Metals in Cyanobacteria: Physiological and Molecular Regulation

Chapter 13

Metals in Cyanobacteria: Physiological and Molecular Regulation Sanjesh Tiwari, Parul Parihar, Anuradha Patel, Rachana Singh, Sheo Mohan Prasad Ranjan Plant Physiology and Biochemistry Laboratory, Department of Botany, University of Allahabad, Allahabad, India

1. INTRODUCTION Among prokaryotes, cyanobacteria (primary producer) are considered the most adaptable and are responsible for creating the oxidized environment by releasing molecular oxygen during oxygenic photosynthesis. They involve three major photosynthetic complexes: phycobilisome (PBS), photosystem I (PS I), and photosystem II (PS II), for harvesting light energy (400–700 nm) required for fixing the atmospheric carbon dioxide (Umena et al., 2011; Shen, 2015) and contribute up to 30% to the yearly oxygen production on the Earth. They also accomplish the nitrogen-fixation process and maintain the biogeochemical cycles (Muro-Pastor et al., 2005). In addition, this group of organisms have also been reported to form symbiotic associations with variety of plants, thereby imparting nitrogen-fixing capacity to the plant system, like Anabaena azollae forms a symbiotic association with the floating fern Azolla, which provides the nitrogen fixation ability to the Azolla and this property has made it economically and sustainably important (Adams et al., 2006; Bergman et al., 2007). Besides this, cyanobacteria are also used for the decomposition of waste materials, metal homeostasis, inhibition of growth of harmful microbes, and production of various bioactive compounds, that is, vitamins, enzymes, antibiotics, hormones, etc., that are used in pharmaceutical industries (Pangestuti and Kim, 2011; Singh et al., 2017). They also serve as an important component of human diet, like various cyanobacterial strains such as Spirulina platensis, Spirulina maxima (now known as Arthrospira sp.), Nostoc commune, Nostoc flagelliforme, Nostoc punctiforme, and Aphanothece sacrum are consumed as the rich sources of proteins, vitamins, antioxidants, etc. (Ferruzzi et al., 2002). Apart from their role in nitrogen fixation and additive food values, they are also being exploited for their efficient role in modulating physicochemical properties of soil, biosorption, and bioremediation properties (Singh et al., 2011, 2016). Environmental contamination due to unrestricted developmental activities such as industrialization and urbanization has gained serious attention worldwide in past few years. Contamination of aquatic ecosystem is perpetually increasing due to release of waste and discharges (having major proportion of toxic heavy metals) from numerous industries that affect the growth and survival of the primary producers of aquatic ecosystem. The sources of heavy metal contamination in the soil and aquatic system are summarized in Fig. 1. Further, some heavy metals like Cu, Fe, Zn, etc., when present in trace amount are useful in several metabolic processes and also act as cofactor in enzyme functioning and at higher concentration pose toxicity (Choudhary et al., 2007); while, heavy metals such as Cd, Cu, Pb, Cr, As, and Hg are toxic at their lower concentration and serves as the major environmental pollutants particularly in areas with high anthropogenic pressure (Nagajyoti et al., 2010). Elevated concentration of such heavy metals significantly causes toxic effects on organisms and the degree of toxicity mainly depends on the duration and type of exposure (Danilov and Ekelund, 2001). A number of studies have been performed to evaluate the heavy metal toxicity in cyanobacteria (Le Jeune et al., 2006; Debelius et al., 2009; Patel et al., 2018). Heavy metals negatively affect the growth of cyanobacteria and the decline in growth has been found to be associated with decreased photosynthetic pigments, nutrient uptake, photosynthesis, and nitrogen-fixation ability (Surosz and Palinska, 2004; Singh et al., 2015a,b). Furthermore, generation of reactive oxygen species (ROS) is a common end point to describe the toxicity level and these ROS disrupts the macromolecules including protein, DNA, and lipids (Singh et al., 2016). To cope up with the oxidative stress caused by heavy metals, cyanobacterial cells employ their antioxidant defense system (enzymatic antioxidants, viz., superoxide dismutase (SOD), catalase (CAT), guaiacol peroxidase (POD), ascorbate peroxidase (APX), glutathione reductase (GR), dehydroascorbate reductase (DHAR), glutathione-S-transferase (GST), and nonenzymatic antioxidants, viz., cysteine, proline, and NP-SH) which scavenges the ROS. In this chapter, we have briefly Cyanobacteria. https://doi.org/10.1016/B978-0-12-814667-5.00013-1 © 2019 Elsevier Inc. All rights reserved.

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FIG. 1  The sources of different heavy metals.

reviewed the research works concerning the response of cyanobacteria to heavy metal stress and adaptation strategy of cyanobacteria at the biochemical and molecular (with special focus on the OMICS approach) levels.

