Critical Reviews in Environmental Science and

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Prospects for Exploiting Bacteria for Bioremediation of Metal Pollution Arif Tasleem Jan Rizwanul Haq

a b

a

a

, Mudsser Azam , Arif Ali & Qazi Mohd.

a

a

Department of Biosciences , Jamia Millia Islamia , New Delhi , India b

National Institute of Immunology , Aruna Asaf Ali Marg , New Delhi , India Accepted author version posted online: 20 Aug 2013.Published online: 05 Feb 2014.

To cite this article: Arif Tasleem Jan , Mudsser Azam , Arif Ali & Qazi Mohd. Rizwanul Haq (2014) Prospects for Exploiting Bacteria for Bioremediation of Metal Pollution, Critical Reviews in Environmental Science and Technology, 44:5, 519-560, DOI: 10.1080/10643389.2012.728811 To link to this article: http://dx.doi.org/10.1080/10643389.2012.728811

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Critical Reviews in Environmental Science and Technology, 44:519–560, 2014 Copyright © Taylor & Francis Group, LLC ISSN: 1064-3389 print / 1547-6537 online DOI: 10.1080/10643389.2012.728811

Prospects for Exploiting Bacteria for Bioremediation of Metal Pollution ARIF TASLEEM JAN,1,2 MUDSSER AZAM,1 ARIF ALI,1 and QAZI MOHD. RIZWANUL HAQ1 1


2

Department of Biosciences, Jamia Millia Islamia, New Delhi, India National Institute of Immunology, Aruna Asaf Ali Marg, New Delhi, India

Metals being recalcitrant to biodegradation process pose a persistent threat to human health and environment. In view of increase in discharge along with improper management of persistent metal pollutants, it is imperative to develop cost-effective and efficient methods for their remediation. As contamination of soil and water has threatened the well being of humans and natural environment, microorganisms play crucial role in combating the widespread pollution of global environment. Clusters of genes coding for catabolic transformation facilitate their detoxification from the environment. Development of effective tools to facilitate environmental cleanup of metal pollutants beyond genetic confines of natural host has resulted in the expressional enhancement of promiscuous enzymes, involved in the transformation of metal compounds. A thorough understanding of microbes that express heterologous proteins for metal transformation would result in economic production and as such its application in bioremediation process. This review summarizes fundamental insights regarding metals in relation to oxidative stress, insights on metal binding proteins/peptides for immobilization, information regarding genetic engineering for enzymes involved in metal transformation, and strategies that can be employed to overcome the bottlenecks associated with microbial based remediation strategies. KEY WORDS: antioxidants, bacteria, bioremediation, genetic engineering, metals, oxidative stress Address correspondence to Qazi Mohd. Rizwanul Haq, Department of Biosciences, Jamia Millia Islamia, New Delhi-110025, India. E-mail: [email protected] Color versions of one or more of the figures in the article can be found online at www.tandfonline.com/best. 519

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1. INTRODUCTION Global pollution that arises as a consequence of unrestrained industrialization and urbanization, together with inadequate measures of emission control and pollution abatement, has raised new challenges especially in the field of environmental protection and conservation. Given our long and intimate association with metals (mercury, arsenic, cadmium, lead, and aluminum), and our continued reliance on them in areas as diverse as medicine, electronics, and catalysis, it is not surprising that their use had lead to significant environmental problems that need to be addressed. Increased influx of metals that are known for their adverse effects on humans, besides being main culprits responsible for environmental deterioration, has raised serious concern regarding their long-term environmental sustainability. Toxic nature of metals (particularly mercury and arsenic) is generally represented by following characteristic features: their toxicity even at concentration of 1.0–10 mg/L, undergo change in valence and type of species but cannot be degraded completely by any methods, ability to get transformed from low toxic to more toxic forms under certain environment conditions as in the case of mercury, and ability to get accumulated in the food chain that hamper normal physiological activities and ultimately endanger human life (McLaughlin et al., 1999; Nair et al., 2008). Concern regarding their potential risk to human population is growing at a steady rate as they enter body from a wide variety of sources including foods, drinks, air, and vaccines. Acute toxicity occurs from a sudden or unexpected exposure to high level of the metal as a result of careless handling, inadequate safety precautions at industrial settings, or from accidental spill during transportation, while as chronic toxicity results from repeated or continuous exposure that leads to its accumulation in the body. Metals such as mercury and arsenic have been recognized as the most toxic elements principally in relation to their series of effects on humans following occupational exposure or from a number of environmental accidents. Environmental pollution is a major problem that arises as a result of natural processes as well as from anthropogenic sources. Although a number of physical processes (e.g., volcanoes, forest fires) release different pollutants in the environment, anthropogenic activities are believed to be the major cause of environmental pollution. Increases in environmental contamination that lead to progressive deterioration of environmental quality presented a challenge before global society to find effective measures of remediation in order to reverse the negative conditions that severely threaten human and environmental health. Physiochemical approaches being expensive, nonspecific, and often leading to secondary metabolites are gradually replaced by eco-friendly treatments commonly referred to as bioremediation. Microbes interact with metals in natural and synthetic environments, altering their physical and chemical state. Microbes and their symbiotic associations with


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each other and higher organisms have contributed actively to geological phenomena that involve metal transformations having beneficial or detrimental consequences with respect to human context (Gadd, 2010). Despite being toxic, microbes grow and flourish in metal polluted locations apparently by adapting themselves through a variety of resistance mechanisms that are active and incidental (Gadd, 2010; Gadd and Griffith, 1978). Over the years, bioremediation has emerged as the most advantageous soil and water cleanup technique for contaminated sites, with bacteria being the most important organism in context to reclamation, immobilization or detoxification of metal pollutants (Fu and Wang, 2011; Malik, 2004). Bacteria developed metal resistance mostly for their survival. Among various strategies through which bacteria resist toxicity of metals, important ones include (a) toxic metal may get sequestered on cell wall components or by intracellular metal binding proteins and peptides such as metallothioneins and phytochelatins along with compounds such as siderophores, and compared to fungi that produce hydroxamate siderophores, bacterial siderophores are mostly catecholates, although some exist as polycarboxylates as well; (b) changing uptake pathways which block uptake of a metal; (c) enzymatic conversion of the metal to a less toxic form; and (4) lowering the intracellular concentration of the metal by a specific efflux system. Considerable interest in exploring metal resistance mechanisms in bacteria serve to highlight the credentials that are particularly advantageous for their practical application in dealing unfavorable metal burden in nature. This review deals with the studies that begin to uncover as how metals particularly mercury and arsenic affect biomolecules, detailed biochemistry behind endogenous detoxification system in microbes, and the ways to genetic engineer microbes for improved detoxification and degradation of toxic pollutants.

2. METALS 2.1 Mercury Mercury (Hg), a transition metal, belongs to group 12 (IIB) of the periodic table. It is the only metal that exists in liquid form at room temperature. For its silver liquid appearance, it has long been known as quick silver. It has been annotated with the symbol Hg, meaning Hydrargyrum (a Latin word, representing watery silver). It occurs in the earth’s crust in elemental form and as a variety of binary minerals such as cinnabar (HgS), metacinnabar, and hypercinnabar (Barkay et al., 2003). Mercury pollution is an emerging problem in the present day world as its contamination in the environment has increased several fold as a consequence of natural as well as anthropogenic activities (Zhang et al., 2012). In nature, mercury exists in three valence states: metallic or elemental form (Hgo), inorganic (Hg2+ or Hg+), and organic form (R-Hg+ or R-Hg-X, where R is methyl, ethyl or phenyl and X is acetate).

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TABLE 1. Joint FAO/WHO evaluation for assessing toxicological profile of different metals on humans (adapted from ftp://ftp.fao.org/codex/meetings/cccf/cccf6/cf06 INFe.pdf) Metals Mercury Agency

Inorganic

Methylmercury

Arsenic (Inorganic)

(JECFA) PTWI 4 μg/kg PTWI 0.0016 3.0 μg/kgbody weight mg/kg body day body weight weight.

Cadmium

Lead

PTMI 25∗ PTWI withdrawn in 2010.

Aluminum PTWI 2#


Note. TWI = provisional tolerable weekly intake; PTMI = provisional tolerable monthly intake; JECFA = Joint FAO/WHO Expert Committee on Food Additives (2006, 2010, 2011).

