Genetic basis and importance of metal resistant genes in bacteria … · 2018. 9. 5. · 2, water, and cellular biomass termed biomineralization. However, inorganic toxic metal re-moval

MINI-REVIEW

Genetic basis and importance of metal resistant genes in bacteriafor bioremediation of contaminated environments with toxicmetal pollutants

Surajit Das1 & Hirak R. Dash1& Jaya Chakraborty1

Received: 5 October 2015 /Revised: 26 January 2016 /Accepted: 28 January 2016 /Published online: 10 February 2016# Springer-Verlag Berlin Heidelberg 2016

Abstract Metal pollution is one of the most persistent andcomplex environmental issues, causing threat to the ecosys-tem and human health. On exposure to several toxic metalssuch as arsenic, cadmium, chromium, copper, lead, and mer-cury, several bacteria has evolved with many metal-resistantgenes as a means of their adaptation. These genes can befurther exploited for bioremediation of the metal-contaminated environments. Many operon-clustered metal-re-sistant genes such as cadB, chrA, copAB, pbrA, merA, andNiCoT have been reported in bacterial systems for cadmium,chromium, copper, lead, mercury, and nickel resistance anddetoxification, respectively. The field of environmental biore-mediation has been ameliorated by exploiting diverse bacterialdetoxification genes. Genetic engineering integrated with bio-remediation assists inmanipulation of bacterial genomewhichcan enhance toxic metal detoxification that is not usually per-formed by normal bacteria. These techniques include geneticengineering with single genes or operons, pathway construc-tion, and alternations of the sequences of existing genes.However, numerous facets of bacterial novel metal-resistantgenes are yet to be explored for application in microbial bio-remediation practices. This review describes the role of bac-teria and their adaptive mechanisms for toxic metal detoxifi-cation and restoration of contaminated sites.

Keywords Bioremediation .Metal resistant genes . Bacterialdiversity . Genemanipulation .Metal resistance

Introduction

Microorganisms are the oldest member of the living systemsand possess higher adaptability to thrive in adverse conditions.They are the primary respondant to the changes in environmentby altering their genetic system, transfer of genetic elements,and many other mechanisms for maintaining the ecosystemstructure and function (Ryan et al. 2009). They serve in manyways for sustenance of the ecosystem such as nutrient cycling,primary production, and catabolism of pollutants produced asby-products of rapid urbanization and industrialization.

Environmental pollution caused by hazardous metals canarise from natural as well as anthropogenic sources. Naturalsources include seepage from rocks into water, forest fires, andvolcanic eruptions. However, vast industrial practices and con-sumerist lifestyle add to the anthropogenic source of metal pol-lution. The most common metal pollutants in the environmentinclude cadmium, lead, mercury, chromium, arsenic, copper,vanadium, nickel, molybdenum, and zinc. The toxicity of thesemetal pollutants causes chronic and degenerative conditions.The general symptoms of acute metal toxication include head-ache, short-term memory loss, mental confusion, gastro-intestinal upsets, vision problems, and allergies in humans(Das et al. 2014a). However, chronic exposure to toxic metalsimpart many dreadful effects to humans, such as increased can-cer risk, disruption of gene expression, and deregulation of cellgrowth and development. Continuous increase in the level ofmetal pollution in the environment at a greater pace worsens thesituation seeking immediate action (Naser 2013). To combat thispersistent environmental problem, recent developments of noveltechnologies involving multidisciplinary approaches utilizingmicroorganisms for enhanced bioremediation capability havebeen proposed. This includes biostimulation, bioaugmentation,bioaccumulation, biosorption, phytoremediation, andrhizoremediation (Niti et al. 2013).

* Surajit [email protected]; [email protected]

1 Laboratory of Environmental Microbiology and Ecology (LEnME),Department of Life Science, National Institute of Technology,Rourkela, Odisha 769 008, India

Appl Microbiol Biotechnol (2016) 100:2967–2984DOI 10.1007/s00253-016-7364-4

In this present scenario of incessantly increasing pollutionlevel, microbial bioremediation is an environment-friendlyapproach to treat noxious environmental conditions. This isway ahead of the conventional methods because it does notalter the natural microenvironment and maintain ecosystembalance. Microorganisms are able to degrade the organic con-taminants present in the environment and subsequently releaseharmless products such as CO2, water, and cellular biomasstermed biomineralization. However, inorganic toxic metal re-moval by the microorganisms is by biosorption to the cellsurface with Van der Waals force, covalent binding, redoxinteractions, extracellular precipitation, or combination ofthese processes. Though many discoveries have taken place,many aspects of exploiting the enormous bioremediation po-tential of microorganisms still remain unexplored. Their highsurface area to volume ratio, presence of extra chromosomalgenetic material, less complex genetic system, higher rate ofadaptation, rapid growth rate, high metabolic activity, super-lative enzymatic, and nutritional versatility play a useful rolein their implication toward effective bioremediation practices(Smith 2005). Genetic system of bacteria provides every op-portunity for their survival, gaining energy for subsequentdegradation of the organic contaminants and bioremediationof inorganic toxic metals from the contaminated environments(Ronchel et al. 1995). Hence, huge diversity of genes presentin bacterial genomes provides them the opportunity to sustainand subsequently contribute toward sustainable development.

This review aims to provide updated information regardingthe exploitation of genetic potential of bacteria for bioremedi-ation practices of most noxious toxic metals. As bacterial en-tities are more prone to genetic changes due to many mecha-nisms such as gene transfer by conjugation, transformation,and transduction as well as transposon-mediated events, grad-ually new genes of varied functions arise which are of primeinterest for use in treating toxic environmental pollution.

Microbial diversity and bioremediation

Bioremediation is a technique that utilizes microbes to re-move, neutralize, or detoxify pollutants from the contaminat-ed environment. There have been many studies regardingcharacterization of bacterial communities, their response topollutants, identification of genes responsible for degradation,and many more (Das 2014). Many reports suggested that theoccurrence of huge varieties of unidentified microbes helpingin bioremediation in the contaminated environment can betraced only by the culture-independent approaches(Marzorati et al. 2010). 16S rRNA gene analysis has revolu-tionized the study of microbial diversity in the natural envi-ronment both by culture-dependent as well as culture-independent approaches (Das et al. 2014b). Though polypha-sic approaches have been widely used for the study of

microbial diversity in the environment, there is no such goldstandard available for the assessment of environmental micro-flora for application in bioremediation practices.

Molecular biology tools have been widely exploited for thestudy of microbial ecology. Moreover, there should be a clearunderstanding of the role of metal-resistant genes from thediverse microbial population prior to their application in con-taminated environments (Dash and Das 2012). The advancedtechniques of culture-independent practices take advantage ofsequencing and in silico approaches for both sequence andfunction-driven screening of genes for bioremediation appli-cation (Khan et al. 2013). In addition, knowing the physico-chemical parameters of the environmental conditions, we canmanipulate the environmental DNA as well as functionalRNA to model the species interaction in a communitystructure.

