Oxidative stress management in the filamentous, heterocystous, diazotrophic cyanobacterium, Anabaena PCC7120.

Reactive oxygen species (ROS) are inevitably generated as by-products of respiratory/photosynthetic electron transport in oxygenic photoautotrophs. Unless effectively scavenged, these ROS can damage all cellular components. The filamentous, heterocystous, nitrogen-fixing strains of the cyanobacterium, Anabaena, serve as naturally abundant contributors of nitrogen biofertilizers in tropical rice paddy fields. Anabaena strains are known to tolerate several abiotic stresses, such as heat, UV, gamma radiation, desiccation, etc., that are known to generate ROS. ROS are detoxified by specific antioxidant enzymes like superoxide dismutases (SOD), catalases and peroxiredoxins. The genome of Anabaena PCC7120 encodes two SODs, two catalases and seven peroxiredoxins, indicating the presence of an elaborate antioxidant enzymatic machinery to defend its cellular components from ROS. This article summarizes recent findings and depicts important perspectives in oxidative stress management in Anabaena PCC7120.

Polyaniline-based highly sensitive microbial biosensor for selective detection of lindane.

A highly sensitive, selective, and rapid, whole-cell-based electrochemical biosensor was developed for detection of the persistent organochlorine pesticide γ-hexachlorocyclohexane (γ-HCH), commonly known as lindane. The gene linA2 encoding the enzyme γ-hexachlorocyclohexane (HCH) dehydrochlorinase (LinA2), involved in the initial steps of lindane (γ-HCH) biotransformation, was cloned and overexpressed in Escherichia coli . The lindane-biodegrading E. coli cells were immobilized on polyaniline film. The rapid and selective degradation of lindane and concomitant generation of hydrochloric acid by the recombinant E. coli cells in the microenvironment of polyaniline led to a change in its conductivity, which was monitored by pulsed amperometry. The biosensor could detect lindane in the part-per-trillion concentration range with a linear response from 2 to 45 ppt. The sensor was found to be selective to all the isomers of hexachlorocyclohexane (HCH) and to pentachlorocyclohexane (PCCH) but did not respond to other aliphatic and aromatic chlorides or to the end product of lindane degradation, i.e., trichlorobenzene (TCB). The sensor also did not respond to other commonly used organochlorine pesticides like DDT and DDE. On the basis of experimental results, a rationale has been proposed for the excellent sensitivity of polyaniline as a pH sensor for detection of H(+) ions released in its microenvironment.

Biochemical and stress responses of rohu Labeo rohita and mrigal Cirrhinus mrigala in relation to acclimation temperatures.

The biochemical and stress responses of two Indian major carps, rohu Labeo rohita and mrigal Cirrhinus mrigala were studied after acclimating them to four preset temperatures (26, 31, 33 and 36 degrees C) for 30 days. The blood glucose and liver glycogen levels showed an inverse trend in both the species and were significantly different in L. rohita at higher temperatures. The decrease in the liver glycogen level of C. mrigala, however, was not significant. Plasma cortisol levels increased significantly whereas the ascorbic acid content in the brain and kidney of both the species decreased significantly with increasing temperatures. Total lipid content in the liver of both the species decreased significantly with increasing acclimation temperatures. The phospholipid concentration decreased in L. rohita with increasing acclimation temperatures, and in C. mrigala the values decreased up to 33 degrees C and increased at 36 degrees C. In C. mrigala, the cholesterol level decreased up to 33 degrees C and then increased at 36 degrees C, but the absolute value was lower in comparison to L. rohita. The cholesterol levels, however, were not significantly different in L. rohita. Triglycerides and free fatty acids concentrations decreased significantly with increasing acclimation temperatures in both the species. The present study indicates species-specific metabolic responses of L. rohita and C. mrigala to thermal acclimation.

Cloning and characterization of the major groESL operon from a nitrogen-fixing cyanobacterium Anabaena sp. strain L-31.