All forms are toxic, but the extent of toxicity varies among different forms of mercury; the organic form being the most toxic and the elemental form the least one. Detrimental fate of organomercurial compounds in humans is accredited to its hydrophobic nature, which it employs while crossing the placental and blood-brain barrier (Jan et al., 2009; Singh et al., 2011). Despite being extremely toxic, there still exists a dilemma regarding their use in chloralkali plants for the manufacturing of chlorine and sodium hydroxide by electrolysis of brine, in paints as preservatives or pigments, in electrical switching equipment and batteries, in measuring and control equipment (e.g., thermometers), as catalyst in chemical processes, as amalgams in toothfilling materials, and as fungicides in agriculture. Enormous rise in mercury levels has become a growing concern for developing countries owing to its high toxicity, translocation, and ability to get accumulated at various steps of food chain. In addition to dental amalgamation and dietary sources, exposure to mercury at occupational and environmental settings poses a continuous threat to human health (ASTDR, 2003). Extent of adverse effects in an organism depends on the form of mercury at the time of exposure, duration and on the route of exposure (Bose-O’Reilly et al., 2010; Dufault et al., 2009). Regardless of the fact that both organic as well as inorganic forms of mercury crosses the membrane with different abilities; both have high affinity for thiol groups of enzymes and proteins, which lead to their inactivation (Gupta and Ali, 2004; Hajela et al., 2002). Difference in the mechanisms involved in the transport and metabolism of inorganic and organic forms of mercury is responsible for disparity in their distribution to tissues and organs, pattern of biological effect, and their toxicity (Zalups and Lash, 1994; Table 1). Molecular mimicry with endogenous substrates plays an important role in facilitating transport of mercury to different cells and tissues. Inside cells, mercury reacts with sulfur atoms of endogenous thiol containing molecules such as glutathione, cysteine, metallothionein, homocysteine, N-acetylcysteine, and albumin, produced by cell in a way to scavenge mercury (Jan et al., 2011). Degradation product of Hg2+-glutathione



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complex (i.e., dicysteinyl-Hg and CH3 -Hg-Cys) that resembles with endogenous cystine and methionine competes with them for their transport into cells and tissues (Zalups, 2000). It is supposed to be the possible explanation for specific transport and tissue specific accumulation of mercury over proximal tubular epithelia into kidneys and endothelial cells of blood-brain barrier. Compared to organic mercury that crosses blood brain barrier and accumulates in the motor regions of the brain and central nervous system (CNS), inorganic mercury exerts its toxic consequences mainly on getting methylated to methylmercury compounds that gets trapped inside the brain (Pedersen et al., 1999). Toxicity of inorganic mercury inside brain lies in that it increases the intracellular calcium level rapidly from the extracellular calcium pools that in turn disturb production of neurotransmitters, whereby creating serious imbalances in the development of brain. In addition, molecular interactions of mercury with sulfhydryl groups of enzymes and proteins results in inhibition of activity of a wide variety of enzymes, interferes with membrane transport protein function, besides disturbing structural integrity of cell through interference in microtubule formation.

2.2 Arsenic Arsenic (As) is a group 15 (V) member of periodic table is a naturally occurring metalloid, with no known nutritional requirement to the biological world. In nature, it occurs mostly as mineral arsenopyrite, a compound of iron, arsenic, and sulfur. It ranks highest in priority on the list of top 20 hazardous substances issued by ASTDR and USEPA (ASTDR, 2005). It exists as As0 (metalloid arsenic), As3+ (arsenates), As5+ (arsenates), and As3− (arsine gas) valence states. However, arsenic compounds are generally categorized as organic (nontoxic) as well as inorganic (toxic) forms. Organoarsenic species, mostly methyl derivatives such as arsenosugars, arsenobetaine, arsenocholine, and arsenolipids, are widespread in aquatic organisms including shrimps, lobsters, fish, seaweeds, and in many species of marine animals. Inorganic arsenic predominantly exists in trivalent (As3+) and pentavalent (As5+) forms, with trivalent compounds having the property to get absorbed more rapidly due to high lipid solubility as more toxic than pentavalent ones (Kaur et al., 2009). They are interconvertible depending on the redox status of the environment, with pentavalent arsenic more stable and predominant under aerobic conditions and trivalent species under anaerobic conditions (Duker et al., 2005). Relative toxicity of arsenic compounds depends primarily on form of arsenic compound, their valence state, solubility, and rate of absorption and elimination (ASTDR, 2007). Generally, forms that are more readily absorbed are more toxic, while as those that are rapidly eliminated are less toxic. Arsenic intoxication in humans occurs primarily after its absorption from the gastrointestinal tract. Inside the body, it exerts its toxic consequences soon after binding to hemoglobin, plasma proteins


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and leukocytes, whereby it gets redistributed to liver, kidney, lung, spleen, and intestines. However, unlike other metals such as mercury and cadmium that get biomagnified through the food chain, arsenic does not appear to get biomagnified. Arsenate (As5+), a phosphate analogue competing with phosphates for adenosine triphosphate, results in adenosine diphosphate monoarsine formation by substituting arsenic for phosphate. Soon after its formation, it interferes with essential cellular processes such as oxidative phosphorylation by hampering ATP synthesis (Jomova et al., 2011). Compared to arsenate, toxicity of arsenite (As3+) is accredited to its propensity to bind sulfhydryl groups of dihydrolipoic acid (a pyruvate dehydrogenase cofactor associated with the conversion of pyruvate to acetyl coenzyme A), thereby resulting in the reduction in citric acid cycle activity. In addition to that, binding of arsenite (As3+) to sulfhydryl groups is associated with the inhibition of gluconeogenesis and production of glutathione, responsible for protecting cells against oxidative stress (Jomova et al., 2011). Cytotoxicity of arsenite (As3+) was found to be greater than that of arsenate (As5+; Bertolero et al., 1987; Jomova et al., 2011). The trivalent arsenicals are preferred substrates for methylation reactions and hence the reduction of arsenic from pentavalent to trivalent may be a critical step in the control of the rate of metabolism of arsenic (Styblo et al., 2000; Styblo et al., 2002). Biomethylation of arsenic is considered as the primary detoxification mechanism during which reactive species of inorganic arsenic, which are potentially more toxic to humans get converted to less toxic methylated forms. In higher organisms including humans, inorganic arsenic is methylated to monomethylarsonic acid (MMA) and finally to dimethylarsinic acid (DMA) in presence of glutathione and methyl donor, S-adenosylmethionine (SAM), in an reaction catalyzed by methyltransferases (Roy and Saha, 2002). These methyl derivatives are thousand-fold less potent as mutagenic agents than the inorganic arsenicals, as evident from a mouse lymphoma assay (Mates et al., 2008).

2.3 Cadmium Cadmium (Cd), the 48th element in the periodic table, occurs naturally in the earth’s crust in association with zinc, copper, and lead ores. It is commonly encountered in cadmium-nickel battery production, nonferrous metal mining and refining, manufacture and application of phosphate fertilizers, and waste incineration and disposal, which constitute the main anthropogenic sources of cadmium in the environment. In humans, major routes of exposure to Cd include cigarette smoke and food especially seafood, mushrooms and chocolate (European Food Safety Authority, 2009). Over the time, accumulation of cadmium salts results in the development of a wide variety of effects such as osteoporosis, anemia, eosinophilia, and irreversible renal tubular injury as evident by increased excretion of low molecular weight proteins such as



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α 1 - and β 2 -microglobulin or enzymes such as N-acetyl-β-D-glucosaminidase (NAG; Jarup, 2003). Cd, a potent carcinogen associated with cancer of lung, prostate, pancreas, and kidney, has been classified as category 1 human carcinogen by International Agency for Research on Cancer (Miura, 2009). Although molecular mechanisms of cadmium induced carcinogenesis are not yet well understood, factors that may be involved in the process of carcinogenesis involve upregulation of mitogenic signaling and replacement of essential metals such as calcium, copper, iron, and zinc in various biomolecules and enzymes. Despite being unable to participate in redox reactions under physiological conditions, indirect generation of radicals such as superoxide radical, hydroxyl radical, and nitric oxide under both in vivo and in vitro conditions, have inhibitory effects on antioxidant enzymes such as catalase, SOD, glutathione reductase, and glutathione peroxidase (Beyersmann and Hartwig, 2008). Replacement of zinc in zinc finger structures was proposed as molecular basis for the inactivation of DNA repair enzymes, besides inhibiting 8-oxo-dGTPase, an enzyme that hydrolyzes mutagenic oxidation products of dGTP species.