The complexity and variability of biological organizationat different levels is known as microbial diversity. This termincludes the amount and distribution of genetic informationwithin microbial species in microbial communities and theirvariation in community structure, complexity associated withinteractions, number of trophic levels, and number of guilds(Hinojosa et al. 2010). Microbes possess the de novo potentialof degrading all naturally occurring compounds through theprinciple of microbial infallibility (Dash et al. 2013).Microorganisms can degrade organic contaminants by oxidiz-ing them to carbon dioxide, whereas in case of toxic metals,microorganisms can only change the speciation of metal con-taminants and mobility (Lovley and Coates 1997). So far, onlya fraction of the total microbial diversity with metabolic po-tential has been explored and further exploitation of theoverlooked genetic resources will accelerate the remediationpractices of toxic metals (Naser 2013). In addition, recentlydeveloped molecular genetics of bioremediation andknowledge-based approaches of rational protein modificationprovides greater insight for the development of designerbiocatalysts for environmental bioremediation (Pieper andReineke 2000; Paul et al. 2005).

Unique mechanisms in bacteria: naturaldevelopment of resistance toward toxic elements

Bacteria are known to thrive in all types of environments,ranging from the frigid poles to the hot deserts, the wetswamps to the aquatic systems. The unique features that theypossess are their small size, high surface area to volume ratio,efficient transfer of genetic traits, and adaptability. In the caseof organic pollutants, bacteria generally degrade them intonon-toxic products by utilizing them as a source of carbon.However, in the case of inorganic metal pollution, microor-ganisms have developed three methods of resistance, namely,efflux of the irritant metals outside the cell by transporters,

2968 Appl Microbiol Biotechnol (2016) 100:2967–2984

transformation of the metals into non-toxic/less toxic forms,and biosorption. Often, biosorption and enzymatic conversionof metal into another form are coupled; i.e., once a metal hasbeen absorbed into the cell, it is acted upon by enzymes,which precipitate the metal as a salt (Williams et al. 2012).The first line of protection from heavy metals is efflux, whichinvolves members of the heavy-metal efflux resistance-nodulation cell division (HME-RND) protein superfamilywith efflux pumps (Nies 2003). Another method by whichbacteria are known to resist as well as remove the harmfuleffects of toxic metals is biosorption (Vieira and Volesky2010).

Biosorption simply means the binding of certain sub-stances, especially contaminants, onto the cellular surface.This involves a solid phase (biosorbent, biological material)and a liquid phase (solvent) with the dissolved species whichis to be sorbed (sorbate, metal ions). The affinity of sorbent-sorbate is maintained till equilibrium is established betweenthe amount of solid-bound sorbate species and its remainingpart in solution, till the sorbate is removed. In bacteria, sorp-tion of metals is generally mediated by a family of proteinscalled metallothioneins (MTs) which are 0.5–14-kDa proteins,rich in cysteine residues (Blindauer et al. 2002). In some cases,they may have histidine residues as well. It has already beenreported that microbial metallothioneins act as a Bstorehouse^for zinc and also protect the cells from cadmium toxicity(Klaassen and Liu 1999). They may also act as a free radicalscavenger and combine with harmful molecules like superox-ide and hydroxide ions. Cysteine undergoes oxidation and isconverted to cystine, and bound metals are released into theenvironment. The process of biosorption is highly efficientwith regeneration of biosorbent and metal recovery possibili-ties (Kratchovil and Volesky 1998). However, early saturationof metal-interactive sites might sometimes be a problem.Bacterial cell wall with different functional groups on the sur-face with variety of polysaccharides and proteins act as activesites for binding different metal ions. This depends on thestatus of biomass (living or non-living), type of biomaterials,properties of metal solution chemistry, and ambient environ-mental conditions (Gee and Dudeney 1998).

Bacteria are known to have the ability to detoxify metals byreducing them to a dissimilar oxidation state. Common reduc-tion mechanisms include the conversion of Hg2+ to Hg0, Cr6+

to Cr3+, and AsO43− to AsO3

3−. Metal precipitation can also beachieved by metallic phosphate precipitation which is a resultof dissimilarity reduction or secondary consequence of meta-bolic processes related to the transformed metals (Valls and deLorenzo 2002). The chemical modification of compounds bybiological agents is termed as biotransformation, and produc-tion of CO2, NH4

+, and H2O is termed as mineralization. Anexample of this phenomena is the reduction of inorganic mer-cury (Hg2+) to elemental mercury (Hg0), mediated by mercu-ric ion reductase (Dash and Das 2012). Other similar reactions

include the conversion of arsenate to arsenite, and chromium(VI) to its less toxic counterpart chromium (III).

However, hazardous waste sites are usually co-contaminated with organic compounds and metal pollutants.Bioavailability of metals is determined by mediumcomposition/soil type and pH and governs the extent to whichmetals contradict biodegradation. In a mixed environment ofvarious pollutants, the non-biodegradable metal component isremoved or stabilized by mobilization, separation and collec-tion, off-site transport, and disposal. Metals inhibit the organicpollutant degradation process by interacting with their specificdegradation enzymes and enzymes involved in general metab-olism. The bioavailability of metals is dictated by their inter-action with organic compounds. In a report by Rosner andAumercier (1990), a common intermediate of aromatic hydro-carbons, salicylate increased uptake of cadmium and toxicityin Escherichia coli. Presence of metals as soluble complexedspecies and ionic solutes in an environment extends acclima-tion periods and reduces the biodegradation rate of com-pounds (Sandrin and Maier 2003). In a co-contaminated en-vironment, the energy requirements to maintain concurrentmetal resistance and organic degradation is too high; there-fore, simultaneous activity of bioremediation by microbes isneeded to perform both activities under environmental condi-tions. Thus, organic pollutant-degrading metal-resistant bac-teria with the properties of biosorption and biotransformationare essential for removal of metal toxicity as well as organicpollution.

Diversity of metal-resistant genes and biotechniquesin metal-resistant bacteria for bioremediation

In the environments contaminated with compounds that arenaturally toxic, microorganisms have devised ways not onlyto withstand such compounds but also to remediate them fortheir own benefits (Guo et al. 2010). The ability of bacteria toresist toxic metals comes from highly modified genetic sys-tems, by means of which they synthesize proteins enablingthem to thrive in the presence of such elements. Bacteria sur-vive by expressing several metal-resistant genes toward toxicmetals such as cadmium, chromium, copper, lead, mercury,and nickel. They survive in the highly toxic environment withthe help of these resistant genes which are recruited further forbioremediation. Specific genes responsible for bioremediationof toxic metals discovered so far have been listed in Table 1.The focus of the metal selection is the relative abundance ofthese metal contaminants in the environments and their degreeof toxicity as indicated by USEPA and the US Department ofLabor (https://www.osha.gov/SLTC/metalsheavy/). Bacteriadevelop resistance to the toxic metals as a part of theirdefense mechanism which may be exploited for cleaning thecontaminated environments (Fig. 1).

Appl Microbiol Biotechnol (2016) 100:2967–2984 2969

Tab

le1

Operon/Geneclustersinvolved

inconferring

toxicmetalresistance/detoxificationin

bacterialsystem

andtheirspecificfunctio

n

Toxicmetal

Gene

Encoded

protein/enzyme

Biologicalfunction

Harboredmicroorganism

sReferences

Cadmium

cadB

Cadmium-binding

protein

Itprotectsthebacterialcellb

ybinding

cadm

ium

atcellmem

brane

Staphylococcus

sp.