The major heat-shock-responsive operon groESL has been cloned from the cyanobacterial diazotroph Anabaena. The bicistronic operon harbors an upstream negative regulatory CIRCE element and is transcriptionally activated upon temperature upshift. The deduced amino acid sequence displays strong identity/similarity with other cyanobacterial GroES and GroEL proteins.

Molecular heterogeneity of ferredoxin:NADP+ oxidoreductase from the cyanobacterium Anabaena cylindrica.

The enzyme ferredoxin:NADP+ oxidoreductase (EC 1.18.1.2) from whole filaments of Anabaena cylindrica can be separated into four major fractions by chromatography on phosphocellulose; chromatography using ferredoxin-Sepharose 4B proved to be less satisfactory in separating the fractions. The purified fractions, designated 1, 2, 3 and 4, all showed diaphorase and ferredoxin-dependent cytochrome c reductase activity. The major fractions present were 2 and 3 which were each obtained in an electrophoretically homogeneous state (forms 2 and 3) and represented 30-37% and 30-42%, respectively, of the total enzyme activity. Each was a monomeric species with a molecular weight of approx. 33 000 as determined by gel filtration and sodium dodecyl (SDS)-polyacrylamide gel electrophoresis. Evidence for the presence of a 70 000 molecular weight dimer was also obtained. Forms 2 and 3 had isoelectric points of 5.75 and 6.0, respectively, had similar kinetic properties and were flavoproteins. Extracts of isolated heterocysts showed no form 2 or 3 activity but contained a single form which closely resembled one of the species present in fraction 4; fraction 1 may have been a purification artifact because it was not detected in crude extracts of the cyanobacterium.

Radioadaptive response in human lymphocytes in vitro.

Exposure to low doses of radiation and/or chemicals can prime an organism to withstand the stress of a subsequent exposure to higher doses of the same agent. In the case of radiation, this phenomenon has been called radioadaptive response. Cytogenetic studies have been undertaken in human lymphocytes to investigate adaptive response (AR) to ionizing radiation, in particular to seek the role of variables such as priming dose, cell cycle stage, and age and gender of the donor. We demonstrated that pre-exposure of lymphocytes in whole blood cultures to very low doses in the range of about 1 cGy (priming or adaptive dose [AD]) reduced the frequency of micronuclei in binucleated cells induced by 100 cGy--that is, produced an AR in these cells in vitro. However, pre-exposure of cells to 10.0 cGy did not reduce the chromosomal damage (micronuclei) induced by the challenging dose (CD) of 100 cGy under the same protocol, thus exhibiting an inverse dose-response relationship. There was marked variability in the AR among the individuals investigated in the study. The extent of AR also depended on the stage of cell cycle exposed to the CD of radiation. Maximum AR was observed when CD of 100 cGy was given 4 hours after AD, 30 hours following the mitogenic stimulation of lymphocytes. The least AR was observed when CD was given 48 hours after stimulation. Interestingly, AR was also found to be dependent on the age of the donor, a decrease in AR being observed with an increasing age. No significant difference in AR was observed between male and female donors. To understand the molecular events underlying AR, protein synthesis patterns were studied in human lymphocytes subjected to mitogen, heat, or radiation stress. Heat shock (45 degrees C, for 15 min) induced 7 proteins with molecular weights ranging from 40 to 80 kDa, while treatment with phytohemagglutinin (40 microg/mL) showed induction of 2 proteins of molecular weights 38 and 48 kDa, respectively. However, exposure of human lymphocyte cultures to gamma radiation did not significantly induce synthesis of any protein, up to 800 cGy dose. Lack of induction of proteins by gamma radiation in human lymphocytes contrasts with the previous reports showing X-ray radiation-enhanced gene expression in melanoma cells and/or human tumor fibroblasts.

Role of alkali cations (K+ and Na+) in cyanobacterial nitrogen fixation and adaptation to salinity and osmotic stress.