2.4 Lead Lead (Pb) is a persistent and common environmental contaminant. Humans are exposed to lead through paint, cosmetics, folk remedies, and food supplements, besides being exposed at occupational settings during the manufacture of batteries, ceramic glazes, and solder. Inorganic Pb can be absorbed following inhalation, oral and dermal exposure through the respiratory tract (submicron size particles), and through GI tract primarily in the duodenum (larger particles >2.5 μm; ASTDR, 2007). Gastrointestinal absorption of inorganic Pb (e.g., lead chloride, lead acetate) appears to be higher in children than in adults. Nutritional status of iron and calcium contributes as a risk factor for Pb intoxication, as their deficiency increases retention of ingested Pb (Ruff et al., 1996). In adults, ∼94% of body burden of lead remains confined to bones compared to ∼73% in children (ASTDR, 2007). Lead (Pb), a ubiquitous environmental toxin that has long been known to amend the hematological system by inhibiting the activities of several enzymes involved in heme biosynthesis, particularly δ-aminolevulinic acid dehydratase (ALAD), results in increased circulating aminolevulinic acid (ALA), a weak γ -aminobutyric acid (GABA) agonist that decreases GABA release by presynaptic inhibition (Hernberg and Nikkanen, 1970; Millar et al., 1970). It inhibits calcium entry into cells thereby interferes with synaptogenesis. Virtually every neurotransmitter system is affected by lead, with dopaminergic, cholinergic, and glutamatergic systems receiving the most attention. In blood, Pb is primarily found in red blood cells. By destabilizing the cellular membranes of red blood cells (RBC), it decreases cell membrane fluidity


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and increases the rate of erythrocyte hemolysis that as a whole led to the development of anemia (Bellinger and Bellinger, 2006; Needleman, 2004). Ability of lead to mimic calcium or perturb calcium homeostasis makes lead a powerful neurotoxin having diverse impacts on the CNS (ASTDR, 2007). At picomolar concentrations, it competes with calcium for binding sites on cerebellar phosphokinase C and thereby affects neuronal signaling. Pathogenesis of lead toxicity is multifactorial. It is plausible that impaired oxidant/antioxidant balance are responsible for the toxic effects of Pb, as it induces oxidative stress through generation of reactive oxygen species, reducing the antioxidant defense system of cells (either by altering activities of SOD, catalase, and glutathione peroxidase or by depleting glutathione), interfering with some essential metals needed for antioxidant enzyme activities and by increasing susceptibility of cells to oxidative attack by altering membrane integrity and fatty acid composition (Gurer and Ercal, 2000). Metabolism of inorganic Pb consists of formation of complexes with a variety of protein and nonprotein ligands. Organic Pb compounds are actively metabolized in the liver by oxidative dealkylation by P450 enzymes.

2.5 Aluminum Aluminum (Al), representing ∼8% of total mineral component following oxygen (47%) and silicon (28%), is the third most abundant element in the earth’s crust. It exists only in one oxidation state (i.e., +3) and does not undergo redox reactions (Exley, 2004). Although exposure of aluminum occurs through consumption of food items, ingestion of water contaminated with aluminum, and inhalation of ambient air, considerable evidence is consistent with the view that Al finds its way into circulation on getting absorbed from the GI tract. Absorption of Al is enhanced in presence of citrate and prevented in presence of phosphate (Fulton et al., 1989). Al shares transporting mechanism with iron for its absorption in intestines and as such is believed to cause decrease in iron content of intestinal cells. Al binds to transferrin at the same site as Fe3+ and as such shares this iron transporter protein for its transport in plasma. It is estimated that ∼90% of Al the plasma is in complex with transferrin and remainder associated with citrate (Ohman and Martin, 1994). In fact, aluminum citrate is the major species present in the extracellular fluid of brain (approximately 60%). In order to prevent Al deposition in brain, it is transported across the barrier through monocarboxylate transporter or by glutamate transporter (Ackley and Yokel, 1998). Schetinger et al. (1999) reported that exposure to aluminum sulfate in mice results in its accumulation in the order: liver > kidney > brain. Robertson et al. (1983) reported that Al induces osteomalacia in rats chronically injected with aluminum chloride, and Bushinsky et al. (1995) reported in their study that incorporation of Al into the bones causes physiochemical mineral dissolution as well as cell mediated bone resorption. Al accumulation


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in renal tissue affects cellular metabolism, besides promoting degeneration of renal tubular cells and inducing oxidative stress (Bertholf et al., 1989). Evidence regarding involvement of Al in oxidative stress is supported by the fact that Al on reacting with superoxide anion generates aluminum superoxide anion, a more potent oxidant than superoxide anion that promotes formation of hydrogen peroxide and hydroxyl radicals that contributes up to a greater extent to oxidizing environment. Although aluminum uptake in the brain is slower compared to other organs, it is believed that there might be a relationship between high level of aluminum and increased risk of a number of neurodegenerative disorders.

3. METAL INDUCED OXIDATIVE STRESS As a step toward development, increased industrial activities contributed significantly to pollution of surrounding environment. Metals soon after their release from contaminated sites migrate through the unsaturated zone of ground water and become culprit for the contamination of drinking water supply. Pollution of water resources is continuously increasing with population growth and industrial development, along with inadequate control measures and proper management practices (World Health Organization, 1996). Toxicity of metal compounds is accredited to their prevalence in close proximity to humans that facilitates their access into living systems. In view of the uncertainties in the estimated fluxes of metals in the environment, maintaining availability of essential and controlled distribution of toxic metal ions within physiological limits requires an extensive regulatory cross-talk between toxic metal homeostasis and homeostatic network of essential nutrients. The existence of a complex metal homeostasis has come into focus as exposure of humans and animals to certain metal derivatives results in a variety of toxic effects ranging from immediate lethality to slow process of carcinogenesis (Kasprzak, 1995, 2000). In addition to interference with the DNA repair system, metal intoxication involves the cumulative generation of reactive oxygen (ROS) and nitrogen species (RNS), which induce a complex series of downstream adaptive and reparative events driven by oxidative and nitrative stress (Figure 1). ROS distinguished by their high chemical reactivity include free radicals such as superoxide (O2 ·−), hydroxyl (OH·), peroxyl (RO2 ·−), and alkoxyl (RO.), along with certain nonradical species such as peroxynitrite (ONOO−) and H2 O2 that are either oxidizing agents or get easily converted into radicals. In biological systems, superoxide (O2 ·−) being chemically more reactive than molecular oxygen itself, gets converted to relatively more reactive species such as peroxyl (RO2 .), alkoxyl (RO.), and hydroxyl (OH·) radicals (Valko et al., 2004). Interaction of superoxide (O2 ·−) with nitric oxide (NO) at physiological conditions results in the generation of highly reactive peroxynitrite (ONOO−). Superoxide (O2 ·−), being poor in


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FIGURE 1. Schematic representation of metals in relation to production of reactive oxygen (ROS) reactive nitrogen species (RNS). Antioxidant system of the body equipped with a variety of antioxidants such as glutathione (GSH) maintains a balance between toxic metal ions and oxidative stress through production of ROS and RNS. Any imbalance in antioxidant system leads to an increase in the cascade of ROS and RNS. It is antioxidant system that coordinately operates formation and propagation of ROS/RNS and network of defences against oxidative stress induced functional alteration and damage to biomolecules.

its ability to cross biological membranes, get converted to easily diffusible H2 O2 , either enzymatically through the involvement of superoxide dismutase (SOD) or nonenzymatic by dismutation of superoxide (O2 ·−) radical. H2 O2 , a potent nonradical oxidizing agent, directly oxidize intracellular ferrous (Fe2+) ions to generate hydroxyl (OH·) radicals (Fenton reaction; Imlay, 2008). In addition to that, interaction of H2 O2 with the superoxide (O2 ·−) also leads to generation of highly reactive hydroxyl (OH·) radicals (Haber-Weiss reaction; Halliwell and Guttering, 1990). O2 + e − → O 2 · −

(1)

2O2 ·− + 2H+ → H2 O2 + O2

(2)

O2 ·− + NO → ONOO− + H+ → ·OH + ·NO2

(3)

−

O2 · + Fe

3+

H2 O2 + Fe

2+

H2 O2 + O2

→ Fe

2+

→ Fe

+ O2

(4) −

+ ·OH+ OH (Fenton reaction)

(5)

→ O2 + ·OH + OH− (Haber − Weiss reaction)

(6)

3+

In view of the essentiality of redox homeostasis, induction of oxidative stress due to fluctuations in the defense machinery appears to be a remarkable phenomenon by which metals cause toxicity in living organisms. Binding of mercury to intracellular thiols, especially glutathione (GSH), either directly or indirectly leads to its depletion followed by increased formation of H2 O2 , associated with the development of oxidative stress (Lund et al., 1993). In a similar manner, arsenic mediates formation of ROS, RNS, and dimethylarsinic peroxyl radical [(CH3 )2 AsO2 ·)], and dimethylarsinic radical [(CH3 )2 As·)] (Lin et al., 1999; Yamanaka et al., 2001). Besides attacking