2.  IMPACT OF HEAVY METAL ON PHYSIOLOGICAL AND BIOCHEMICAL PROCESSES Cyanobacteria are the first oxygen-evolving prokaryotes and are considered as major biomass producers in aquatic ecosystem and contribute around 50% of the total biomass in several ecosystems. Due to the increased industrialization, urbanization, and uncontrolled use of various agrochemicals, aquatic systems often get contaminated with excessive concentrations of various toxic elements including heavy metals which negatively influence the cyanobacterial growth and development (Sacan et al., 2007; Peralta-Videa et al., 2009; Kovacik et al., 2010; Patel et al., 2018; Tiwari et al., 2018). A number of metals, viz., Fe, Cu, and Zn are essential for the growth of cyanobacteria, since they are important parts of various enzymes and play crucial roles in different metabolic processes. However, these metals sometimes exhibit adverse impacts if present at high concentrations (Kovacik et al., 2010; Shanmugam et al., 2011). Unlike the above metals, As, Cr, Cd, Pb, etc., exert detrimental impacts on the cyanobacteria even at very low concentrations. Due to heavy metal contamination, physiological processes like growth, pigment contents (chlorophyll a, β-carotene, and phycocyanin (PC)) (Arunakumara and Xuecheng, 2009), photosynthetic activity get affected. Generation of ROS is common end point of aerobic life exposed to various abiotic and biotic stresses (Gill and Tuteja, 2010) and they subsequently damage the macromolecules like amino acids, protein, DNA, and RNA (Singh et al., 2016). Toxicity of ROS depends on their intracellular concentrations since they play both positive and negative roles. At low concentrations, they may act as signaling molecule and enhances the developmental process as well as improve productivity of aquatic ecosystem whereas at high concentrations they are detrimental to the cells. The impact of heavy metals on the growth and important physiological parameters has been discussed in the following sections and represented diagrammatically in Fig. 2.

2.1  Impact of Heavy Metal Stress on Growth and Photosynthetic Pigments It is inferred that cell growth/biomass accumulation is negatively affected by the presence of the heavy metals. Decline in biomass accumulation of cyanobacteria under heavy metal stress has been reported by several authors (Thompson et al., 2002; Gupta et al., 2014; Mota et al., 2015; Patel et al., 2018). The reason for decline in the growth has been attributed

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FIG. 2  Sites of action of heavy metals and their toxicity in cyanobacteria.

to the competition of heavy metals with essential nutrients for the binding sites of enzymes and transporters, thereby ­impairing the cell functioning and eventually causing cell death (Nies, 1999; Tottey et al., 2012). Moreover, the toxicity of the heavy metal depends on several factors like concentration of the metal, oxidation state, and exposure time. In a study, it has been shown that addition of 1 mg L−1 of Pb resulted in growth impairment of Cyanothece cells and this concentration was also lethal for Anabaena (Heng et al., 2004), while the concentration less than 0.2 mg L−1 negatively affected the growth of S. platensis-S5 (Choudhary et al., 2007). Similarly, Al-Mousawi (2010) reported that Cu2+ inhibited the growth of Chroococcus sp. Decline in the growth could be correlated with negative effect on the growth-regulating processes like photosynthesis, nitrogen metabolism, etc. Pigment contents are directly associated with the photosynthetic capability and therefore with the biomass accumulation. Interference of heavy metals also reduces the contents of light-harvesting photosynthetic pigments (chlorophyll, carotenoids, and PC), which directly and indirectly influence the photosynthesis process, and disorganize the chlorophyll structure (Kana et al., 2009). The detailed investigations by some authors suggested that decline in the pigment contents due to heavy metals could be attributed to direct damage by the ROS, inhibition in the activity of enzyme involved in Chl biosynthesis, and replacement of Mg atom from the Chl molecule by the heavy metal (Singh et al., 2012; Gill et al., 2012). Tiwari et al. (2018) reported that exposure of Nostoc to Cr declined the Chl a content which directly altered the photosynthetic activity. The PBSs which constitute the major pigment part are water-soluble complexes and attached to the thylakoid membranes; it has three components, that is allophycocyanin (APC) core and PC rods attached to the core and phycoerythrin (PE), and heavy metals have been reported to severely affect APC and PC contents, which declines the photosynthetic process and eventually the biomass production (MacColl, 1998). Moreover, the decline in PC content has been suggested to the exterior localization in the thylakoid membrane, thereby making it more prone to direct damage by the ROS (Singh, 2014). Apart from declining the pigment contents, heavy metals also decline the surface area of thylakoids which leads to inhibition of photosynthetic activity due to the modification in the functioning of QB which is the primary electron acceptor of PS II, thereby impairing the PS II activity (Ayyaraju, 2016). The decline in the pigment contents could also be correlated with the photochemistry of PS II as well as photosynthesis that has been discussed in the following section.