Cyanobacteria occupy almost every possible ecological niche on earth, being tolerant to a large number of environmental stresses, including salinity and drought. Many of them also fix atmospheric nitrogen. They are responsible for a significant share of biosolar energy conversions on this planet and make substantial contributions to the carbon and nitrogen status of both oceans and soils. Sodium and potassium are two of the most prevalent cations on this planet. While K+ is an essential macronutrient in most life-forms, Na+ is strongly discriminated by means of highly selective alkali cation transport systems, favouring K+ over Na+. Although a nutritional requirement for K+ has not been specifically investigated, rapid accumulation of K+ during salt/osmotic stress has been observed in several cyanobacteria. Genes and proteins constituting a membrane-bound, turgor- and osmo-inducible, Kdp-ATPase-like system in Anabaena strains that may help in their early K+ responses to salt/osmotic stress have been identified. An unusual, specific and absolute requirement for trace quantities of sodium has been documented in cyanobacteria. Work done in our laboratory, and elsewhere, has elucidated the mechanisms underlying such a unique requirement. It has long been believed that cyanobacteria scavenge and immobilise sodium. We have, however, shown that sodium exclusion brought about by curtailment of influx and active efflux of Na+ forms the basis of salt tolerance in these microbes and that the inherent salt tolerance can be modified by factors that modulate Na+ fluxes in cyanobacteria. Identification of genes affecting the cation relationships in nitrogen-fixing cyanobacteria is currently in progress.

Interaction of uranium with a filamentous, heterocystous, nitrogen-fixing cyanobacterium, Anabaena torulosa.

The filamentous, heterocystous, diazotrophic cyanobacterium, Anabaena torulosa was found to bind uranium from aqueous solutions containing 100 µM uranyl carbonate at pH 7.8. The uranyl sequestration kinetics exhibited (a) an initial rapid phase, binding 48% uranium within 30 min resulting in a loading of 56 mg U g(-1) of dry wt, followed by (b) a slower phase, binding 65% uranium with resultant loading of 77.35 mg U g(-1) in 24h. Energy Dispersive X-ray fluorescence spectroscopy of uranium loaded biomass revealed all components of UL X-rays (UL(l), UL(α), UL(ß1) and UL(ß2)). Heat killed cells or extracellular polysaccharides derived from live cells exhibited limited uranyl binding (~26%) highlighting the importance of cell viability for optimum uranyl binding. The present study revealed the involvement of acid soluble polyphosphates in uranium accumulation by this brackish water cyanobacterium.

Uranium sequestration by a marine cyanobacterium, Synechococcus elongatus strain BDU/75042.

A marine, unicellular cyanobacterium, Synechococcus elongatus strain BDU/75042 was found to sequester uranium from aqueous systems at pH 7.8. The organism could remove 72% (53.5 mg U g(-1) dry weight) of uranium from test solutions containing 100 microM uranyl carbonate within 1h. The equilibrium data fitted well in the Langmuir isotherm thus suggesting a monolayer adsorption of uranium on the cyanobacterial biomass and predicted the maximum adsorption capacity of 124 mg U g(-1) dry weight. Light and scanning electron microscopy coupled with energy dispersive X-ray fluorescence (EDXRF) spectroscopy confirmed the uranyl adsorption by this organism. Most of the bound uranium was found to be associated with the extracellular polysaccharides (EPS) suggesting its interaction with the surface active ligands. Fourier transform infrared (FT-IR) spectroscopy suggested the amide groups and the deprotonated carboxyl groups on the cyanobacterial cell surface were likely to be involved in uranyl adsorption. The cell bound uranium could be released by washing with ethylene diamine tetraacetic acid (EDTA) or 0.1N HCl. The X-ray diffraction (XRD) analyses revealed the identity of uranium deposits associated with the cell biomass as uranyl carbonate hydrate. The study revealed the potential of this cyanobacterium for harvesting uranium from natural aquatic environments.