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nitrogenous bases and deoxyribose sugar in DNA that cause DNA strand breakage, they hamper repair mechanisms by binding to sulfhydryl groups of enzymes that lead to their inactivation (Shanker, 2008). Cadmium that is capable of replacing iron and copper from various cytoplasmic and membrane proteins, leads to an increase in unbound amount of the respective metal, having the capability to induce oxidative stress via Fenton reaction (Casalino et al., 1997; Koedrith and Seo, 2011). Redox inactive metals such as cadmium that are unable to perform redox reactions in biological systems, stimulate oxidative stress mainly by inhibiting antioxidant enzymes through interaction with their thiol groups. Metal ion generated oxidative damage via, ROS/RNS that is associated with the perturbation of redox homeostasis, is maintained primarily by an extensive network of enzymatic antioxidants such as catalase and SODs, as well as by naturally occurring antioxidants such as glutathione (GSH). However, as counter-productive and beneficial defensive mechanisms of antioxidants are not present in excess, it is plausible that an increase in ROS/RNS production or decrease in ROS-scavenging capacity (redox imbalance) due to exogenous stimuli alter cellular functions through direct modifications of biomolecules (proteins, nucleic acids, and lipids). Metal catalyzed damage of proteins includes loss of histidine residues, introduction of carbonyl groups, bityrosine cross links, and generation of carbon centered alkyl radical (R·), alkoxyl radical (RO·), and peroxyl radical (ROO·; Valko et al., 2005; Valko et al., 2006) R − H + ·OH → R· + HOH ROOH + Fe2+ → RO· + − OH + Fe3+ R· + O2 → ROO· Side chains of amino acid residues such as histidine, arginine, proline, and cysteine, being most vulnerable to attack by ROS and RNS, result in the formation of following products (Dean et al., 1997): Histidine → 2-oxyhistidine Arginine → glutamic semialdehyde Proline → 2-pyrrolidone-4-hydroxyproline Cysteine → Cys-S-S-Cys Methionine → Methionine sulfoxide By interacting with polyunsaturated fatty acids in membrane phospholipids, ROS can elicit peroxidation of lipids followed by their subsequent degradation and fragmentation (Novo and Parola, 2008). Subjected to oxidant challenges from endogenous as well as exogenous sources, interaction of polyunsaturated fatty acids with ROS or other free radicals in pathological


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FIGURE 2. ROS/RNS induced damage of DNA represented through formation of different nitrogen base adducts. (a) (6-amino-9H-purin-9-yl) mercury. (b) 2-amino-1-methyl mercury6,9-dihydro-1H-purin-6-one. (c) 7-oxa-1,5-diaza-8-mercurabicyclo[4.2.0]octa-2,5-dien-4-one. (d) {5-amino-7-oxo-3H,6H,7H-imidazo[4,5-b]pyridin-6-yl}(3-methyl-2,6-dioxocyclohex-3-en1-yl) mercury.

conditions like atherosclerosis, ischemia etc, is exacerbated by the presence of divalent metal ions. Compared with proteins and lipids, nuclear DNA is less susceptible to oxidative modifications due to its double helix structure and protective shield from histone proteins. However, under physiological conditions, cellular DNA is constantly subjected to damage in nearly all of its components by ROS and RNS (Figure 2). In biological systems, generation of hydroxyl (·OH) radical leads to formation of an adduct 8-OH-dG, a reliable marker of ROS-dependent damage to DNA (David et al., 2007; Haghdoost et al., 2005). Owing to their vulnerability to toxic metals, studies that involve ROS and RNS (inadvertent by-products of aerobic metabolism) interaction with biomolecules, have not only helped in elucidating the mechanisms that are involved in the generation of oxidative stress but have also illuminated the path in discovering additional protective strategies capable of complementing inherited scavenging enzymes in protecting biomolecules from metal induced oxidative stress.

4. MICROBIAL INTERACTION WITH METALS IN THE ENVIRONMENT Metals, in particular mercury and arsenic that represent widespread environmental pollutants of great concern, have severely threatened human and



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environmental health by escaping to environment through natural and anthropogenic activities (D’Souza and Peretiatko, 2002; Garbisu and Alkorta, 2001). Their concentrations are modified by biogeochemical processes, by natural inputs such as dust particles derived from soil, rocks, and volcanic ash and most importantly by anthropogenic inputs (i.e., pollution). Sensing chemicals in the environment and responding to changes in their concentrations has led to selection of a whole repertoire of adaptive mechanisms that ensures better adaptation of microorganisms to frequently changing hostile environment (Gadd and Griffith, 1978). Microbes, being prime mediators of biochemical cycling, are ubiquitous. Their activity strongly influences metal speciation and transport in the environment. By having extraordinary enzymatic diversity, they interact with metals in many ways and mediate reduction, oxidation, methylation, and alkylation reactions, involved in chemical cycling, biosorption, complexation, and mineralization (Gadd, 2010). Although most organisms are endowed with detoxification abilities (i.e., mineralization, transformation, and/or immobilization of pollutants), microorganisms, particularly bacteria, owing to their vast catabolic potential inherited by expressing catabolic genes encoding enzymes involved in the biodegradation of toxic pollutants, play a crucial role in sustainable development of biosphere and in biogeochemical cycles. Their abundance, together with the ability for horizontal gene transfer and high growth rates, allows them to evolve strategies for quick adaptation to changing environmental conditions (Brown et al., 1998). However, their efficiency to diminish the risk associated with remediation processes to human health depends on many factors: chemical nature, and the concentration of pollutants, their availability to microorganisms, and on the physicochemical characteristics of the environment.

5. BIOCHEMICAL BASIS OF BACTERIAL METAL RESISTANCE Metal pollution in association with increased incidence of environmental and health problems has brought the possibility of long-term environmental disasters into public conscience. Wide array of microbial strains with distinct characteristics (degradative enzymes for biodegradation of contaminants) possesses an extraordinary ability to cope with metal stresses and to treat environments charged with different metals. Natural attenuation is a passive remedial approach that depends on natural processes for transformation or immobilization of metal contaminants. It is believed to be the major process involved in the reduction/transformation of contaminant concentrations, spurred by the addition of nutrients (biostimulation) or through the addition of bacterial strains with desired catalytic capabilities (bioaugmentation) that enhance resident microbial population’s ability of transforming

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TABLE 2. Bioremediation agents in National oil and hazardous substances pollution contingency Plan (NCP) product schedule (adapted from USEPA, 2011) S. No

Product

Composition

Manufacturer

1. 2.

INIPOL EAP 22 WMI 2000

NA MC

3.

OPPENHEIMER FORMULA

MC

4.

MICRO-BLAZER

MC

5.

VB591TM, VB997TM, BINUTRIXR STEP ONE SYSTEM E.T. 20

NA

Societe CECA, S.A., France WMI International, Inc. Houston, TX Oppenhemer Biotechnology, Inc. Austin, TX Verde Environmental, Inc. Houston, TX BioNutra Tech, Inc. Porter, TX


6. 7. 8. 9. 10. 11.

MC MC MC EA MC NA

12.

BET BIOPETRO OIL SPILL EATER II PRISTINE SEA II LAND & SEA RESTORATION PRODUCT 001 S-200

13. 14.

SPILLREMED JE1058BS

MC NA

15. 16. 17.

BIOWORLD BHTP MUNOX SRR SOIL RX

MC MC MC/NA

18. 19.

MC/NA MC

21. 22. 23. 24.

PRO-ACT BIOREM 2000 OIL DIGESTOR TM DRYLETTM MB BIOREMEDIATION DUALZORBR REMEDIADE TM ERGOFIT MICRO MIX AQUA SHAMANTRA GREEN

MC NA MC/EA/NA NA

25.

SUMP SAFE BIO-RECLAIM

MC

20.

NA

MC

B&S Research, Inc. Embarrass, MN Environmental Restoration Service, Windsor, CA BioEnviro Tech. Tomball, TX OSEI Corporation. Dallas, TX Fluid Tech, Inc. Las Vegas, NV Land & Sea Restoration LLC. San Antonio, TX International Environmental products LLC, Villanova, PA Sarvo Bio Remed LLC, Trenton, NJ Japan Energy Corporation, Saitama, Japan BioWorld Products, Visalia, CA Osprey Biotechnics, Sarasota, FL 3 Tier Technologies LLC, Orlando, FL Pro-Act Microbial, Inc. Warren, RI Clift Industries, Inc. Charlotte, NC DryLet Technologies, Inc. Prosper, TX. LBI Renewable, Buffalo, WY JDMV Holdings, LP. Houston, TX Ergofit USA LLC, Newark, DE Molecular Mediation PLC, Wilmington, DE Teamwork Distributing, Stony Plain, Alberta

Note. MC = microbial culture; EA = enzyme additive; NA = nutritive additive.

contaminants (Rosenberg et al., 1992; Tyagi et al., 2011; Table 2). Addition of rate limiting nutrients in the form of nitrogen and phosphorus allow native bacterial population to develop and augment, for metabolism or cometabolism of the pollutant in order to accelerate biodegradation process (Boopathy, 2000). Generally, bioaugmentation is best suited for sites that either do not have sufficient bacteria or the native population does not possess metabolic routes that are necessary to metabolize the compounds under concern (Gentry et al., 2004; Leahy and Colwell, 1990). A critical factor in