Perry

andSilv

er1982

Metallothionein

Binds

cadm

ium

underhigh

cadm

ium

stress

conditions

Synechococcus

Robinsonetal.1990

cadD

Cadmium

binding

CadDconfersamodestlevelof

cadm

ium

resistance

bysimilaractio

nto

thatof

CadB

Staphylococcus

aureus

Crupper

etal.1999

Chrom

ium

chrA

Chrom

atereductase

BiotransformsCr(VI)to

thenon-toxicCr(III)

with

NADHor

NADPHas

co-factors

Som

etim

esCr(VI)isused

asan

electron

acceptor

intheelectron

transportchain

under

anaerobicconditions

Arthrobacteraurescens,Bacillus

atrophaeus,

Pseudom

onas

putid

a,Rhodococcus

erythropolisDesulfotomaculum

reducens

Parketal.2000;

Patraetal.2010

Tebo

andObraztova

1998

Copper

cusF

Copperaccumulation

Bindto

copper

intheperiplasmicspace

andenhances

copper

accumulation

inside

thecells

E.coli

Yuetal.2014

cueO

Copperdetoxificatio

nOxidizesCu(I)to

aless

toxicCu(II)

E.coli

Djoko

etal.2010

Lead

pbrD

Leadbindingprotein

Putativeprotein,bindswith

thePb2

+intracellularly

andthus

reducing

itstoxiceffect

Cupriavidus

(Ralstonia)metallid

urans

Borremansetal.2001

Mercury

merA

Mercuricionreductase

Helps

inthereductionof

Hg2

+to

volatileHg0

Bacillus

subtilis,Pseudom

onas

putida

Ham

lettetal.1992;

Zhang

etal.2012;

Dashetal.2014

merB

Organom

ercuriallyase

Responsibleforreducing

toxicorganomercurial

compounds

(methylm

ercury

andphenyl

mercuricacetate)

into

nontoxicvolatile

elem

entalm

ercury,i.e.,lysis

ofC–H

g+bond

Desulfovibrio

desulfu

ricans,G

eobacter

sulfu

rreducens,Streptom

yces

sp.

Osbornetal.1997;

Raveletal.2000;

Schaeferetal.2011

Nickel

NiCoT

Nickelcobalttransferase

Nickelb

ioaccumulation

Clonedin

E.coli

Dengetal.2003;

Zhang

etal.2007


Cadmium

Cadmium is a highly toxic non-essential metal, and its effect canbe disastrous even when present in trace amounts. First reporton cadmium genetics suggested that there are no special mech-anisms for cadmium resistance in bacteria (Silver and Misra1984). However, this was widely disproven by Trevors et al.(1986), who studied cadmium transport, resistance, and toxicityin bacteria, algae, and fungi. Cadmium resistance is conferredby plasmid-borne operon named the cad operon, generallyfound in Staphylococcus spp., and czc operon inPseudomonas aeruginosa located on a 3972-bp element(Crupper et al. 1999; Chakraborty and Das 2014). The cadoperon has been found to be a two-component operonconsisting of the cadA and cadB operons (Zhang et al. 2015).The cadA operon is carried by the plasmid pI258 (Nucifora et al.1989). It carries two genes, cadA and cadC. CadA protein isanalogous to the ArsB protein of the ars operon and providesprotection by forming an energy-dependent ATPase which ef-fluxes cadmium from the bacterial cell. The CadC protein is thetranscriptional regulator of the operon (Hsieh et al. 2010).

The cadB operon encodes a 204-residue polypeptide; themechanism of action is not yet well elucidated completely. It ishypothesized that cadB, located on the plasmid pII147, causescellular binding of cadmium, most probably at the plasmamembrane (Smith and Novick 1972). Crupper et al. (1999)reported the presence of another new mechanism of cadmiumresistance through the cadD system in the Staphylococcusaureus plasmid pRW001 containing two genes, cadD andcadX*. cadD has been found to be similar to the gene cadB.cadX* encodes an inactive transcription regulator. However,this operon provides only low-level resistance to cadmium.Chaouni et al. (1996) reported the presence of another cadmi-um resistance operon in the Staphylococcus lugdunensis plas-mid pLUG10. This comprised a cadB-like resistance gene andanother transcription regulatory gene named cadX. These two

genes together provide high level of resistance to cadmium.cadX was found to be 40 % similar in sequence to cadC. Inturn, the cadX* gene from the pRW001 plasmid is similar insequence to the cadX gene, but only over the first 78 nucleo-tides. Beyond this, it is truncated and hence loses its transcrip-tion regulation ability. The cadX protein also displays about30 % homology to the ArsR protein of the ars operon (Yoonand Silver 1991). These are the genetic adaptations by whichbacteria resist the cadmium-rich environment.

In the practical sense, metals cannot be degraded; therefore,most biological metal remediation approaches are based ondetoxification and immobilization of metal which can reduceits biological toxicity and also hinder metal transportation.Cadmium remediation by bacteria initially involves metalbinding with the bacterial cell wall. In vicinity with cadmium,calcium ions and protons are releasedwhich is an indication ofthe competitive binding nature of the cell wall. On surround-ing with bivalent ions, the cell wall becomes positivelycharged based on pH-dependent charging and metal bindingdata as reported by Plette et al. (1996). Carboxylic and phos-phatic sites present on the cell wall of bacteria are used forstrong coordination with the cadmium ions helping in theirsite remediation. Gram-negative bacterial cell wall is com-posed of peptidoglycan, phospholipids, and lipopolysaccha-rides, of which the highly anionic character and charged na-ture of lipopolysaccharides render metal binding on the cellenvelope. Mechanisms include a combination of ion ex-change, complexation, coordination, adsorption, electrostaticinteraction, chelation, and microprecipitation (Vijayaraghavanand Yun 2008). In a report by Roane et al. (2001),Pseudomonas strain H1 and Bacillus strain H9 showed anintracellular mechanism of cadmium sequestration (36 %)for reducing cadmium toxicity. These strains showed the pro-duction of exopolymers (EPS) which accumulated cadmiumand reduced soluble cadmium levels by 22 and 11 %,respectively.

Fig. 1 A generalized illustrationof the genetic mechanism ofresistance to toxic metals bybacteria. Resistance to toxicmetals in bacteria is attributed toefflux systems, presence of metal-resistant genes, detoxificationgenes, biosorption, orbioaccumulation


Another alternative is the usage of microbial biomass forcadmium bioremediation which is cost effective and environ-ment friendly. This mechanism is due to independent extra-cellular adsorption by surface complexation, ion exchange, orelectrostatic interaction leading to intracellular accumulationby the cell surface (Vargas-García et al. 2012). A study byKhan et al. (2015) showed the removal of 18.8, 37, and56 % Cd+2 from aqueous medium after 48, 96, and 144 h,respectively. But an increase in Cd2+ concentration in the me-diumwas observed after 192 h, with a decrease in intracellularaccumulation of Cd2+. It is due to the activation of effluxsystem in E.coli for its survival. This illustrates the interde-pendence of metal accumulation and efflux system in bacteria.Another strategy of cadmium remediation is the use of CdSnanoparticles synthesized by functionalized EPS ofPseudomonas aeruginosa JP-11, which removed 88.66 % ofcadmium from aqueous solutions (Raj et al. 2016).

Chromium

Chromium is the seventh most abundant metal on earth andexists in two stable states in the environment, trivalent Cr3+

and hexavalent Cr6+. Chromium causes oxidative damage andinhibits sulfate membrane transport in bacteria. To fight chro-mium toxicity, microbes have developed two mechanisms ofchromium resistance. The first is a method of chromate effluxfrom the cells, and the second method involves enzymaticreduction of toxic Cr6+ to less toxic Cr3+. The chromate effluxprotein is encoded by a chrA gene, which has homologs ineubacteria, archaea, and even eukaryotes. Nies et al. (1998)described two chromate efflux pumps with six transmembranesegments. However, Diaz-Magana et al. (2009) showed that inE. coli, these two pumps are not separate entities, but togetherform a heterodimer of 12 segments.