2.2  Impact of Heavy Metal Stress on Photosynthesis (Photosynthetic Activity and PS II Photochemistry) and Respiration Photosynthesis is a highly regulated, multistep process and depends on light-harvesting complexes that comprise photosynthetic pigments. It encompasses the harvest of solar energy, transfer of excitation energy, energy conversion, and electron

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transfer from water to NADP+, ATP generation, and a series of enzymatic reactions that assimilate carbon dioxide and synthesize carbohydrate (Tanaka and Makino, 2009). Photosynthesis is negatively affected due to heavy metal contamination. The reduced photosynthetic efficiency is associated with the impacts of heavy metals on the important components of p­ hotosynthetic machinery. The foremost is water-splitting complex, which has been reported to be the most labile component for metal toxicity at lower doses and at higher concentrations the toxicity is mediated beyond the OEC (oxygenevolving complex) and PS II (Prasad et al., 1991). Moreover, heavy metals affect the synthesis of proteins needed for the functioning of PS I, PS II, and the light-harvesting components and also downregulate the genes of different enzymes involved in carbon fixation (Ali et al., 2006; Perreault et al., 2009). Biochemical process of photosynthesis initiates from the absorption of light by antenna pigments and then its transfer to the reaction center (RC) where the photochemical reactions take place. The transfer of energy from one photosystem to another results in the generation of ATP and NADPH which further act as the major energy source during carbon dioxide fixation. The excess energy absorbed by the photosynthetic pigments is dissipated in the form of heat or radiation known as fluorescence (Strasser et al., 2000). In cyanobacteria, absorption of light is accompanied by PBS and thereafter transfers to the RC complex (Singh et al., 2015a,b). The toxicity imposed by heavy metal is also analyzed with the help of the fluorescence transient test which provides information about the impact on energy fluxes and electron transport through and beyond PS II. Heavy metals such as Ag, Pb, Cr, and Hg, etc., interrupt the electron transport chain and as a result, energy is dissipated in the form of heat. Chlorophyll fluorescence (JIP-test) involves both fluorescence kinetics and energy flux parameters which describe the efficiency of PS II under stressful condition. Various fluorescence kinetic parameters such as quantum yield of primary photochemistry (ϕP0), size and number of active RC (FV/F0), quantum yield of electron transport from QA to plastoquinone (ϕE0), yield of electron transport per trapped excitation (Ψ0), and performance index of PS II (PIABS), have been found to decrease under heavy metal exposure (Paunov et al., 2018; Patel et al., 2018; Tiwari et al., 2018). Furthermore, energy flux parameters, absorption of photon (ABS/RC), trapped energy flux (TR0/RC), electron transport flux (ET0/RC) and energy dissipation flux (DI0/RC) per active RC have been reported to increase under heavy stress condition due to decline in the number of active RC (Ali et al., 2006; Gong et al., 2008; Wang et al., 2012; Patel et al., 2018; Tiwari et al., 2018). In a study by Wang et al. (2012), it was demonstrated that treatment of Microcystis aeruginosa with 1 mg L−1 As(III) declined the number of active RCs and to maintain the balance between absorption and utilization of excess energy, the load on the active RC is increased, which results in increased values for energy flux parameters. Moreover, the in vitro experiments also suggested that heavy metals have two target sites: one at the donor side of PS II and other toward the acceptor side and also toward PS I (Zhang et al., 2017). The PS II activity has also been reported to be decreased under heavy metal stress due to the inhibition of D1 protein (Zsiros et al., 2006). High susceptibility of PS II than PS I to heavy metals was also described by other research works (Kupper et al., 1996; Dixit et al., 2002; Cox and Saito, 2013; Dixit and Singh, 2015). Apart from the PS II activity, the process of phosphorylation either cyclic or noncyclic is also affected by heavy metal stresses. Overall, it can be stated that a general decline in proteins related to carbon metabolism and photosynthesis is a common response under heavy metal stress and is associated with the downregulation of FutA2. The main target of heavy metal is to reduce the photosynthetic process (Aggarwal et al., 2012). Study also suggested that heavy metal stress also affected the proteins related to photosynthesis, as some proteins were downregulated, for example, the ferredoxin-NADP oxidoreductase, and some were upregulated including the 12 kDa extrinsic protein of PS II (PsbU), and a putative thylakoid lumen peptidyl-prolyl cis-trans isomerase (sll0408), important for the stabilization of PS II (Cox and Saito, 2013). Moreover, the respiration rate has been found to show differential impact under heavy metal stress condition, like Vega et al. (2006) reported that 4 mM As(III) decreases the respiratory activity at 4 h as well as at 24 h in Chlamydomonas, while, 0.30 mM As(III) concentration did not affect the respiratory activity. Moreover, studies by Patel et  al. (2018) and Tiwari et  al. (2018), reported increased respiration rate under As and Cr stress and the possible explanation for this increase was suggested to be the energy management process as well as the overproduction of ROS. The negative impact on photosynthesis is mediated by decline in the carbohydrate content, and Bakiyaraj et al. (2013) reported that with increasing concentration of Cu at 1, 2, 5, 10, 25, 50, and 100 ppm, there was a decrease in carbohydrate content of Phormidium tenue.