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deciding whether bioremediation is appropriate cleanup remedy for a site depends on the susceptibility of contaminants to biodegradation by native organisms or by organisms that could be added so as to enhance their remediation from the site. Therefore, in order to increase the rate of success of any bioremediation process, it is important to have a better understanding of how microbial populations respond to elevated metal concentrations, for enumerating the role of existing methods in metal detoxification. Adoption of a suitable bioremediation strategy for metabolizing different metal contaminants depends on three basic principles (i.e., the accessibility of the contaminant to microorganisms [bioavailability], amenability of the pollutant to biological transformation [biochemistry], and on the prospect for optimization of biological activity [bioactivity]). As microorganisms inhabiting native habitats evolved towards ecological fitness rather than to biotechnological competence, cleaning contaminated sites by bacteria through the process of natural selection is a challenging task to accomplish. In order to exploit the synthetic capacity of biological systems and broaden the creation of microbial chemical factories, it is necessary to go beyond natural pathways for the synthesis of natural products towards de novo design and assembly of biosynthetic pathways for both natural and unnatural compounds (Chauhan and Jain, 2010; Doong et al., 1998; Mohan and Pittman, 2007; Patel et al., 2010). Compared to natural attenuation, engineered bioremediation relies on accelerating the growth of microorganisms as well as on optimizing the environment in which they carry out the detoxification reactions. The decision to implement either or both of these techniques for bioremediation depends on the degrading capability of the indigenous microbes and the extent of contamination of the site to be treated. To meet the challenges presented by environmental pollution, researchers have designed several strategies to tackle the environmental burden of metals using microbes that not only are well adopted with changing environment conditions but also possesses the capability to transform the contaminant present (de Carvalho and da Fonseca, 2005; de Carvalho et al., 2009; Khomenkov et al., 2008; Ron and Rosenberg, 2002; Van Hamme et al., 2003). As resistance to toxic metals is commonly acquired by (a) sequestration at the cell surface, (b) intracellular precipitation, and (c) redox transformation, strategies such as ability to modify the cellular membrane through production of surface active compounds (biosurfactants) and/or increase the expression of metal binding peptides and proteins either on the surface or inside the cell followed by efficient transformation of toxic forms into less toxic ones plays an important role in the remediation of toxic pollutants.

5.1 Biosurfactant Application for Immobilization Despite being advantageous over conventional treatment technologies, the ever emerging field of microbial remediation technologies is lagging in its


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application. Major constrain to any remediation strategy appears to be nonavailability of contaminant to degrading organisms. Bioavailability of pollutant depends largely on its physicochemical properties: more complex the structure, less soluble is the substance and more recalcitrant is pollutant to biodegradation (Goyal et al., 2003; Pacwa-Płociniczak et al., 2011; Wang and Chen, 2009). Being recalcitrant to degradation, sequestration, and/or immobilization seems to be an ideal remediation strategy. Biosurfactants are surface-active microbial products that are produced extracellularly or as part of the cell membrane by virtue of which surface tension of the liquid is decreased so as to make metals more available for remediation (Ron and Rosenberg, 2001). Production of rhamnolipid by Pseudomonas spp. (Pseudomonas aeruginosa ATCC 9027), cyclic lipopeptide surfactin by Bacillus subtilis and biodispersan by Bacillus sp. strain IAF-343 are examples of growth-associated biosurfactant production (Asci et al., 2010). They are characterized by their properties: (a) change in surface active phenomena, such as lowering of surface and interfacial tensions, (b) wetting and penetrating actions, (c) microbial growth enhancement, and (d) metal sequestration. Amphiphilic structure of biosurfactants that includes a hydrophilic moiety consisting of amino acids, peptides, anions, or cations; mono-, di-, or polysaccharides; and a hydrophobic moiety consisting of unsaturated or saturated fatty acids that leads them to accumulate metals at the interfaces, thereby increasing their transport across membrane or immobilization at the cell surface. Removal of the toxic metals from the polluted environments has previously been investigated using native microorganisms, but recombinant DNA technology offers the possibility of improving the metal-binding capacity of the bacteria (Doong et al., 1998; Gupta et al., 2000). Enhancement in metal binding capacity through the generation of superactive microbial strains for immobilizing metal contaminants would result in economic gain of production and as such its application in the bioremediation process.

5.2 Genetic Engineering for Metal-Sequestering Proteins and Peptides Nonbiodegradability of metal ions that result in increased accumulation followed by pronounced toxic effects in animals at higher trophic levels has necessitated development and implementation of economic alternative with promising potential to remove hazardous metals from the environment. Among the various mechanisms that are involved in detoxification and transformation of metals, engineering bacterial for proteins and peptides that possess the potential to efficiently immobilize metals has emerged as a technique useful for their removal from the environment (Beveridge and Murray, 1980). Bacterial surface display of metal-binding peptides and proteins to remove the toxic metals from water and waste water has become


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an increasingly active area of research with a wide range of biotechnological and industrial applications. Surface expression of novel metal binding peptides and proteins offer higher metal-binding capacity and/or specificity and selectivity for a target metal ion, thereby enabling rapid binding and as such their immobilization for improved metal resistance of growing cultures (Pazirandeh et al., 1998). Surface exposure of metal binding peptides and proteins is fortuitous and relative efficient, as it improves metal binding properties of microorganisms that are employed in various systems for their removal from the environment.


5.2.1 METALLOTHIONEIN Metallothioneins (MTs) are intracellular, low molecular weight, cysteine-rich metal binding proteins encoded by the mt genes, endowed with wide range functional capabilities in biosystem (Hamer, 1986; Sousa et al., 1998). They have been expressed in bacteria with the purpose of increasing metal resistance via, immobilization. They possess high metal content comprising predominantly Zn, Cu, or Cd, bound by sulfur atoms in thiolate clusters with a tetrahedral geometry, and have highly conserved cysteine residues (18–23) that bind metal ions and sequester them in biologically inactive form (Bell and Vallee, 2009; Vasak, 2005). The number and position of the cysteine residues that remains highly conserved forms Cys-x-Cys, Cys-x-y-Cys, and Cys-Cys sequences (where x and y are noncysteine amino acids). Five cysteines bridge four zinc atoms bound to eleven cysteines in the C terminal of α-domain, whereas N terminal β-domain is formed by three zinc atoms bound to nine cysteines. These arrangements (28 intramolecular zinc-sulfur bonds) account for the extremely tight zinc binding in both clusters and for the zinc-donating properties of MT (Krezel and Maret, 2007). As many as 18 different metals may associate with MT, but only Cu+, Cd2+, Pb2+, Ag+, Hg2+, and Bi2+ can displace Zn (Coyle et al., 2002; Kagi and Kojima, 1987). In view of its role in metal detoxification, attempts have been made to express metallothioneins in bacteria. However, bacterial expression of MT was shown to be unstable and had to be fused with glutathione-S-transferase (GST; Berka et al., 1988; Chen and Wilson, 1997a, 1997b). Explanations for its instability in bacteria includes rapid degradation of the transcripts and small peptide, low protein expression, nonspecific binding to a variety of metals, and interference with redox pathways (Chen et al., 1998; Yang et al., 2007). One promising approach to circumvent this problem involves expression of MTs on the cell surface of environmentally robust organisms, such as Pseudomonas. On expressing MT in the permissive site 153 of the maltose binding protein (LamB) sequence, Sousa and co-workers (1998) observed a 15–20-fold increase in Cd2+ binding by MT. An alternative to this strategy is to simultaneously express MT with a metal transport system. In accordance with the previous study by Pazirandeh et al. (1995), Chen and Wilson (1997a) on co-expressing MTs with the Hg2+ transport proteins (MerT and

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A. T. Jan et al.

MerP) in E. coli found a significant increase in the bioaccumulation of Hg2+. As overexpression of MTs in bacterial cells results in enhanced metal accumulation, and it offers a promising strategy to achieve remediation of metals at contaminated sites.

5.2.2 PHYTOCHELATINS Phytochelatins (PCs) are polypeptides that contain an increased repetition of γ Glu-Cys dipeptide residues with a terminal Gly [(γ Glu-Cys)n -Gly], synthesized by PC synthase (PS), via transfer of a γ Glu-Cys unit from glutathione (GSH; γ Glu-Cys-Gly) to another GSH molecule or to an elongating PC polypeptide (Cobbett, 2000; Pal and Rai, 2010; Vatamaniuk et al., 2000). Downloaded by [Jamia Millia Islamia] at 00:58 07 February 2014

γ ECS

Glu + Cys −→ γ Glu − Cys GS Cys γ Glu − Cys −→ γ Gly − − Gly GSH Gly PS Cys)n Gly GSH −→ (γ Glu PCs γ Glu−Cys The reaction is strictly dependent on the presence of some metal ions, including Cd2+, Zn2+, and Cu+/2+ (Beck et al., 2003). One important similarity between MTs and PCs is their high content of cystein (Cys), an amino acid containing sulfur (S) atom, to which metals bind. An important finding regarding prokaryotic PS homologs include lack of variable C-terminal domain and the second acylation (γ Glu-Cys acceptor) site that is present in the eukaryotic PS (Harada et al., 2004; Tsuji et al., 2004; Vivares et al., 2005). It is reported that E. coli with cell surface-expressed synthetic phytochelatin (EC) exhibits higher cadmium (Cd2+) and mercury ion (Hg2+) accumulation than cells with intracellularly expressed EC by 12- and 20-fold, respectively (Bae et al., 2001; Bae et al., 2002).