In another study, the presence of a chromium resistance op-eron in Ochrobactrum tritici was described which conferredresistance up to 50 mM of Cr6+ (Branco et al. 2008). This op-eron, present on the 7189-bp transposable element TnOtChr,comprised four genes in chrBACF. These genes were not foundto be continuous, but interspaced with other genes. chrB wasfound to be the regulator of the operon, which induces its ex-pression in the presence of chromium (Chihomvu et al. 2015).chrA encodes a chromate ion transporter, which is sensitive toC r 6 + b u t n o t C r 3 + . C h r A c o n t a i n s a m o t i fGGX12VX4WX16PGPX9/8G (X = any amino acid) homologousto many species. ChrC is a 202-amino acid protein, similar toiron/manganese superoxide dismutase; however, the exact func-tion is yet not well elucidated (Morais et al. 2011). Though ChrFshowed some similarity to putative superoxide dismutase pro-teins (Branco et al. 2008), the exact function of ChrF is not wellunderstood. Deletion of chrF2 also did not affect chromate re-sistance levels (Juhnke et al. 2002). Gonzalez et al. (2005) iden-tified ChrR as chromate reductase bearing the signature

sequence LFVTPEYNXXXXXX-LKNAIDXXS, which could al-so provide additional protection against H2O2. The general chro-mate transport reaction involves the family of chromate iontransporters. Three other genes, chrJ, chrK, and chrL, wereidentified in Arthrobacter sp. strain FB24 (Henne et al. 2009).Viti et al. (2013) proposed that chrJ encodes a putative malate/quinone reductase protein, chrK, which specifies a YVTN beta-propeller repeat-containing protein, and chrL forms a probableconserved lipoprotein of the LppY/LpqO family.

Another method of chromate detoxification is the enzymat-ic reduction of Cr6+ to Cr3+ (Batool et al. 2012). Presence ofchromate-reducing enzyme and fast reduction of Cr6+ inchromium-resistant Lactobacillus strain have proven to beuseful for bioremediation of Cr6+ from contaminated environ-ment (Mishra et al. 2012). Cervantes and Campos-García(2007) broadly classified Cr6+ reduction by three mecha-nisms—aerobic reduction by reductases in presence ofNADH or NADPH, usage of Cr6+ as an electron acceptor inthe electron transport system, or by reaction of Cr6+ with or-ganic compounds. During anaerobic reduction, when Cr6+ ismade to act as an electron acceptor, the catalyzing enzymesgenerally show a flavin oxidoreductase activity. Examples ofsuch enzymes include ferric reductase from Paracoccusdenitrificans, which reduces both Fe3+ and Cr6+. Other en-zymes include YieF Cr6+ reductase from E. coli which is sim-ilar in structure with the ChrR of Pseudomonas putida(Ackerley et al. 2004). Many bacterial strains such asEnterobacter sp. andPseudomonas sp. have also been isolatedand utilized in Cr6+ reduction under anaerobic conditionsusing chromate reductase (Kamaludeen et al. 2003). TheYieF reaction involves transfer of four electrons. Three elec-trons are used to reduce Cr6+ to Cr3+, while the single electroncombines with O2 and generates reactive oxygen species(ROS) (Viti et al. 2013). Since these ROS are harmful to thecell, superoxide dismutases act and convert these ROS to ox-ygen or water. Similarly, ArsH from Synechocystis sp. PCC6803 has also been found to be capable of reducing Cr6+ (Xueet al. 2014). These are the genetic adaptation mechanisms inbacteria for chromium toxicity.

Copper

Copper is the third oldest Bhuman used^ metal having wideapplication in wires, motors, architecture, and medicines.Excess copper in the body causes copperedius, leading toproduction of ROS, which have the potential to damage pro-tein, lipids, and DNA. Copper cycles between two oxidationstates, Cu(I) and Cu(II), and can displace iron (Fe) from ac-cessible Fe–S clusters in dehydratases and other iron–sulfurproteins (Macomber and Imlay 2009). Tetaz and Luke (1983)first reported a plasmid pRJ1004-mediated copper resistancein bacteria. Later, Bender and Cooksey (1986) identified in-digenous pPT23D plasmids in Pseudomonas syringae pv.


tomato helping from copper toxicity. Subsequently, Mellanoand Cooksey (1988) established the presence of a copper-inducible cop operon in the 35-kb pPT23D plasmid. It showedconsiderable similarity to the copper resistance genes ofP. cichorii and P. fluorescens (Cooksey et al. 1990).Wunderli-Ye and Solioz (1999) described the copper resis-tance mechanism in the bacterium Enterococcus hirae, whichcontained four genes: copYABZ. copY and copZ encode acopper-responsive repressor and a chaperone protein, respec-tively. copA was postulated to encode a protein which helpscopper uptake in limiting conditions, and CopA and CopB areP-type ATPases. In this regard, Streptomyces sp. AB2Ashowed early copper retention followed by drastic metal ef-flux process suggesting the extracellular cupric reductase ac-tivity in the organism (Albarracin et al. 2008).

Copper resistance in E. coli has also been extensivelystudied. E. coli has been reported to have a double regula-tory mechanism of copper resistance. The first is a two-component system called the cus (copper sensing) locus(Munson et al. 2000). It has two regulator genes cusR andcusS, which forms a regulator-sensor pair and regulates theexpression of cusCFBA. The CusCBA proteins are similarto cation/proton antiporter complexes which export metalions outside the cell in exchange of influx of H+. CusF isknown to bind to copper in the periplasmic space enhancingcopper accumulation inside the cells (Yu et al. 2014). CusFis a small 10-kDa protein located in perplasmic space andbinds one copper per polypeptide. It is involved in copperresistance and contains several methionine, aspartate, andhistidine residues required for copper binding. However,site-directed mutagenesis deduced the involvement of me-thionine residues for CusF binding with copper. Predictionof sequence analysis of CusF revealed an N-terminal leadersequence and a potential signal peptidase cleavage site sig-nifying the periplasmic location of CusF (Franke et al.2003).

The second copper homoeostasis system is called cue, orcopper efflux system. The regulatory gene cueR regulates twogenes, copA and cueO. CopA, as described, is a P-typeATPase, and cueO encodes a multicopper oxidase, which ox-idizes Cu+. CueO oxidizes Cu(I) to a less toxic Cu(II) andreduces dioxygen to water through four single-electron trans-fer steps (Djoko et al. 2010). In the case of multicopper oxi-dases, three types of copper atoms, type 1 (T1), type 2 (T2),and two type 3 (T3), are present, of which T1 is buried in theinterior of the protein and catalyzes oxidation of the substratewhereas, T2 and two T3 form a trinuclear center (TNC) wheredioxygen is reduced. However, CueO has an extramethionine-rich helix that blocks the solvent access to theT1 site and forms an additional copper-binding site accumu-lating copper (Singh et al. 2004). CsoR is another regulatorygene, which in the presence of Cu+ derepresses copper resis-tance genes (Chang et al. 2014).