2.3  Impact of Heavy Metal on Nitrogen Metabolism Nitrogen is an essential nutrient for all living beings and is important component of amino acids, nucleic acids, pigments such as chlorophyll, and other biomolecules. In atmosphere, it is present as dinitrogen (N2) and acquisition of N is therefore a major ecological challenge for all organisms. Cyanobacteria are able to fix atmospheric N2 in a special cell structure called heterocyst. Biological nitrogen assimilation firstly involves nitrate assimilation where nitrate

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­reductase (NR) and nitrite reductase (NiR) reduce the nitrate to nitrite and ammonium, respectively (Ohashi et  al., 2011). The genes that positively regulate the expression of nitrate transporters (NRTs), NR, and NiR are influenced by level of nitrate in cell (Fernandez and Galvan, 2008; Krouk et al., 2010) while ammonium exerts negative effects on nitrate assimilatory genes. Mechanism of nitrate assimilation is same in cyanobacteria as well as in plants but the difference lies in NRT. In ­cyanobacteria, two distinct types of NRT transporters are present, the first is ABC (ATP-binding cassette)-type NRT encoded by four proteins NrtA, NrtB, NrtC, and NrtD (Omata, 1995) and the second one is MFS (major facilitator protein superfamily)-type transporter encoded by nrtP gene (Wang et al., 2000; Aichi et al., 2006). The nirA and narB gene promotes the activity of NiR and NR, respectively (Gonzalez et al., 2006). NtcA and NtcB are two transcription factors that positively regulate the nitrate assimilation and are activated in response to excessive accumulation of 2-oxoglutarate and nitrite (Tanigawa et al., 2002; Vazquez-Bermudez et al., 2002). Furthermore, atmospheric nitrogen is fixed by an oxygen-sensitive protein complex nitrogenase which is composed of two subunits: Fe protein coded by NifH and Mo-Fe protein coded by NifDK (Berman-Frank et  al., 2003). Chromium at elevated concentrations significantly reduces the activity of nitrogenase enzyme but these responses may vary from genus to genus, as it has been reported in a study that exposure of Cr at 5–20 mg L−1 to some cyanobacterial strains showed varied responses, as some strains, viz., Anabaena oryzae HH-20, Cylindrospermum michailovskoense HH-22, and Nostoc spongiaeforme HH-27 showed sensitivity, while Nostoc piscinale HH-21, Anabaena khanne HH-23, Anabaena circinalis HH-24, and N. punctiforme HH-26 were resistant and N. spongiaeforme HH-18, N. punctiforme HH-19 were found to be acclimatized under the Cr stress. In metal-tolerant species, increased nitrogenase activity was observed and this may be due to the secretion of exopolysaccharide (EPS) (for complex formation with metals) (Kiran and Kaushik, 2008) and extracellular metal binding proteins (to act as chelators), which helped in mitigating the effects of heavy metals (Duncan et al., 2006; Huang et al., 2009).

2.4  Impact of Heavy Metal on Oxidative Biomarkers and Antioxidants Status Generally, cell metabolic processes end with the production of ROS. The production and elimination of ROS is balanced in normal condition but under stressful environment the balance is disturbed (Karuppanapandian et al., 2011). The ROS include singlet oxygen (1O2), the superoxide anion (O2 •−), hydrogen peroxide (H2O2), and hydroxyl radical (•OH) that can oxidize the thiols of the cysteine residues of proteins (SH) into sulfenic (SOH), disulfides (SS), sulfinic acids (SO2H), and sulfonic acids (SO3H) (Cheloni and Slaveykova, 2018). In cyanobacteria, the varieties of ROS are formed at different sites of electron transport chain located in the cell membrane as well as in thylakoids (Latifi et al., 2009). Heavy metals are known to generate not only the ROS but also reactive nitrogen species (RNS) that in turn damage the lipid membranes and DNA (Volko et al., 2005). The ROS play detrimental roles as they inactivate several enzymes which are involved in various physiological and biochemical processes, damage cellular membrane structure, degrades and reduces photosynthetic pigment contents, proteins, lipids, and nucleic acids (Singh et al., 2016), which ultimately results in the death of organisms. Contrary to this, ROS at low concentration act as secondary messengers and activate signal transduction pathway (Vogel et al., 2014; Dietz, 2016). Cellular system is equipped with an array of antioxidant system and in response to ROS generation enzymatic and nonenzymatic antioxidants quickly act to protect cells from oxidative damages and detrimental effects of ROS are brought under control (Foyer and Noctor, 2005; Navrot et al., 2007). However, the rapid generation of ROS may disturb the balance between oxidants and antioxidants hence leads to severe damage of the cell. Enzymatic antioxidants, that is, SOD, CAT, APX, GR, MDHAR, DHAR, guaiacol peroxidase (GPX), and GST and nonenzymatic antioxidants, that is, AsA (ascorbate), GSH (reduced glutathione), phenolic compounds, alkaloids, nonprotein amino acids, and α-tocopherols, which work synergistically to control the cascades of uncontrolled oxidation and protect cells from oxidative damage (Latifi et al., 2009; Gill and Tuteja, 2010); Fig. 3. The actions of antioxidants are not only the protective mechanism adapted by cyanobacteria but they also have molecular mechanisms that in turn help these organisms survive under heavy metal stress.