5.2.3 DE

NOVO SYNTHESIS OF METAL BINDING PEPTIDES

Unlike organic contaminants, metals are intrinsically persistent in nature. Detoxification of metals from metal-polluted environments relies on their removal from contaminated sites primarily by microorganisms (Nriagu and Pacyna, 1988). As intracellular expression of Cys-rich proteins for accumulation of metals may interfere with the redox pathways in the cytosol (Raina and Missiakas, 1997), incorporating multiple binding sites within a single metal-binding motif and expressing them on the surface of bacteria seems to be a versatile strategy for the removal of mixed metal contaminants. Overexpression of metal-binding peptides on the surface of microbial cells increases the rate and capacity of adsorption, while as minimizing the toxicity to host cells. As peptides with an abundance of cysteine or histidine residues (amino acids able to establish coordination bond) are known to bind Cd2+ and Hg2+ with a very high affinity, they can either be designed de novo



537

or selected by screening peptide libraries (Sousa et al., 1996; Sousa et al., 1998). The de novo design of metal-binding peptides is an attractive alternative to MTs as they offer the potential of better affinity and selectivity for metals. Expression of a repetitive metal-binding motif containing Cys–Gly and Cys–Cys–Gly repeats as a fusion with the calcium binding (LamB) protein in E. coli enhanced Cd2+ and Hg2+ binding by 10-fold (Sousa et al., 1998). Similarly, cells expressing hexa-histidine (6His) peptides as a fusion to the calcium binding (LamB) protein showed a fivefold increase in cadmium accumulation for one hexahis peptide and an 11-fold increase when two hexahistidine peptides were expressed in tandem (Sousa et al., 1996; Table 3). Caulobacter crescentus is nonpathogenic to humans and other organisms, and they thrive in nutrient-limiting environments. Xu et al. (2010) reported the construction of a recombinant C. crescentus strain JS4022/p723–6H with 6His inserted at a permissive site of the S-layer protein RsaA. As engineered S-layer fusion protein RsaA-6His is displayed at the cell surface, strain JS4022/p723–6H can be directly used as a whole cell adsorbent for sequestration of dissolved metals from water (Patel et al., 2010). Novel metal binding peptides could also be selected from phage display library. On expressing the peptide His–Ser–Gln–Lys–Val–Phe that exhibit strongest affinity for Cd2+ in E. coli as a fusion to the cell-surface-exposed outer membrane protein OmpA, Mejare and co-workers (1998) observed increased survival of cells in growth medium containing toxic levels of CdCl2 .

5.3 Genetic Engineering for Improved Enzymatic Capabilities Plasticity of the genome is a prerequisite for evolvability and dynamic adaptation to new pollutants in a particular ecosystem. Genes associated with the biodegradation pathways that are usually clustered on mobile genetic elements, such as transposons and plasmids; facilitate horizontal transfer of respective genes and as such rapid adaptation of microorganisms to changing environment (Eccles, 1999). The gene clusters usually consists of catabolic genes encoding enzymes catalyzing various steps of catabolic pathway, transport genes that are involved in the active uptake of compounds as well as regulatory genes responsible for the controlled expression of the catabolic and transporter genes for the compound to be degraded (Figure 3). The detoxification machinery for efficient remediation of metals can be best explained by an example of various mer operon genes, involvement in imparting resistance against mercury in bacteria. Owing to their involvement in resistance against mercury, mer operon usually consists of structural genes encoding functional proteins associated with various functions such as transport (mer T and mer P), regulation (mer R and mer D), and reduction (mer A and mer B; Mathema et al., 2011; Ruiz and Daniell, 2009). In addition to the previously mentioned genes, mer C, mer F, mer E, and mer H (all membrane proteins)

538

— Copper, Cadmium, Zinc Mercury Cadmium, Nickel Cadmium — Copper, Nickel Mercury — Arsenic Cadmium

Cadmium —

Heavy metal

Escherichia coli — — — — Staphylococcus xylosus Ralstonia eutropha CH34 Escherichia coli Saccharomyces cerevisiae — — — Caulobacter crescentus

Representative Bacteria Lam B — — Ice nucleation protein SPA IgA β-domain Lpp-OmpA α-Agglutinin Lpp-OmpA Ice nucleation protein Outer membrane S-layer protein

—

Expression site (Outer membrane) Hexa-histidine MT MT (α-domain) Metal binding peptide Mer R Polyhistidyl peptides MT Phytochelatin Hexa-histidine EC20 Mer R MT RsaA-6his fusion protein

Metal binding protein/peptide

Reference Sousa et al. (1996) Sousa et al. (1998) Kotrba et al. (1999) Kotrba et al. (1999) Schembri et al. (1999) Samuelson et al. (2000) Valls et al. (2000) Bae et al. (2000) Kuroda et al. (2001) Bae et al. (2001) Bae et al. (2003) Singh et al. (2008) Patel et al. (2010)

TABLE 3. Microorganisms engineered for different metal binding proteins on their outer membrane for sequestration of metals




539

FIGURE 3. A schematic overview of mechanisms involved in the detoxification of metals. Expression of metal binding proteins and peptides such as metallothionein (MT) and phytochelatin (EC) on the surface of microbial system improves metal binding properties of microorganisms employed in achieving bioremediation of contaminated sites. Along with this, overexpression of genes such as those encoding organomercurial lyase (Mer B), mercuric reductase (Mer A), and arsenic reductase (Ars C) involved in the reduction/transformation of different metals compounds along with transporter proteins such as Mer, P., Mer, T., Mer G, and Ars A&B involved in transport across the membrane results in the improvement of metal resistance of growing cultures.

are believed to assist in transport function (Sasaki et al., 2005; Wilson et al., 2000) and mer G involved in conferring resistance to phenyl mercury (Kiyono and Pan-Hou, 1999; Kiyono et al., 2009; Schue et al., 2009). Uptake of mercury required to confer resistance is considered as the rate limiting step in resistance against mercury encoded by mer determinants. In the same way, resistance to both arsenite (As3+) and arsenate (As5+) in bacteria results from the simultaneous function of minimum of three or five co-transcribed genes of ars operon: ars R and ars D (determining the regulatory repressor), ars A and ars B (determining the membrane transport pump), and ars C (determinant of small intracellular arsenate reductase) (Kaur et al., 2009). Increase in the expression of metal ion transporters encoded by different genes of mer and ars operon leads to extend the range of substrates that an organism can utilize, avoiding substrate misrouting into unproductive routes or to highly reactive intermediates, increasing bioavailability of metal pollutants and in improving the process-relevant properties of microorganisms.


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Despite the fact that microorganisms have acquired the ability to use pollutants as carbon and energy sources, their natural bio-transforming efficiency would take a long time to achieve complete remediation of polluted sites. Rapid progress in genetic engineering and recombinant DNA techniques have provided us with the capability to design, modify and engineering natural degradative pathways by assembling catabolic modules responsible for transport and transformation from different origins in the same host cell. Hence, studying the physiology, biochemistry, and genetics of the catabolic pathways becomes crucial to recreate and accelerate natural processes as well as to accomplish their rational manipulation in order to design more efficient biocatalysts for different biotechnological applications (Okino et al., 2000). As the precise composition of the mer operon varies between bacterial strains, strains resistant to high levels of mercury have developed a two-stage two enzyme detoxification strategy for transformation of organomercurials. Organomercurial lyase (Mer B) catalyzes protonolytic cleavage of carbon-mercury bond of organomercurials to generate alkyl moiety (-R) and ionic form of mercury that directly get transferred to the mercurial reductase (Mer A) (Murtaza et al., 2002; Murtaza et al., 2005). It makes the use of physiological thiols, glutathione and cysteine for its activity but is strongly inhibited by nonphysiological dithiol, DDT. The second enzyme of this system (i.e., mercuric reductase reduces inorganic [Hg2+] to metallic [Hgo] form at the expense of oxidation of NADPH coenzyme; Haq et al., 2010; Walsh et al., 1987). In a similar way, bacteria have evolved resistance mechanisms for arsenic based on reduction of As5+ to As3+ mediated by arsenic reductase (Ars C), followed by efflux via Ars A and Ars B. Although As3+ is more toxic, it serves as substrate for the Ars B transport protein. Arsenate (As5+) sharing tetrahedral structure and bonding sites as phosphate (PO4 3−) gets inside cells by hijacking phosphate transport mechanism, that in turn leads to high arsenic toxicity in most of organisms (Wolfe-Simon et al., 2011). As5+ conversion to As3+ serves as a counter-productive mechanism to differentiate As5+ from PO4 −3 in order to avoid extrusion of phosphate ion, PO4 3− that plays an essential role in maintaining structure of DNA and RNA, cell membranes, and in the generation of adenosine triphosphate (ATP). In short, ars operon functions as a detoxification mechanism by lowering the intracellular arsenic concentration, thus conferring resistance to As5+ and As3+. Effectiveness of remediation strategies that offer the advantage of partial or complete transformation of contaminants at a site is evaluated by disappearance of the chemical of interest. Genetic engineering of biodegradative pathways offers the potential of expanding the existing capabilities found in nature. Development of new genetic tools and a better understanding of microorganism’s natural transformation ability at the genetic level have accelerated the progress to genetically engineer microbes with enzymes such as organomercurial lyase, mercuric reductase and arsenic reductase for improved detoxification and degradation of mercury and arsenic pollutants