A Streptococcus strain was seen to have a copper transportoperon named copYAZ in which copY and copZ wereestablished as heavy metal-binding proteins (Vats and Lee2001). Pseudomonas fluorescens has been reported to possessa copRSCD operon (Hu et al. 2009). In contrast, Helicobacterpylori contain two separate operons for copper export andimport, hpcopA and hpcopP (Ge and Taylor 1996). Bacillussubtilis has another copper regulatory system, mediated byYcnJ and regulated by YcnK and CsoR (Chillappagari et al.2009). Together, these genes maintain a state of copperhomoeostasis in the cell. However, ATPase-driven copper ef-flux system is the main mechanism responsible for cytoplas-mic copper removal. Periplasmic copper handling,multicopper oxidases, metallochaperones, and RND systemsare involved in this process (Bondarczuk and Piotrowska-Seget 2013).

Grass et al. (2004) reported the multicopper oxidase CueOin E.coli which expressed in presence of copper and oxidizesCu(I) to Cu(II) in the periplasm and also catecholate-containing ligands such as enterobactin. As the siderophore,enetrobactin is able to reduce Cu(II), it was proposed thatenterobactin oxidation by CueO plays a supplementary func-tion in Cu resistance not generating more toxic Cu(I) ions. Inaddition, 2,3-dihydroxybenzoic acid (DHB), an intermediatein enterobactin biosynthesis, stably binds Cu ions, acting as aCu sink. In another uropathogenic E.coli, yersiniabactin wasreported to sequester Cu(II) outside bacterial cells and pre-vents its catechol-mediated reduction to Cu(I) (Chaturvediet al. 2012), thus protecting the bacteria from intracellularkilling. Furthermore, the Cu(II)–yersiniabactin complex hassuperoxide dismutase activity protecting bacteria from oxida-tive stress inside phagocytic vesicles (Chaturvedi et al. 2014).

Lead

Lead (Pb) is a non-essential, persistent, and hazardous toxicmetal pollutant. Several bacteria, such as Arthrobacter spp.,Bacillus megaterium, Pseudomonas marginalis, Citrobacterfreundii, Staphylococcus aureus, and E. coli have been foundto be resistant to lead. Perhaps the most studied lead resistanceoperon is found in the endogeneous pMOL30megaplasmid ofthe bacterium Cupriavidus (Ralstonia) metallidurans CH34(Borremans et al. 2001). This operon, named pbr operon,was found to contain many structural genes and one regulato-ry gene (pbrR), of which pbrT encodes Pb(II) uptake protein,pbrA encodes P-type Pb(II) efflux ATPase, pbrB encodes pre-dicted integral membrane protein of unknown function, andpbrC encodes predicted prolipoprotein signal peptidase. pbrAwas proposed to export Pb(II) from the cytoplasm, whichwould then be converted to a phosphate salt by the inorganicphosphate produced by PbrB. Hynninen et al. (2009) showedthat pbrB encoded an undecaprenyl pyrophosphate phospha-tase. A Pb(II)-binding protein encoded by pbrD is located


downstream of pbrC vital for lead sequestration. In the pres-ence of lead, the regulator pbrR induces the transcription ofpbrABCD operon.

Regulation of the pbr operonwas done by pbrR, which wasfound to be similar to the merR family of heavy metal ion-sensing regulatory genes. pbrR induced the expression ofpbrABCD as a single transcriptional unit in the presence ofPb2+. pbrABC combination was believed to be involved inlead efflux, and pbrD is thought to be responsible for Pb2+

accumulation (Borremans et al. 2001). PbrD possesses a po-tential metal-binding motif, rich in cysteine residues (Cys-7X-Cys-Cys-7X-Cys-7X-His-14X-Cys), and also bears a largenumber of proline and serine residues (Jarosławiecka andPiotrowska-Seget 2014). Monchy et al. (2007) showed thepresence of another gene pbrU, which is induced in the pres-ence of Pb2+. However, pbrU codes for a permease belongingto the major facilitator superfamily (MFS) which is present inthe inner membrane ofC. metallidurans (Taghavi et al. 2009).Hither to this, C. metallidurans was identified as Ralstoniametallidurans. However, the name of some of the genes inthe Deutsche Sammlung von Mikroorganismen undZellkulturen (DSMZ) database are still starting with BRme^which stands for R. metallidurans (von Rozycki and Nies2009).

Roane (1999) investigated the intracellular sequestration of0.6 mM of lead by B. megaterium by metallothionein-likeproteins. P. aeruginosa strain WI-1 isolated from Mandoviestuary also possesses bacterial metallothionein (BmtA) whichbioaccumulated 26.5 mg lead/g dry weight of cells intracellu-larly to reduce the toxic effect of lead (Naik et al. 2012a).Metallothionein protein from the smtAB gene was amplifiedfrom Salmonella choleraesuis and Proteus penneribioaccumulating 19 and 22 mg of lead, respectively (Naiket al. 2012b). Therefore, presence of metallothioneins in bac-teria may be employed for lead bioremedaition in contaminat-ed environmental sites.

Metal immobilization by the process of extracellular se-questration is important for regulating metal toxicity.Extracellular polymeric substances (EPS) consisting of poly-saccharides, proteins, nucleic acids, humic substances, andlipids with diverse functional groups of hydroxyl, carboxyl,amides, and phosphoryl exhibit high affinity toward heavymetals with high specificity and affinity (Bhaskar andBhosle 2006; Bramhachari et al. 2007). Lead binding by thenegatively charged components of EPS of P. aeruginosaCH07 was also investigated by De et al. (2007).Pseudomonas marginalis was able to resist 2.5 mM lead bysequestering lead in an exopolymer (Roane 1999). Similarly,EPS of Paenibacillus jamilae biosorbed 303.03 mg lead/gEPS from lead solution (Morillo et al. 2008). There are manyenzymatic activities in the bacterial EPS which assist in toxicmetal transformation by chemical reaction, precipation, or en-trapment (Paul 2008). Therefore, EPS producing lead-

resistant bacterial strains may serve as potentialbioremediative agent (lead biosorbent) in lead-contaminatedenvironmental sites.

Bioprecipitation of toxic metals to insoluble complex for-mation is another strategy which reduces metal bioavailabilityand toxicity. Bacillus iodiniumGP13 and Bacillus pumilus S3were reported to precipitate lead as lead sulfide (PbS) (Deet al. 2008). A phosphate-solubilizing bacterium E. cloacaewas found to resist lead by immobilizing lead as insoluble leadphosphate mineral named pyromorphite (Park et al. 2011).This method of lead reclamation is an effective, eco-friendlyapproach for lead bioremediation. Siderophore induction inresponse to lead stress is also another detoxification strategy.Lead-resistant P. aeruginosa strain 4EA showed lead-inducedsiderophore (pyochelin and pyoverdine) production (Naik andDubey 2011). Therefore, genetic adaptations rendering leadresistance followed by various biotransformation techniquesfor lead remediation may be employed for microbial remedi-ation of lead.

Mercury

Mercury is one of the most toxic elements in the universe andis found to have severe health concerns in comparison to othertoxic metal pollutants. It is concentrated in sediments, soils,atmosphere, and water. Consumption of fish has been shownto be the most potent source of mercury ingestion. Mercuryalso enters the environment as industrial wastes. Two differentoperons are present in bacteria for mercury resistance. One is anarrow-spectrum mer operon, while the other is the broad-spectrum mer operon (Silver and Phung 2013). The simplestmer operons are those found on the transposons, Tn5037(Kalyaeva et al. 2001) and Tn5070 (Mindlin et al. 2001),whereas the most complex is the mer operon of Tn5718(Schneiker et al. 2001).