Persistent sub-lethal chlorine exposure augments temperature induced immunosuppression in Cyprinus carpio advanced fingerlings.

Apart from increased temperature, thermal effluents discharged through cooling systems of nuclear power plants may often contain chlorine (used against bio-fouling), which may affect the immune status of fish. Therefore, a 28-day trial was undertaken to delineate the effect of high temperature and a persistent sub-lethal chlorine exposure on immunomodulation in Cyprinus carpio advanced fingerlings. Fish were acclimated to four different temperatures (26, 31, 33 and 36 degrees C) and maintained for 30 days in two different groups. One group was exposed to persistent chlorine (0.1mgL(-1)) and was compared with their respective temperature control groups (without chlorine exposure). Expression of heat shock proteins (hsp 70) was tested in muscle after 28 days using Western blotting. Haematological parameters (erythrocyte count, leucocyte count, haemoglobin), serum parameters (total protein, albumin, globulin, A/G ratio) and respiratory burst activity were tested to assess immuno-competence of C. carpio in response to temperature and chlorine exposure. Results indicated that hsp 70 was induced at 36 degrees C in temperature control groups but not in their respective temperatures in the presence of chlorine. Haematological parameters such as haemoglobin, erythrocyte and leucocyte counts appeared depressed in chlorine treated groups as compared to their respective temperature control groups. Serum protein and globulin were affected due to chlorine exposure at different acclimation temperatures. A decrease in NBT activity was recorded in chlorine treated groups as compared to their respective temperature control groups. Overall results indicate that increasing acclimation temperatures alters the immune status of C. carpio advanced fingerlings and persistent sub-lethal exposure to chlorine augments this temperature induced immunosuppression.

Membrane electrogenesis and sodium transport in filamentous nitrogen-fixing cyanobacteria.

Transport of Na+ and its relationship with membrane potential (delta psi m) was examined in Anabaena L-31 (a fresh water cyanobacterium) and Anabaena torulosa (a brackish water cyanobacterium) which require Na+ for diazotrophic growth. The data on the effect of N,N'-dicyclohexylcarbodiimide indicated that delta psi m was generated by electrogenic proton extrusion predominantly mediated by ATPase(s). In addition, operation of a plasmalemmabound, non-ATP-requiring, H+-pumping terminal oxidase was suggested by the sensitivity of delta psi m to anaerobiosis, cyanide and azide, all of which inhibit aerobic respiration. The response of delta psi m to external pH and external Na+ or K+ concentrations indicated that a diffusion potential of Na+ or K+ may not contribute significantly to delta psi m. Kinetic studies showed that Na+ influx was unlikely to be a result of Na+/NA+ exchange but was a carrier-mediated secondary active transport insensitive to low concentrations (less than 10 mM) of external K+. There was a close correspondence between changes in delta psi m and Na+ influx; all the treatments which caused depolarisation (such as low temperature, dark, cyanide, azide, anaerobiosis, ATPase inhibitors) lowered Na+ influx whereas treatments which caused hyperpolarisation (such as 2,4-dinitrophenol, nigericin) enhanced Na+ influx. Remarkably low intracellular Na+ concentrations were maintained by these cyanobacteria by means of active efflux of the cation. The basic mechanism of Na+ transport in the fresh water and the brackish water cyanobacterium was similar but the latter demonstrated less influx, more efficient efflux, more affinity of carriers for Na+ and less accumulation of Na+, all attributes favouring salt tolerance.

Transient expression of multiple genes in salinity-stressed young seedlings of rice (Oryza sativa L.) cv. bura rata.