3.  MECHANISM OF STRESS TOLERANCE IN CYANOBACTERIAL SYSTEM The phenomenon of stress tolerance is the ability of organism to adjust with the changing environmental conditions. Cyanobacteria are a group of interesting photosynthetic prokaryotes that have a high capability to tolerate various kinds of abiotic and biotic stresses (Singh, 2014). They are highly adaptable organisms that can respond to changing environmental conditions such as high or low temperature, light, and metal concentration. The mechanisms by which cyanobacterial cells minimize the harmful effects of heavy metal and maintain the cellular homeostatic condition are: (1) exopolysaccharide production, (2) biosorption and bioaccumulation, and (3) modulations in molecular indices.

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FIG. 3  Role of antioxidant defense system under heavy metal stress.

3.1  Exopolysaccharides—First Barrier for Protection at Cellular Level Physical barrier of organism against stress is considered as the first protective mechanism, for example, plants have thick epidermis whereas cyanobacteria have thick outer mucilage covering (synthesized from extracellular polymeric substances, mainly of polysaccharide) named exopolysaccharides (EPSs), which serves as protective covering against antimicrobial agents (Heindl et al., 2014) as well as act as primary protective barrier against heavy metals (Cd, Co, Fe, and CeO2, TiO2) (Zeyons et al., 2009; Planchon et al., 2013). The EPS are natural polymers of high molecular weight secreted by microorganisms into their environment which maintain the structural and functional integrity of biofilms. In heavy metal contaminated area, the EPS mediate the detoxification of heavy metal by acting as metal-binding agents (Gutnick and Bach, 2000). They actively participate in metal detoxification due to the presence of hydroxyl groups, ionizable functional groups as well as flexible polymer chains (Yin et al., 2011a,b). Cimini et al. (2011) reported the presence of various functional groups (carboxyl, amide, and hydroxyl) that actively participate in the sorption of Cu(II) as well as metal sequestration mediated by uronic acid (glucuronic acid), an acidic functional group of EPS.

3.2  Biosorption and Bioaccumulation Heavy metal sequestration is also done by the process of biosorption and bioaccumulation. Biosorption is an approach evolved by cyanobacterial as well as algal system to maintain the balance of excess concentration of heavy metals. It is a passive immobilization of metals and this property of cyanobacteria is subjugated to excrete heavy metals that are not biodegradable from polluted water and soil. Biosorption includes array of mechanism such as adsorption, absorption, ion exchange, surface complexation and precipitation (Wang et al., 2010) and accumulation. Basically two phases, that is, one is solid phase (biomass or biosorbent) and another is a liquid phase (solvent) having metal, have to be implemented in the mechanism of biosorption. The biological membranes of living organisms having property of ion exchange act as chemical substance that absorbs the heavy metals on its external surface. Interaction between microbial surface and heavy metals depends on the physicochemical interactions between the metal and functional groups present on the cell wall. Since the cell wall of cyanobacteria is made up of polysaccharides, lipids, and proteins which provide the binding

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sites for metals, the process is completed in minutes and is reversible due to noninvolvement of metabolism of organisms (Veglio and Beolchini, 1997). There are various factors that influence the biosorption process including contact time, incubation temperature, pH, biomass and initial metal concentration (Chojnacka, 2010). In past years, the known mechanisms for biosorption were surface complexation and precipitation, physical adsorption, ion exchange, etc. However, in recent time ion exchange is the dominating one of all. This mechanism involves competition between proton and metal ion for the binding site and the process is operated by the endogenous pH of the cell (Schiewer and Volesky, 2000). The pH influences the availability of the site to absorb/release metal ions either by protonation or de-protonation from the binding sites. On lowering the pH, metal ions are released from the binding site and this property is used for the recovery of metal cations and regeneration of the biosorbent (Chojnacka, 2010). Besides biosorption, another mechanism is bioaccumulation which is most complex process for metal tolerance. In this process, heavy metals are accumulated inside cells lining the inner membrane spaces or inside vacuoles and their detoxification occur in two stages: the first process is similar to biosorption which is very quick, and the subsequent, a slower process includes transport of metals inside cells by active transport system that requires metabolic energy. The process of bioaccumulation involves transportation of metals across the membrane and binding with the intracellular structures (Kujan et al., 1995) which ultimately results in their intracellular storage. In the cell interior, metals play oxidation or reduction reactions which lead to the loss of cellular integrity and function (Yilmazer and Saracoglu, 2009). Bioaccumulation is nonequilibrium process (Aksu and Dönmez, 2000, 2006) and results in the precipitation of toxic metals and support wastewater treatment processes known as bioremediation.

3.3  Regulation at Molecular Level Under the influence of heavy metal ions the survival strategy of cyanobacteria increases up to a certain level through the involvement of molecular approaches that gives a more comprehensive analysis. The “OMICS” tools are very useful in understanding the regulation of heavy metal stress which is a modern technology in the field of molecular science. Regulatory process from metal uptake to translocation and its toxicity is constricted under control at different subcellular levels (Schirmer et al., 2010) including abundance of RNA transcripts (transcriptome) and expression of proteins (proteome).