The narrow-spectrum mer operon consists of the genesmerR, merT, merC, merF, merP, and merD (Dash and Das2012). This operon is inducible by inorganic mercury (Hg2+)and provides resistance to inorganic mercury salts only. merRacts as the positive transcriptional regulator of the operon andis transcribed separately. In the presence of extracellular mer-cury, or absence of intracellular mercury, it binds to thepromoter/operator region of the operon to positively and neg-atively regulate the expression of the functional genes. Theoutermost protein of the operon is MerP, located in the peri-plasmic space. This is a 72-amino acid protein with aβ-α-β-β-α-β fold. The two α helices are found to overlaythe four-strand antiparallel β sheet (Eriksson and Sahlman1993). The mercury-binding site contains the GMTCAACconsensus sequence. MerP scavenges inorganic mercury ionsand transports them to the MerT protein (Hamlett et al. 1992).MerT, encoded by the transposon Tn501, is a 116-amino acidprotein, which receives the inorganic mercury from MerP at


the plasma membrane and transports it inside the cell by bind-ing to cysteine residues on its transmembrane helices.Removal or mutation of these residues results in loss of mer-curic resistance.

The exact mechanism ofMerC is obscure. However, MerChas been shown to play a part in mercury transportation andaccumulation (Kiyono et al. 2013). merC expression inArabidopsis thaliana and Nicotiana tabacum has led to theirdoubling ability to accumulate mercury (Sasaki et al. 2006).MerF, on the other hand, is a 8.7-kDa protein with two trans-membrane helices. It also functions as a broad-spectrum mer-cury transporter. The ∼1600-nucleotide-longmerA gene codesfor a protein calledmercuric ion reductase (Moore et al. 1990).This dimeric enzyme functions to reduce Hg2+ in the cell toform volatile Hg0, which is then easily released from the cell.MerA protein is a flavoprotein, which requires NADPH as anelectron donor to perform the reaction. MerD forms a second-ary regulator protein, which binds, albeit weakly, to the samepromoter/operator region as MerR and negatively regulatesthe operon (Nascimento and Chartone-Souza 2003). In a re-cent study, merH has been identified as a mercuric ion trans-porter (Schue et al. 2009). Located upstream of themerA gene,it has been found to transport Hg2+ ions by cysteine residuesand is co-expressed with merA itself. merH putatively func-tions as a metal-trafficking protein to merR, which in turninduces the mer operon for merA-mediated volatilisation ofHg2+ (Schelert et al. 2013). merI has also been identified im-mediately downstream of themerA gene (Schelert et al. 2006).However, its exact function has not yet been deciphered, andthe huge diversity of arrangement and sequence ofmer operoncan be explored further for bioremediation application(Rebello et al. 2013).

The broad-spectrum mer operon contains similar genes asthe narrow-spectrum operon. In addition to the genes alreadypresent, additional genes likemerE,merG, andmerB are pres-ent. The broad-spectrum operon provides protection to organ-ic mercury (Barkay et al. 2003). These compounds are moretoxic as they can easily enter the cell without transporter mol-ecules. Organic mercury (R-Hg) enters the cell by passivediffusion or is transported inside by MerE or MerG. merEwas originally located in the transposon Tn21 and can mediatethe transport of both methyl-mercury (CH3Hg

+) and inorganicmercury (Boyd and Barkay 2012). Phenylmercury resistanceis conferred by the MerG (∼20 kDa) protein, which is theproduct of a 654-bp gene (Kiyono and Pan-Hou 1999).Located between merA and merB on the operon, it protectsthe cell from phenylmercury by preventing it from enteringthe cell (Schneiker et al. 2001). The other enzymatic moleculeof the mer operon is merB, coding for the enzymeorganmercurial lyase. MerB protein catalyzes the protonolysisof the carbon–mercury bond resulting in the formation of ionicmercury and a reduced hydrocarbon. The ionic mercury isthen reduced to the elemental form Hg0 by the mercuric ion

reductase (Dash and Das 2012). Hg0, due to its high vaporpressure, is volatilized out of the bacterial cell.

Mercury bioremediation by microbes is mediated by vari-ous enzymatic transformations like reduction of Hg2+ to Hg0,breakdown of organomercury compounds, methylation ofHg2+, and oxidation of Hg0 to Hg2+. In a study by Dash andDas (2015), a transgenic bacterium Bacillus cereus BW-03(pPW-05) was constructed by transforming a plasmid-harboring mer operon of the marine bacterium Bacillusthuringiensis PW-05 isolated from Bay of Bengal (Dashet al. 2014) into another mercury-resistant marine bacteriumB. cereus BW-03 with mercury biosorption ability. This couldremove >99 % of mercury supplement in vitro by simulta-neous volatilization (>53 %) and biosorption (∼40 %). Manymercury-resistant mer gene-incorporating bacterial isolateswere isolated which could volatilize and reduce Hg2+ to Hg0

(De et al. 2008). Reduction of mercury and breakdown oforganomercury compounds are performed by proteins of themer operon. In addition to that, the electrochemical potentialof Hg2+/Hg0 at pH 7 is +430 mV, and this indicates that theliving cells reduce Hg2+ to the elemental form, which is non-toxic to bacteria. Themelting point/boiling point of mercury islow (−39/357 °C); therefore, metallic mercury leaves the cellby passive diffusion and is volatilized into the air or precipi-tates due to its low solubility in water removing toxic Hg2+

(Wagner-Dobler 2003). Methymercury production in the en-vironment is controlled by both microbial and abiotic trans-formations. Direct transformation includes Hg2+ methylationand MeHg degradation. Hg2+ reduction to Hg0 and its cyclicoxidation affect MeHg formation indirectly by controllingHg2+ levels, the substate for methylation (Barkay andWagner-Dobler 2005). Moreover, large-scale cleanup ofmercury-containing wastewater by mercury-resistant mi-crobes is an environment friendly and cost-effective treatmenttechnology practiced nowadays. This was evidenced by theconstruction of pilot plants for mercury removal which treated100 m3 of 50 % mercury-containing wastewater per day(Wagner-Dobler 2003).

Besides enzymatic transformation, mercury can also bebioremediated by using metallothioneins and polyphosphatesby sequestering mercury ions in a biologically inactive form.ppk gene encodes polyphosphate kinase which is involved inpolyphosphate biosynthesis. These negatively charged ortho-phosphate polymers are capable of binding mercury ions(Kornberg 1995). ppk genes expressed in many transgenic bac-teria were shown to withstand and accumulate up to 16 μM ofmercury from solutions (Pan-Hou et al. 2002). Another ap-proach is the mercuric sulfide (HgS) formation by the directreaction of Hg2+ with H2S produced anaerobically byClostridium cochlearium (Pan-Hou and Imura 1981). Similarwork reported that Klebsiella aerogenes NCTC418 producedHgS when grown in continuous aerobic culture with mercurychloride. Mercury was also biologically removed by a mercury-


reducing biofilm which had both the natural and engineeredmercuric reductase (Brunke et al. 1993). Use of sulfate-reducing bacteria is also used as source of H2S for precipitatingmetal as sulfides. The high-solubility product of HgS rendersmercury removal by H2S (Hakansson et al. 2008).