541


TABLE 4. Microorganisms genetically modified for different metal ion transporters and enzymes involved in the detoxification of metals Heavy metal Mercury — — —


— — — — — —

Representative bacteria Deinococcus radiodurans E. coli — — — E. coli JM109 Acidithiobacillus ferrooxidans Pseudomonas K-62 — Achromobacter sp. AO22

Modified gene expression

Reference

Mercuric reductase

Brim et al. (2000)

Organomercurial lyase Mer A and ppk Hg transporters (Mer T, Mer P), Mer B and ppk Regulatory protein (ArsR) Hg2+ transporter and MT Hg2+ transporter

Murtaza et al. (2002b) Pan-Hou et al. (2002) Kiyono et al. (2003) Kostal et al. (2004) Zhao et al. (2005) Sasaki et al. (2005)

Hg2+ transporters and organomercurial lyase Hg2+ transporter (Mer C) Mercuric reductase

Kiyono and Pan-Hou (2006) Kiyono et al. (2009) Ng et al. (2009)

(Table 4). Degradative enzymes with new or improved activities and stability under selected conditions can be generated by genetic engineering strategies such as rational site-directed mutagenesis and by DNA-shuffling methods. Assessing effect of different combination of expression levels for organomercurial lyase and/or mercuric reductase and arsenic reductase by installing different promoters upstream of each gene in a pathway seems a better alternative to this approach. Although enzymes are folded proteins with robust structures, stable in the environment in which they have evolved, a small modification of genes encoding biodegradative enzymes or promoter instead of catabolic enzymes would result in the enhancement of biodegradative capability of an organism (Ho and Ellermeier, 2012). Therefore, deciphering bacteria with improved capability for degradation of different mercury or arsenic compounds along with the study of specific promoters that associated with the increased expression of desired genes under stress conditions can open up new perspectives to genetically engineer microbes with enzymes such as organomercurial lyase, mercuric reductase, and arsenic reductase using holistic, multifaceted approach for improved detoxification of mercury and arsenic pollutants.

6. STRATEGIES TO OVERCOME BOTTLENECKS ASSOCIATED WITH MICROBIAL BASED REMEDIATION STRATEGIES Contaminants causing environmental troubles leading to inequity in environment require worldwide denunciation. As principal concern associated with contaminants such as metals is toxicity and health risk to humans, it is


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therefore essential to develop strategies in order to mitigate inorganic contaminants, so as to prevent contamination of natural environment (Evans and Furlong, 2003; Gomez and Bosecker, 1999; McLaughlin et al., 2000). Occurrence of higher numbers of resistant bacteria in polluted habitats compared with unpolluted habitats gives an indication regarding contribution of metal resistance to increased proportion of resistant bacteria in the aquatic environment. Problem of environmental pollution resulting from industrialization and urbanization, escalating day by day requires a model resolution based on microbial systems for the abatement of toxic wastes (Fulekar et al., 2009). As introduction of bacterial biomass in an existing niche create a palatable habitat for protozoa, thereby preventing bacterial population to grow beyond a certain level (Iwasaki et al., 1993). Natural engineering processes for the most part involve mutations that broaden the substrate range of pre-existing enzymes, shuffle sequences, and horizontally transfer DNA pieces between members of a microbial community to form new hybrid genes and metabolic operons (Van der Meer, 1997). However, it is readily apparent that an optimal clean-up agent displays maximum catalytic ability with minimum of cell mass. Development of new genetic tools and a better understanding of microorganism’s natural transformation ability at genetic level have accelerated the progress to genetically engineer microbes so as to acheive improved transformation of toxic pollutants. However, as soon as the prospect of releasing genetically modified microorganisms (GMOs) for bioremediation became a reality, it have ignited a scientific and public debate on the possible ecological risks of such applications (Lindow et al., 1989). In reality, bottleneck that are associated with the efficacious use of GMOs in the environment has not been the body of legal regulations that limit their release; instead, it is the lack of knowledge regarding monitoring of recombinant strains along with the threat of horizontal transfer of recombinant genes that have restricted their use for achieving decontamination of polluted environments.

6.1 Monitoring of Recombinant Strains Monitoring of recombinant strains that possess potential risk to human health as well as to environment has resulted in the enactment of various approaches for their assessment in the environment. Constructing bioreporter (comprising of cellular sensory and regulatory components) bacteria capable of translating chemical detection by a cell into quantifiable reporter protein signal (e.g., luminescence or fluorescence) is an important accomplishment to achieve bioremediation of metals in the environment (Van der Meer and Belkin, 2010). Attempts have been made to use reporter gene fused to pollutant responsive genes (encoding transcriptional regulator plus a promoter/operator), whereby toxicity of the analyte or target compound is measured as a decrease in the activity of reporter protein (Shin, 2011; Yagur-Kroll



543

and Belkin, 2011). In such promoter-reporter constructs, promoter dictates the detection spectrum of the sensor, whereas reporter protein determines the type of signal emitted by the sensor and hence the instrumentation required for its detection. The most frequently used reporter genes that are effective for on-line as well as off-line monitoring of metal contaminants are lux/luc (encoding bacterial/firefly luciferase), gfp (encoding green fluorescent protein), and lac Z (encoding β-galactosidase; Ron, 2007). In presence of oxygen, bacterial luciferase catalyzes the oxidation of long-chain fatty aldehydes and reduced flavin mononucleotides to form the corresponding fatty acid and FMN In this reaction, free energy is emitted in the form of light (bioluminescence) with a wavelength of 490 nm. Of the bacterial luciferase system, lux A and B genes are required for luminescence, whereas lux C, D and E that encodes fatty acid reductase, are responsible for synthesis and recycling of fatty aldehydes (Close et al., 2012). Firefly luciferase in presence of oxygen and adenosine triphosphate (ATP) catalyzes the oxidation of benzothiazole-thiazole luciferin to bioluminescent oxyluciferin. Firefly luciferase based reporter system is more sensitive than bacterial luciferase, but requirement for addition of a substrate and ATP for bioluminescence makes them less favorable over bacterial luciferase system (Ron, 2007). Increased public awareness regarding environmental contamination and its potential impact on human health; has resulted in an upsurge in the interest for development of highly sensitive and more reliable bioreporter. Bioreporter strains developed for the detection of mercury and arsenic were among the first metal responsive systems to be developed (Selifonova et al., 1993). A number of lux/luc (encoding bacterial/firefly luciferase) based recombinant bacterial sensors for the detection of Hg, As, Cd, Zn and Pb have been developed (Table 5). The sensor construct used in most of these studies is a plasmid borne lux/luc system fused with mercury inducible promoter Pmer , in combination with its regulatory gene mer R from transposon Tn21 (Hakkila et al., 2004; Park et al., 1992; Petanen et al., 2001). However, in case of arsenic responsive bioreporter strains, lux/luc system is fused with arsenic inducible promoter from plasmid R773 and Staphylococcus aureus plasmid p1258, in combination with its transcriptional repressor ArsR and autoregulated arsenite derepressor arsRp (Tauriainen et al., 1999). Lowering of high background expression due to ArsR-arsRp construct is achieved by placing a second ArsR binding site downstream of arsR as well as upstream of the reporter gene cassette (Stocker et al., 2003). Bondarenko et al. (2008) constructed nine luminescent metal sensor bacteria, belonging to both Gram-negative (Pseudomonas fluorescens and Escherichia coli) as well as Gram-positive (Bacillus subtilis and Staphylococcus aureus) genera, to analyze the bioavailable fractions of Cd, Zn, and Hg in soil. They found that the detection limit of sensor is dependent on the type of the metal response element, whereas toxicity on the type of bacterium is used for the construction of the sensor. Ivask et al. (2009) performed a comparative study using 19

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TABLE 5. Microorganisms engineered for fusion of enzymes involved in metal detoxification and genes responsible for luminescence for their traceability in nature


Microorganism

Reporter plasmid

Analyte

Promoter

References Ivask et al. (2001); Ivask et al. (2009) Hakkila et al. (2004); Shin (2011) Hakkila et al. (2004); Shin (2011) Tauriainen et al. (1999); Diplock et al. (2009) Petanen et al. (2001); Diplock et al. (2009) Selifonova et al. (1993); Rasmussen et al. (2000) Petanen et al. (2001) Stocker et al. (2003); Baumann and Van der Meer (2007) Trang et al. (2005); Van der Meer and Belkin (2010) Petanen et al. (2001); Ivask et al. (2009) Petanen et al. (2001); Ivask et al. (2009) Ivask et al. (2009)