3.3.1  Genomics and Proteomics Genomics includes regulation at the gene and transcript level whereas translational regulation can be studied by analyzing the change in the protein expression. It can be said that genomics and proteomics work in consonance to establish heavy metal tolerance in cyanobacteria. In cyanobacteria cofactors are essentially required to carry out various metabolic processes, such as Fe (which is involved in cytochromes), Mn (which acts as an electron carrier in the water-splitting complex), Cu (in plastocyanin), and Mg (for chlorophylls) (Rahman et al., 2007). Excess metal availability generates a stress in cyanobacteria. Being an important metal, Fe homeostasis is regulated by ferric uptake regulator FurA in nitrogenfixing cyanobacteria. The FurA significantly enhances the synthesis of siderophores by upregulating proteins belonging to TonB and Dps family. FurA also regulates several enzymes involved in chlorophyll biosynthesis (Hernández et al., 2007; González et al., 2010, 2012). Among various heavy metals, As is a ubiquitous toxic metalloid which adversely affects the metabolic processes including oxidative phosphorylation as well as cause damage to the proteins, lipids, and nucleic acids (Shi et al., 2004; Requejo and Tena, 2005). To minimize the toxic effects of arsenic, cyanobacteria actively regulate an operon system (arsBHC operon) which involves different proteins that convert the arsenate to arsenite and also enhance the efflux of arsenic (Wang et al., 2009). It constitutes three genes: (i) arsB (arsenite efflux protein), (ii) arsH unknown protein, and (iii) arsC (arsenate reductase) regulated by the transcriptional repressor arsR. Furthermore, arsenite-­stimulated ATPase was found to be enhanced and encoded by arsA and arsD which is involved in arsenic efflux (Pandey et al., 2012). On the other hand, in Anabaena, asr1102 and alr1097, homologues of arsB (multidrug efflux protein), homologue for arsenate reductase is alr1105 that actively participate in arsenic stress management (Pandey et al., 2012). In cyanobacteria, genes, that is isiA (forms additional antenna system), isiB (codes flavin-containing protein that replaces ferredoxin) and idiA (codes integral subunit of PS II) were found to be enhanced under Fe limitations (Kunert et al., 2003; Lax et al., 2007). For iron acquisition, there is overexpression of ABC-type ferric ion transporter protein in Synechocystis (Katoh et al., 2001). In Cyanothece sp. CCY 0110, there is an overexpression of extracellular polymeric substance proteins under influence of Cu and Cd and acts as primary barrier for metal entrance (Mota et al., 2015). Genome of Anabaena PCC 7120 consists of 27% hypothetical proteins and 28% unknown proteins having no function. Among these, 174 proteins are described as transporter proteins involved in the transport of various heavy metals (Pandey et al., 2012). Under cadmium

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and other heavy metal exposure there is an upregulation of hypothetical protein All3255 involved in the resistance of heavy metal (Singh et al., 2015a,b). The ROS particularly singlet oxygen significantly enhance the synthesis of antioxidant proteins such as GPX-H or GPX5 and GSTS1 (Fischer et al., 2006). In Anabaena variabilis, a hypothetical protein named alr4050 was overexpressed, which leads to the formation of akinete marker protein (a resistant structure) and confers heavy metal resistance (Zhou and Wolk, 2002). Under As stress, two major proteins named peroxiredoxin (Prx) that detoxify the H2O2 (Dietz et al., 2006) and thioredoxin (Trx) were maximally upregulated in Synechocystis sp. PCC 6803 (Lindahl and Florencio, 2003). Copper is an essential metal and is involved in the synthesis of plastocyanin, encoded by petE, while Fe is involved in cytochrome b6f complex synthesis, encoded by petJ (Zhang et al., 1992). Cyanobacteria under elevated concentrations of heavy metals like Zn, Ca, and Cu enhance the synthesis of protein SmtA which is cysteine-rich and involved in metallothionein synthesis that subsequently chelates the metal in cytosol. The SmtB protein is transcribed from smtA and is mediated by DNA-binding repressor SmtB (Busenlehner et al., 2003). Mutants of smtA and smtB provide tolerance against heavy metals (Turner et al., 1993). Furthermore, for metal export in Synechocystis PCC 6803 ziaA gene was found to be upregulated under Zn exposure and acts in a manner similar to P1-type ATPases metal-transporting proteins. The regulators of SmtA and ZiaA (SmtB and ZiaR) are very similar in amino acid sequence (Thelwell et al., 1998). Some detailed studies on the transporters involved and regulation at the genomics and proteomic level has been provided in Tables 1 and 2.