Nickel

Nickel exists as five stable isotopes 58Ni, 60Ni, 61Ni, 62Ni, and64Ni with 58Ni being the most abundant metal in the environ-ment. Nickel has an important role in the biochemistry ofmicrobes and plants. The enzyme urease contains nickel.Other enzymes like hydrogenases, superoxide dismutase,and glyoxalase enzyme contain Ni-Fe clusters or use nickelas a co-factor. However, nickel is highly toxic to animals andhumans due to its potential to cross the placenta and affect thefetus. Nickel resistance in bacteria is generally mediated byefflux pumps. One such resistance mechanism has been stud-ied in Cupriavidus (Ralstonia) metallidurans CH34 by Grasset al. (2000). They reported the presence of a cnrCBA effluxpump encoded by the cnrYHXCBAT gene system. Once nickelenters the periplasm, transcription is initiated at the cnr pro-moter by cnrY and cnrC. The products of the three genescnrYXH regulate the expression of the whole gene cluster.cnrH is an extracytoplasmic function (ECF) sigma factor(Grass et al. 2000) which constitutively activates cnrCBA ex-pression. cnrX and cnrY located in the periplasm aremembrane-bound proteins, which putatively function as anti-sigma factors. The cnrCBA encodes a highly efficient pumpwhich is activated only in micromolar concentrations of nick-el. These gene products form an efflux pump to efflux excessnickel outside the cell.

Grass et al. (2005) also characterized the nre (nickel resis-tance) operon in Achromobacter xylosoxidans 31A. They foundthe nre locus on the plasmid pTOM9, and only one gene, nreB,was responsible for conferring the entire nickel resistance.However, similar to the cnr operon, this operon did not detoxifyNi2+, instead effluxed it outside the cell. Earlier, Schmidt andSchlegel (1994) reported another operon on the same pTOM9plasmid termed ncc operon, which provided combined nickel,cobalt, and cadmium resistance. However, these nickel resis-tance mechanisms do not contribute much to nickel bioremedi-ation. Seven open reading frames (ORFs) were studied and des-ignated nccYXHCBAN. Nucleotide sequence revealed signifi-cant similarity to the cnr and czc operons of Alcaligeneseutrophus CH34 (Tibazarwa et al. 2000). The NccX protein iscomposed of 76 amino acids and has many His residues, indi-cating that it may serve as a metal-binding protein utilized innickel remediation (Trepreau et al. 2011). In E. coli, Rodrigueet al. (2005) performed studies on the yohM gene and found thatit encoded amembrane-bound polypeptide which had the abilityto confer resistance to nickel and cobalt. Higher copies of yohMled to reduced intracellular nickel. yohM was, thus, renamed as

rcnA, the first nickel efflux system discovered inE. coli. Anotherefflux pump was identified in Helicobacter pylori and namedcznABC, for cadmium–zinc–nickel (Stahler et al. 2006). Out ofthe three genes, cznA and cznC were found to confer nickelhomoeostasis.

E.coli JM109 was genetically engineered for the simulta-neous expression of nickel transport system and metallothio-nein gene to remove as well as recover Ni2+ from aqueoussolution. It showed a sixfold increase in nickel-binding capac-ity than the host cells following the linearized Langmuir iso-therm (Deng et al. 2003). In another study, the nickel/cobalttransferase gene, NiCoT, from Staphylococcus aureusATCC6538 was amplified and ligated into vector pET-3cwhich established high bioaccumulation of nickel of11.33 mg/g which was three times more than that of the orig-inal E. coli BL21 strain (Zhang et al. 2007). Genetic engineer-ing of bacterium E. coli by genomic suppression of the RcnAnickel (Ni) and cobalt (Co) efflux system was combined withthe plasmid-controlled expression of a specific metallic trans-porter, NiCoT from Novosphingobium aromaticivorans. ThisNi/Co buster strain resulted in enhanced nickel (II) and cobalt(II) uptake with metal accumulation of 6 mg/g bacterial dryweight in the first 10 min of treatment. Additionally, a syn-thetic adherence operon was introduced into the plasmid ableto form thick biofilm structures in the presence of nickel (II)and cobalt (II). Therefore, genetic engineering demonstratedincreased metal sequestration and biofilm formation by E.coliwhich can be used for biofiltration of nickel as well as cobaltin an industrial scale by the immobilized cells (Duprey et al.2014).

Nickel metallochaperones are responsible for shuttlingnickel and transferring it to enzyme precursors through pro-tein–protein interactions in a complex stepwise process. Thesemetallochaperones can be engineered for nickel bindingand removal. Infact, nickel removal was reported to be234.4 μg/ml by Pseudomonas cepacia 120S (living and deadbiomass) and 117.2 and 351.6 μg/ml by living and dead bio-mass, respectively, by B. subtilis 117S (Abdel-Monem et al.2010). Soil-inhabiting Ni-resistant Bacillus thuringiensis wasreported to tolerate up to 10mMNi and found to remove 82%of Ni from the medium by the process of biosorption (Daset al. 2014c). A recent approach incorporated the synthesisof nickel oxide nanoparticles from Microbacterium sp.MRS-1 utilized for the treatment of nickel electroplating in-dustrial effluent which showed nickel removal efficiency of95 % (Sathyavathi et al. 2014).

Discovery of novel metal-resistant genes involvedin bioremediation

In response to toxic metal stress, bacteria have developedsome amazing survival mechanisms incorporated into their


genome. They produce diverse group of enzymes and proteinswhich help them to overcome these adversities (Johnsen et al.2005). Bacteria may also be genetically modified or metabol-ically engineered to yield products that confer special featuresto the host cells. Currently, scientists are examining the possi-bility of finding Bnew^ adaptation strategies in microorgan-isms for removal and subsequent detoxification of pollutants.Introduction of genes encoding enzymes is required that canchange the oxidation state of heavy metal from highly toxic toless toxic forms; e.g., for bacterial merA encoding, mercuricreductase was introduced into other bacterium for better bio-remediation (Dash and Das 2015). Thus, introduction of mod-ified genes may be helpful in obtaining the new mechanismsof detoxification of heavy metals (Arora et al. 2010).

Another approach which can be undertaken is the use of insilico methods. Nowadays, with the advent of computers andsoftwares, it is possible to get information on any object from asingle source. Various databases exist which provide informa-tion on the toxicity of compounds and their occurrence, proper-ties, and pathways by which they may be degraded. Some of theimportant databases include USEPA (http://www.epa.gov/),ATSDR (http://www.atsdr.cdc.gov/), and KEGG PATHWAYDatabase (http://www.genome.jp/). An interesting comparisonof utilizing such computational sources for carrying outbioremediation virtually before testing it on site has beenreviewed by Khan et al. (2013). They listed 11 software toolsfor predicting the toxicity of compounds. They also described 10databases with toxicity information on several compounds. Inaddition, environmental degradability of compounds could also

be tested by means of 15 programs. Two of the most efficientprograms described were Biodegradability Evaluation andSimulation System (BESS) and Biochemical NetworkIntegrated Computational Explorer (BNICE). With this knowl-edge in hand, and with the help of biological pathway predictionsoftwares like Scansite 2.0 (Obenauer et al. 2003), BioCyc(Karp et al. 2005), SMART5 (Letunic et al. 2006), STRING7(Von Mering et al. 2007), and KEGG (Kanehisa and Goto2000), it is now possible to study the detailed interactions be-tween various biomolecules in silico and to deduce novel pro-teins and genes whichmay be used for bioremediation purposes.