To date only about 15 abundantly expressed osmoresponsive genes/proteins have been described in rice cultivars. Using in vivo radiolabeling followed by two-dimensional electrophoresis and autoradiography, a record number of at least 35 salinity stress-induced polypeptides (14-90 kDa) and 17 salt stress-repressed polypeptides were detected in a halotolerant Indica rice cultivar Bura Rata. These include > 20 hitherto unreported rice polypeptides that exhibited a low abundance, short-term expression during NaCl stress. Prolonged exposure to NaCl decreased their synthesis. These findings have widened the scope of further investigations into new osmoresponsive genes, especially those with possibly transient regulatory functions in rice halotolerance.

Cloning of salinity stress-induced genes from the salt-tolerant nitrogen-fixing cyanobacterium Anabaena torulosa.

A subtractive hybridization procedure was used to clone genes of the cyanobacterium Anabaena torulosa expressed in response to salt stress. The method uses total RNA from salt-treated cells, labeled in vitro, as the probe. Hybridization to restriction digests of total DNA was used for interspecies comparison; the procedure was also successful for isolation of cosmids by colony hybridization, semiquantitative dot blots, and cosmid characterization by Southern blotting. Analysis of eleven independent cosmids containing genes whose transcription is abundantly induced by salt suggests that a substantial portion of the A. torulosa genome, probably in excess of 100 kilobases, responds to salt.

Comparative analysis of proteins induced by heat shock, salinity, and osmotic stress in the nitrogen-fixing cyanobacterium Anabaena sp. strain L-31.

Heat, salinity, or osmotic stress influenced protein synthesis in nitrogen-fixing Anabaena sp. strain L-31. Salinity and osmotic stresses were identical and specifically induced 15 polypeptides. Four polypeptides were unique to heat shock, and four other polypeptides were induced under every stress. The results demonstrate a commonality and a stress specificity of protein synthesis regulation.

Salinity-stress-induced proteins in two nitrogen-fixing Anabaena strains differentially tolerant to salt.

Salinity altered the protein synthesis patterns in two cyanobacterial strains: Anabaena torulosa, a salt-tolerant brackish water strain, and Anabaena sp. strain L-31, a salt-sensitive freshwater strain. The cyanobacterial response to salinity was very rapid, varied with time, and was found to be correlated with the external salt (NaCl) concentration during stress. Salinity induced three prominent types of modification. First, the synthesis of several proteins was inhibited, especially in the salt-sensitive strain; second, the synthesis of certain proteins was significantly enhanced; and third, synthesis of a specific set of proteins was induced de novo by salinity stress. Proteins which were selectively synthesized or induced de novo during salt stress, tentatively called the salt-stress proteins, were confined to an isoelectric pI range of 5.8 to 7.5 and were distributed in a molecular mass range of 12 to 155 kilodaltons. These salt-stress proteins were unique to each Anabaena strain, and their expression was apparently regulated coordinately during exposure to salt stress. In Anabaena sp. strain L-31, most of the salt-stress-induced proteins were transient in nature and were located mainly in the cytoplasm. In A. torulosa, salt-stress-induced proteins were evenly distributed in the membrane and cytoplasmic fractions and were persistent, being synthesized at high rates throughout the period of salinity stress. These initial studies reveal that salinity-induced modification of protein synthesis, as has been demonstrated in higher plant species, also occurs in cyanobacteria and that at least some of the proteins preferentially synthesized during salt stress may be important to cyanobacterial osmotic adaptation.

Regulation of potassium-dependent Kdp-ATPase expression in the nitrogen-fixing cyanobacterium Anabaena torulosa.

The KdpB polypeptides in the cyanobacterium Anabaena torulosa were shown to be two membrane-bound proteins of about 78 kDa, expressed strictly under K(+) deficiency and repressed or degraded upon readdition of K(+). In both Anabaena and Escherichia coli strain MC4100, osmotic and ionic stresses caused no significant induction of steady-state KdpB levels during extreme potassium starvation.

Salinity and osmotic stress-regulated proteins in cowpea Rhizobium 4a (peanut isolate).

