KCN, DNP, NaN3 also supports the involvement of oxidative phosphorylation in the regulation of active Na+ influx. Thus, it appears that the synthesis of SSPs, accumulation of compatible solutes, and exhibition of low Na+ influx in the NaClr strain made this organism NaCl tolerant.

NO 2 ? Efflux and its regulation in cyanobacterium Nostoc MAC

NO2−efflux and its regulation have been studied in the cyanobacterium Nostoc MAC. 3-(3,4-Dichlorophenyl)-1,1-dimethylurea (DCMU), carbonyl cyanide-m-chlorophenyl hydrazone (CCCP), sodium azide, p-chloromercuribenzoate (PCMB), and dicyclohexylcarbodiimide (DCCD), a specific inhibitor of bacterial ATPase, inhibited the NO2−efflux activity singificantly. No NO2−efflux activity was observed under dark-aerobic as well as under dark-anaerobic conditions; however, the addition of ATP resulted in NO2−efflux even under dark-aerobic condition. Maximum NO2−efflux activity was observed when NO3−served as the sole nitrogen source. However, NH4+ions inhibited the NO2−efflux activity when both NO3−and NH4+wer simulatneously available to the cells. The NO2−efflux was freed from NH4+repression by l-methionine-dl-sulfoximine (MSX), an irreversible inhibitor of glutamine synthetase (GS). Chloramphenicol, a protein synthesis inhibitor, inhibited the derepression of NO2−efflux system when NH4+-incubated cells were transferred to NO3−medium. Tungstate-treated cells lacking functional NO3−reductase but having NO3−uptake activity also lacked NO2−efflux activity. These results suggest that (i) NO2−efflux in Nostoc MAC is NO3−dependent and an energy-dependent process that can be regulated at the levels of NO3−uptake and NO3−reductase; (ii) NO2−efflux system is NH4+repressible; however, the product of NH4+assimilation via GS is being required for repression to occur; (iii) de novo protein synthesis is required for derepression of the NO2−efflux system; and (iv) the catalytic activity of NO2−reductase also seems to play an important role in the regulation of NO2−efflux system.

Energy-dependent Ca2+ efflux from the cells of Nostoc calcicola Bréb : Role of modifying factors

Abstract. Energy-dependent Ca2+ efflux and its regulation from the diazotrophic cyanobacterium Nostoc calcicola Bréb has been investigated. Like Ca2+ uptake, Ca2+ efflux pattern also reflected a rapid phase for the first 10 min followed by a slower one lasting up to 1 h with a total of 80 nmol Ca2+ mg−1 protein (31% of the Ca2+ concentration taken in by such cells at 1 h). Ca2+ efflux kinetics remained hyperbolic with a Km of 1.9 mM and Vmax 5.5 nmol mg−1 protein min−1. Ca2+ efflux to a major extent depended on photosynthetic energy generation as the cells facing dark incubation and addition of 3-(3,4-dichlorophenyl)-1-dimethyl urea (DCMU) to light-grown cells showed significant reduction in Ca2+ extrusion. The strong inhibition in Ca2+ efflux by addition of metabolic inhibitors like carbonyl cyanide-p-nitrofluoromethoxylphenyl hydrazone (FCCP) and N,N,-dicyclohexylcarbo-diimide (DCCD) suggested the vital role of membrane potential and ATP hydrolysis in driving this process. Verapamil (Ca2+ antagonist) had insignificant effect on Ca2+ efflux, whereas the addition of Calmodulin antagonists like trifluoroperazine, W-7 and compound 48/80 resulted in the enhancement in Ca2+ efflux over control sets, thus suggesting that this increase may be owing to the additional extrusion of intracellular free calcium that was unable to bind with calmodulin in the presence of these antagonists.

Glutamine synthetase and arginine inhibition of nitrate reductase activity in Anabaena cycadeae

Wild-type Anabaena cycadeae with normal glutamine synthetase (GS) activity utilized arginine as sole N source whereas a mutant strain lacking GS activity did not. Nitrate reductase (NR) activity, higher in the mutant strain than the wild-type strain, was inhibited by arginine though arginine-dependent NH4+generation was higher in the mutant strain than in the wild-type. This suggests that (1) NR activity is NOinf3sup--inducible and arginine-repressible; and (2) while GS activity is required for the assimilation of arginine as sole N-source, it is not required for arginine inhibition of NR activity.