3.3.2  Heat Shock Proteins (Molecular Chaperones) Molecular chaperones are a family of proteins which assist in the correct folding of the polypeptides or their assembly into oligomeric structures (Rajaram et al., 2014). Chaperonins are a distinct group of chaperones which include subunits of a chloroplast protein complex involved in the assembly of ribulose bisphosphate carboxylase/oxygenase (Rubisco) (Zhao and Liu, 2017) the 60-kDa heat shock protein (hsp60) of yeast (Kalderon et al., 2015) and the proteins encoded by groESL operon in Escherichia coli. One type of chaperonins, called chaperonin-60 (cpn60), is homologous to groEL protein of E. coli (Goyal and Chaudhuri, 2015) and has molecular weight(s) in the range 56–61kDa. Chaperonin-60 from plastids of higher plants (Rbu-P2 carboxylase-binding protein) and from bacteria (groEL proteins) share several structures. They are involved in the assembly of oligomers into multimeric structures (Goyal and Chaudhuri, 2015). Molecular sequencing of genes encoding these chaperonins has revealed a high conservation in the amino acid sequences in chaperonins from E. coli and higher plants. There are, however, some differences between these two chaperonins. The groEL protein of E. coli is active as a functional complex with groES protein (chaperoninl0), while the existence of chaperonin-10 in higher plants is yet to be demonstrated. The plant chaperonin complex contains two distinct cpn60 subunits, that is, a and p. These two types of subunits are different from each other. Two such divergent types of cpn60 proteins are not found in bacteria. Heat shock proteins with molecular masses of approximately 70 kDa (hsp70) have been identified and characterized from several organisms (He et al., 2008). It has been known for a long time that cellular concentrations of these highly conserved proteins increase rapidly under stress conditions. Their role during normal growth conditions has been only recently realized. They function as chaperones in the transport of certain secreted or mitochondrial proteins. Members of hsp70 family have been shown to be present in mitochondria (Böttinger et al., 2015) and chloroplasts (Zhong et al., 2013). Photosynthetic processes in cyanobacteria are functionally and structurally similar to those in chloroplasts of higher plants. Therefore, cyanobacteria and plants may have similar mechanisms for assembling their photosynthetic protein complexes. Examining the role of molecular chaperones in cyanobacteria, that are model species for molecular genetics, might help in elucidating the function of these proteins in plastids. Therefore, some works have been initiated to clone and characterize genes encoding important chaperones from cyanobacteria and so far two genes, dnaK and cpn60, encoding homologues of hsp70 and cpn60 proteins, respectively, from the cyanobacterium Synechocystis sp. PCC 6803 have been characterized (Chitnis and Nelson, 1991). The overall physiological, biochemical, and molecular mechanisms of heavy metal tolerance in cyanobacteria have been shown in Fig. 4.

4. CONCLUSION In conclusion, the increasing concentration of heavy metals in the environment not only restricts the growth of cyanobacteria but also adversely affects cellular biochemical processes that are important from ecological point of view. Cyanobacteria employ various defense mechanisms to survive or mitigate heavy metal stress. Among these, synthesis of EPS, metallothioneins, chaperone proteins are most common. Moreover, induction of enzymatic and nonenzymatic antioxidants is also a common feature exhibited by cyanobacteria under heavy metal stress.

TABLE 1  Transporters Involved Under Heavy Metal Stress in Cyanobacteria and Other Microorganisms Name

Metals

Localization

Functions

Organisms

References

CDF-family (cation diffusion facilitator)

CrMTP1-CrMTP5

Cd, Zn, Co

Integral membrane proteins and vacuoles

Ion uptake, Act as efflux pumps

Chlamydomonas reinhardtii, Staphylococcus aureus

Hanikenne et al. (2005), Xiong and Jayaswal (1998)

ZIP-family (ZRT-IRT) like protein, iron regulated transporters

ZRT1-ZRT5 ZIP1-ZIP7

Zn, Fe, and Mn

Vacuoles, organelles and secretary pathways

Active uptake of ions influx and efflux

Chlamydomonas reinhardtii

Guerinot (2000), Merchant et al. (2006)

CAX-family (cation antiporters)

Cr CAX1-Cr CAX5

Ca/proton

Membrane protein, Vacuoles and organelles

Calcium: proton antiporter activity and cellular calcium ion homeostasis

Chlamydomonas reinhardtii, Chlamydomonas smithii

Merchant et al. (2007)

MRP-family Multidrug resistance-associated protein

CrMRP1-CrMRP7

Organic anions

Vacuolar membrane

Xenobiotic detoxification of organic anions (glutathionated compounds) efflux activity

Cyanidioschyzon merolae

Sharma et al. (2002)

NRAMP-family (natural resistanceassociated macrophage protein)

CrNRAMP1CrNRAMP3

Fe/Mn transport

Vacuoles and secretary pathway

Transport divalent metal cations into cytosol

Chlamydomonas reinhardtii, Chlamydomonas smithii, Cyanidioschyzon merolae

Blaby-Hass and Merchant (2012)

P-type ATPases

Na+/K+-ATPase, H+ATPase, H+/K+-ATPase, Ca++-ATPase

Cu

–

Transport cations across membranes against their electrochemical gradient using ATP

Anabaena variabilis, Nostoc sp., Acaryochloris, Prochlorococcus, Synechococcus, Synechocystis.