E. coli MC1061

pmerRBS BPmerlux

Hg, Cd

mer R,B

E. coli MC1061

pmerRluxCDABE

Hg

mer R

E. coli MC1061

pmerBRBS Luc

Hg, Me-Hg,Ph-Hg

mer R,B

E. coli MC1061

pT0031

As, Sb, Cd

ars R

E. coli MC1061

pT0011

Hg, Cd

mer R

E. coli HMS174

pmerTLuxCDABE

Hg

mer R

E. coli HB101 E. coli DH5α

pRB28 pJAMAarsR

Hg As, Sb

mer R,T ars R

E. coli DH5α

parsRluxAB

As, Sb

ars R

P. fluorescens OS8

KnmerRBS BPmerlux

Hg, Cd

mer R,B

P. fluorescens OS8

pDNmerRBS BPmerlux

Hg, Cd

mer R,B

P. fluorescens OS8

KnzntRPzntAlux

Hg, Cd, Zn, Pb

znt R,A

recombinant (13 metal induced and six constitutive strains as control) bacterial strains representing Gram-positive (Staphylococcus aureus and Bacillus subtilis) and Gram-negative (Escherichia coli and Pseudomonas fluorescens) bacteria, to express lux based reporter system. Comparison of Gram-negative and Gram-positive strains revealed that Gram-positive strains were remarkably more sensitive than Gram-negative strains. Despite the fact that both are Gram negative, Escherichia coli was found slightly more sensitive than Pseudomonas fluorescens. Green fluorescent protein (GFP), a reporter protein from the jellyfish Aequorea Victoria, produces fluorescence, following irradiation at its excitation wavelength, without requiring any exogenous substrate or ATP. The autofluorescence activity of GFP is due to the presence of a covalently bound imidazolinone chromophore inside the protein. An E. coli based arsenic biosensing system that was developed by coupling arsR and arsD along with their promoter with gene encoding for GFP from A. Victoria was found capable of detecting both As(III) and As(V) with a range of 1–10,000 ppb (Roberto et al., 2002). Tauriainen et al. (1999) reported that gfp based reporter systems allows real time detection without any requirement for addition of substrate and without causing any disturbance to host cell metabolism. Keeping in view, ease of detection and the minimal metabolic cost to host cell, it appears well suited in selecting gfp over other systems for requirement of substrate in order to detect a signal. However, its use as a reporter is



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hampered by the presence of intrinsic fluorescence in bacterial cells as well as due to delay in response during which it undergoes a series of autocatalytic reactions in order to become fully active fluorophore. In order to overcome such limitation, it appears crucial to design strategies for modifying GFP that should mature more quickly and emit brighter fluorescence for subsequent detection of sub-toxic levels of various metals in different environmental matrices. β-gal is a well-known bacterial enzyme that is widely used to monitor the transfection efficiency of plasmid. Among the various methods that employ a variety of substrates to detect β-gal activity, using O-nitrophenyl β-D-galactopyranoside as substrate is most commonly used for the colorimetric detections. Development of bioreporters allows not only detection of the target chemicals but also helps in the toxicological screening of these chemicals on the living system. Compared to Capital intensive and less user-friendly instrumental techniques such as graphite furnace atomic absorption spectrometry (GF-AAS), constructed E. coli (MC1061) based luc bioreporter under the control of merR exhibited detection limit for mercury as low as 0.1fM (Virta et al., 1995; Wanekaya et al., 2008). To meet the challenging need for detecting chemicals with great sensitivity, Yagur-Kroll et al. (2010) reported that by modifying the promoter region through manipulation of promoter fragment length, random or site-directed mutagenesis of the promoter region, and by promoter duplication, enhancement in the speed of sensor response and lower detection threshold for different chemicals can be achieved. The main justification of using bioreporter for bioremediation is their advantage of being cost effective than existing analytical approaches along with their ability to allocate effective assessment of physiochemical constraints that allows optimization of bioremediation process by alleviation of such constraints. In spite of the previously mentioned advantages, these systems have some drawbacks associated with them: (a) as these systems require large instruments for determining light emission that are operated only at laboratory setups, their practical application for on-line and on-site monitoring cannot be achieved; and (b) at biological front, light emission system that requires considerably high energy sources cannot function in anaerobic condition (Ron, 2007). However, technological advancement that has dramatically accelerated the pace for discovering molecules possessing the property to bioluminescence, have come up with new horizons in screening them as a tool of visual analysis. To address the prevailing problem associated with the bioreporters, researchers have developed a rare Rhodovulum sulfidophilum (a purple photosynthetic bacterium) based sensing system that utilizes crtA gene that is involved in the synthesis of carotenoid as reporter to detect environmental pollutants. This system indicates presence of pollutant by bacterial color change without any requirement of specific reagent or substrate for color development (Fujimoto et al., 2006; Yagi, 2007). Although significant progress has been made for the development of sensitive and highly

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specific biomonitoring methods that could monitor recombinant strains in their target environments, they have neither been widely adopted nor been commercialized successfully as the optimum conditions under which they perform differ from the real environment, which may be harsh. In short, for real time monitoring of genetically modified microorganisms for use in bioremediation, there is an urgent need for the development of a suitable system capable of predicting cellular toxicity or genotoxicity in the environmental samples of unknown composition with high degree of sensitivity and specificity.


6.2 Suicidal Mechanism to Reduce Horizontal Transfer of Recombinant Genes Bioremediation process that involves use of microorganisms to aid destruction of contaminants being cost efficient and reliable method for removing hazardous wastes has led to an increase in its application for environmental protection within the last decade. However, its implementation for sites contaminated with multiple metals requires a combination of many different approaches such as introduction of specific genes or alteration of genes, if present through genetic engineering, that significantly affect productivity of bioremediation approaches. Genetic transfer among bacteria probably accounts for much of the spread of resistance. This process, known as horizontal gene transfer (HGT) often confers new metabolic capabilities to the recipient, allowing its adaptation to new ecological niches. The mobile genetic elements mediating HGT through transformation, conjugation and transduction, consist of plasmids, transposons, and gene integrating integrons. Exogenous DNA acquired through HGT either integrate into recipient’s chromosome or replicate independently (Aminov and Mackie, 2007; Martinez et al., 2009a, 2009b). From a genetic perspective, localization of genes encoding resistance to metals together with genes responsible for antibiotic resistance on mobile genetic elements (e.g., plasmids) allows their frequent and facile transport among bacterial strains and species. Emergence of drug-resistant bacteria leads to a significant burden on global economies and public health. Knowledge on source and fate of antibiotics and their metabolites in the environment is important to estimate their potential impacts on ecology and human health. In view of horizontal transfer of genes responsible for metal resistance, process behind acquisition of resistance genes (metal as well as antibiotic) requires a global comprehension of genetic causes along with timely evaluation of physiological consequences of its acquisition. Keeping the sustainability issues and environmental ethics in mind, for any microbial based technology encompassing bioremediation process to be adopted, it is essential to monitor implanted recombinant strains of bacteria and design strategies to program cell death once the biocatalyst had



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fulfilled its mission, or in the event genetically modified genes get accidentally transferred. The most commonly studied approach for control of GEM survival in the environment is the incorporation of an inducible suicide gene such as DNases and RNases, bacteriophage lysis genes, and cell membrane-destabilizing genes into the microorganism, with the property to get expressed itself when the target contaminant is eliminated. Contreras et al. (1991) developed a conditional-suicide systems based on two elements contained on separate plasmids: Pseudomonas putida promoter (Pm) from TOL plasmid meta-cleavage pathway, Lac repressor (lacI ) gene from E. coli along with the gene for positive activator (xylS) of Pm on one plasmid and Ptac promoter, and a gef gene encoding a porin like protein possessing the ability to kill host by destabilizing the cell membrane from E. coli on another plasmid. Cells remain viable as long as XylS effector (i.e., 3-methylbenzoate) is present. In presence of 3-methylbenzoate, production of XylS positively regulates production of the Lac repressor (lacI ) that in turn results in the negative regulation of the Ptac promoter as because of which no Gef production occurs. In contrast, when 3-methylbenzoate is absent as would be the case when bioremediation is complete, production of Lac repressor (lacI ) is stopped, allowing transcription of gef gene, performing the killing function (Ramos et al., 1990; Ramos et al., 1997; Ruiz et al., 2001). To avoid loss of lethal function due to mutation, Ronchel and Ramos (2001) improved killing by gef -based system by using a asd mutant Pseudomonas putida strain, provided with an alternate asd gene (essential for cell wall synthesis), inserted under the control of Pm promoter. Expression of asd gene was positively regulated by the same XylS that negatively regulates gef expression via, transcription of Lac repressor (lacI ). The asd mutant Pseudomonas putida strain deprived of 3-methylbenzoate would die not only through production of gef protein, but also due to its inability to synthesize diaminopimellic acid. By using this dual containment system, the level of survival of this strain was below the limit of detection (