The effect of salinity and osmotic stress on protein synthesis was studied in cowpea Rhizobium 4a. Osmotic component of salinity stress has been shown to induce ten proteins in a salt tolerant/osmosensitive cowpea Rhizobium. 4a (groundnut isolate). Of these seven polypeptides were induced only by salt/osmotic stress while two were induced by heat shock. The results demonstrate a commonality as well as stress specificity of protein synthesis regulation.

A role for osmotic stress-induced proteins in the osmotolerance of a nitrogen-fixing cyanobacterium, Anabaena sp. strain L-31.

The molecular basis of tolerance to osmotic stress was investigated with a cyanobacterium, Anabaena sp. strain L-31. The inherent osmotolerance of this strain (50% growth inhibition at 350 mM sucrose) was enhanced by adaptation with 100 mM sucrose for 30 min. Addition of 10 mM KNO3 during growth also conferred significant osmoprotection, but addition of 3 mM NH4Cl did not. Exposure of cells to 350 mM sucrose induced the expression of at least 12 osmotic-stress-induced proteins (OSPs) within 30 min, in the molecular mass range of 11.5 to 84 kDa. Exposure of cells to 100 mM sucrose or to 10 mM nitrate also induced all the OSPs, but addition of ammonium did not. The observed correspondence between the presence of OSPs and osmotolerance strongly suggests a role for OSPs in osmotolerance of Anabaena sp. strain L-31.

Differential responses of nitrogen-fixing cyanobacteria to salinity and osmotic stresses.

Two nitrogen-fixing Anabaena strains were found to be differentially tolerant to salinity and osmotic stresses. Anabaena torulosa, a brackish-water, salt-tolerant strain, was relatively osmosensitive. Anabaena sp. strain L-31, a freshwater, salt-sensitive strain, on the other hand, displayed significant osmotolerance. Salinity and osmotic stresses affected nitrogenase activity differently. Nitrogen fixation in both of the strains was severely inhibited by the ionic, but not by the osmotic, component of salinity stress. Such differential sensitivity of diazotrophy to salinity-osmotic stresses was observed irrespective of the inherent tolerance of the two strains to salt-osmotic stress. Exogenously added ammonium conferred significant protection against salinity stress but was ineffective against osmotic stress. Salinity and osmotic stresses also affected stress-induced gene expression differently. Synthesis of several proteins was repressed by salinity stress but not by equivalent or higher osmotic stress. Salinity and osmotic stresses induced many common proteins. In addition, unique salt stress- or osmotic stress-specific proteins were also induced in both strains, indicating differential regulation of protein synthesis by the two stresses. These data show that cyanobacterial sensitivity and responses to salinity and osmotic stresses are distinct, independent phenomena.

Enhancement of cyanobacterial salt tolerance by combined nitrogen.

Presence of certain nitrogenous compounds in the growth medium significantly enhanced the salt tolerance of the fresh-water cyanobacterium Anabaena sp. strain L-31 as well as the brackish water cyanobacterium Anabaena torulosa. Among these, nitrate, ammonium, and glutamine were most effective followed by glutamate and aspartate. These nitrogenous compounds also inhibited Na(+) influx in both Anabaena spp. with the same order of effectiveness as that observed for protection against salt stress. The inhibition of Na(+) influx on addition of the nitrogenous substances was rapid; nitrate and ammonium inhibited Na(+) influx competitively. Proline and glycine did not affect Na(+) influx and also had no influence on the salt tolerance of either Anabaena sp. The observed protection was not consequent to a stimulatory effect of combined nitrogen on growth per se. Uptake of NO(3) (-) and NH(4) (+) increased during salt stress but was not correlated with growth. Intracellular levels of NO(3) (-) and NH(4) (+) were found to be inadequate to constitute a major component of the internal osmoticum. The results suggest that inhibition of Na(+) influx by combined nitrogen is a major mechanism for protection of cyanobacteria against salt stress.