Kühlbrant (2004), Babayan (2010)

HMA-family (heavymetal-associated domain)

CrHMA1-CrHMA3

Cu and Cu/Zn for superoxide dismutase and Cd

Plastids

Cu/Ag supply and play a role in Cu supply to plastocyanin in alga

Cyanidioschyzon merolae, Chlamydomonas reinhardtii

Williams and Mills (2005)

FTR-family

FOX 1

Iron (Fe)

Chloroplast and Secretory pathway

Involves in iron homeostasis

Chlamydomonas reinhardtii

Terzulli and Kosman (2009)

Metals in Cyanobacteria Chapter | 13  269

Subfamily

270 Cyanobacteria

TABLE 2  Showing Regulation of Heavy Metal Stress Tolerance in Cyanobacteria at Genomic and Proteomic Level Organism

Heavy Metal

Synechocystis PCC 6803

Cd and Co

Transcription Factors Involved

Cyanobacterial Response

References

sll0923, sll1581, slr1875, and sll5052

Sequestration of heavy metals, bio mineralization, and encountered stress

Jittawuttipoka et al. (2013)

Ce and Ti

sll0923, sll1581, and slr1875

Shield Synechocystis from direct contacts with the oxidative stressgeneration

Von Moos and Slaveykova (2014)

Ni, Co, and Zn

ZiaA, (slr0798), ZiaR, (sll0792)

ZiaA gene regulates Zn-repressor, which is a homologue of smtB. Both the gene ZiaA and ZiaR are responsible for homeostasis of heavy metal

Nakamura et al. (1998), Thelwell et al. (1998)

Mn

mntC (sll1598) (Sll0649)

Protection against oxidative stresses

Chen et al. (2014)

As

arsBHC (slr0944slr0945-slr0946), arsR gene sll1957) ArsH, ArsC

Tricistronic operon, encoding quinone reductase activity ArsC is an arsenate reductase, which uses the glutathione/glutaredoxin system for reduction

Lopez-Maury et al. (2009), Yin et al. (2011a,b)

Hg and U

slr1849

Codes for mercuric reductase MerA enzyme protects against and NADPH-driven reduction of, mercuric ions This enzyme is also capable to reduce uranyl ions thereby reducing U induced toxicity

Marteyn et al. (2013)

Synechococcus PCC 7942

Zn and Cd

smtA gene which is transcribed from smtB

smtA act as a metal responsive repressor

Blindauer (2011) Huckle et al. (1993)

Anabaena sp. PCC 7120

Co and Zn

furA gene

Homodimeric metalloprotein and constitutive proteins, that complex with heavy metal under high intracellular concentrations and its expression increases under oxidative stress conditions

González et al. (2016)

Synechocystis spp.

Cd, Co, As, Mn, and Hg

Fed proteins ferredoxin-encoding genes designated as fed1 (ssl0020), fed2 (sll1382), fed3 (slr1828), fed4 (slr0150), fed5 (slr0148), fed6 (ssl2559), fed7 (sll0662), fed8 (ssr3184), and fed9 (slr2059)

Fed genes are highly conserved in cyanobacteria and decrease the tolerance to oxidative stress (superoxide radical, SOR and H2O2) and metal stresses Fed proteins are also involved in promoting growth

Cassier-Chauvat and Chauvat (2014)

Synechocystis sp.

As, Hg, Co, Cd, and Zn

Fur-Like proteins (FUR), Slr1738, PerR proteins, zinc uptake regulator (ZUR)

PerR protein senses reactive oxygen species through catalyzing oxidation and dissociates them, similarly ZUR involved in repressing the transcription which involved in metal homeostasis

Fillat (2014)

Metals in Cyanobacteria Chapter | 13  271

FIG. 4  Schematic representation illustrating the regulation of heavy metal stress at physiological, biochemical, and molecular levels.

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FURTHER READING Chan, H., Babayan, V., Blyumin, E., Gandhi, C., Hak, K., Harake, D., Kumar, K., Lee, P., Li, T.T., Liu, H.Y., Lo, T.C., Meyer, C.J., Stanford, S., Zamora, K.S., Saier, M.H., 2010. The p-type ATPase superfamily. J. Mol. Microbiol. Biotechnol. 19, 5–105. Gayomba, S.R., Jung, H.I., Yan, J., Danku, J., Rutzke, M.A., Bernal, M., Krämer, U., Kochian, L.V., Salt, D.E., Vatamaniuk, O.K., 2013. The CTR/COPTdependent copper uptake and SPL7-dependent copper deficiency responses are required for basal cadmium tolerance in A. thaliana. Metallomics 5, 1262–1275. Glatz, A., Vass, I., Los, D.A., Vigh, L., 1999. The Synechocystis model of stress: from molecular chaperones to membranes. Plant Physiol. Biochem. 37, 1–12. Pengfu, L., Stephen, E., Liu, H.Z., 2001. Cyanobacterial exopolysaccharides: their nature and potential biotechnological applications. Biotechnol. Genet. Eng. Rev. (1), 375–404.