Manipulation of bacterial genetic systemfor enhanced bioremediation

Most pollutants have high persistence in the environment,mainly due to sub-optimal degradation pathways (Timmisand Pieper 1999). Many organic pollutants are degraded bybiological pathways, whereas inorganic toxic metals are trans-formed by various enzymes. There exist many intracellularand extracellular events in bacteria involving microbial biore-mediation of toxic pollutants from the environment. In re-sponse to the presence of toxic metals in the environment,resistant bacteria synthesize many intracellular and extracellu-lar enzymes to remove/degrade the toxic form of metals tonon-toxic/less toxic forms (Fig. 2). Each enzymatic pathwayhas a rate limiting step. Manipulation of this rate limiting stepcould be a solution to increase their bioremediation potential.

Fig. 2 Intracellular and extracellular events involving microbialbioremediation of toxic metals from the environment. In response to thepresence of toxic metals in the environment, resistant bacteria synthesizemany intracellular and extracellular enzymes to remove/detoxify the toxicform of metals to non-toxic/less toxic forms. These processes include A

oxidation-reduction of metals, Bmetal sequestration by metallothioneins,C conjugate formation with organic compounds/precipitation, D metalefflux by metal transporters followed by bioremoval by microbialproducts, and E bioremoval of metals by microbial products(biosurfactants or EPS)


There are many techniques, involving the manipulation ofbacterial systems, which have been discussed below in details.The modern technology of genetic engineering allows design-ing of microorganisms capable of transforming specific metalcontaminants. The opportunity of creating artificial combina-tion of genes that are not present in nature offers huge out-come for these GMOs to be used for in situ removal of metalpollutants. The most common techniques include engineeringwith a single gene or operon, alteration of existing gene se-quences, and pathway switching.

Engineering single gene or gene cluster/operon

Microorganisms prevalent in the polluted sites with high con-centration of heavy metals adapt to resist the harmful effectsby modulating various genetic mechanisms. These strainswith their intrinsic ability to survive in polluted sites are pre-ferred for bioremediation application. Thus, construction of abacterium by introduction of a single gene or gene cluster intothe intrinsic bacterium will be suitable for bioremediation ap-plication due to the de novo adaptation of these strains. Sandaaet al. (1999) found that mostly Gram-positive and α-proteobacteria is found in heavy metal-contaminated soils.Since majority of the indigenous bacteria are incapable ofsurviving in such adverse conditions, genetically modifiedrecombinant strains may be used to enhance bioremediation.It is preferred to alter the indigenous bacterial population ge-netically, but the major problem that arises is the instability ofthe cloned genes and subsequent transfer onto the futuregenerations. Lorenzo et al. (1998) suggested the use of mini-transposons, derived from the naturally occurring Tn5 andTn10 transposons. These mini-transposons have the specialitythat only the functional segments of a DNA can be isolatedand cloned into the Tn5 vector, following which it may beinserted into the chromosome of Gram-negative bacteria.

Ruiz et al. (2011) transformed E. coli JM109 with vectorsresulting in enhanced expression of metallothionein (mt1) andpolyphosphate kinase (ppk) genes. Metallothioneins aremembrane-bound proteins involved in binding of metals andtheir subsequent reduction. This engineered bacterium wasfound to accumulate more than 100 μM Hg. Brim et al.(2000) transformed the radiation-resistant Deinococcusradiodurans with the mercuric ion reductase gene (merA)from E. coli BL308 host. The resulting mutant strain wasfound to grow both in the presence of high radiation andmercury. They were also able to volatilize inorganic Hg(Hg2+) to elemental Hg (Hg0). In a similar approach, Dashand Das (2015) developed a transgenic strain B. cereus BW-03(pPW-05) that harbors the mer operon-containing plasmidfrom a wild strain of B. thuringiensis PW-05. The transgenicstrain was reported to possess both the mechanisms of mercu-ry resistance, i.e., Hg volatilization as well as Hg biosorptionwhich can be widely used in situ for efficient removal of

mercury from the contaminated environments. The heavymetal-resistant bacterium C. metallidurans strain MSR33was genetically modified to resist mercury (Rojas et al.2011). This bacterium was found to be tolerant to both inor-ganic mercury and methylmercury, in addition to copper andchromate.

In a study by Chaturvedi and Archana (2014), the expres-sion of two metal-binding peptides was expressed inDeinococcus radiodurans R1 which became an attractivestrategy for developing metal tolerance. A synthetic gene(EC20) encoding a phytochelatin analog and cyanobacterialmetallothionein (MT) gene, smtA, was constructed by overlapextension and expressed in DR1 under the native groESLpromoter. This recombinant strain demonstrated 2.5-foldhigher tolerance to Cd2+ and accumulated 1.21-fold greaterCd2+. Therefore, engineering of desirous genes into compati-ble bacteria bestows the environment with better approachesfor remediation.

Alteration of intrinsic genes

There are many disadvantages of using a geneticallyengineered microorganism in environmental conditions.Some of the introduced genes may not be stable in variousenvironmental conditions to carry out the desired function(Dixit et al. 2015). In certain instances, the expression levelof the foreign genes is highly affected in the presence of con-taminants and adversely affects the bioremediation practice(Kiyono and Pan-Hou 1999). Thus, instead of using the for-eign genes, the intrinsic genes may be targeted or manipulat-ed. This technique allows the growth of the microorganisms intheir natural environment in addition to carrying out the de-sired function in terms of metal bioremediation.

There are many studies on targeting the alteration/modification of the existing gene clusters of indigenousmicroflora for bioremediation application. Kermani et al.(2010) isolated 3 cadmium-resistantPseudomonas aeruginosafrom industrial sludge. Mutation in these strains by exposureto the dyes acridine orange and acriflavine increased the toler-ance of these strains upto 7 mM of Cd2+. Phytochelatin syn-thase from Schizosaccharomyces pombe was cloned andoverexpressed in E. coli. As a result, the genetically modifiedE. coli strain was found to accumulate 25-fold higher concen-tration of Cd2+ as compared to the control strain (Kang et al.2007). Wu et al. (2006) found that overexpressing a syntheticphytochelatin EC20 in the soil bacterium Pseudomonas putida06909 resulted in triple cadmium binding capability and alsoimproved cell growth by 2-fold. The close relative of theradiat ion-resistant Deinococcus radiodurans andDeinococcus geothermalis was engineered by Brim et al.(2003). With the help of plasmids, D. geothermaliswas foundto reduce not only Hg2+ but also Fe3+, U6+, and even Cr6+.Ackerley et al. (2004) overexpressed nfsA in E. coli and found


increased chromate reduction upto 1.5-fold. Overproductionof ChrR in Pseudomonas putida increased Cr6+ reduction by2.4 times (Gonzalez et al. 2005). A detailed list of the bacterialstrains which have effectively been engineered for bioremedi-ation purpose has been summarized in Table 2.

Pathway switching

Pathway switching involves the construction, extension, andregulation of certain novel genetic mechanisms for bioreme-diation applications. In the case of toxic metals constructing amicrobial consortium each performing a specific path, it canbe an effective measure for achieving complete bioremedia-tion. Limited studies have been carried out so far for the im-provement of the bacterial strains for metal removal by con-struction of novel consortium. Pathway switching approachmay be employed for the metal removal using GMOs as well.As the uptake of Cu–Mb (copper–methanobactin) bymethanotrophic bacteria is quite evident, the mechanism canbe developed for simultaneous uptake of Cu–Mb complexesrather than Cu dissociating from Mb prior to uptake(Balasubramanian et al. 2011). Hence, the identification of asuitable pathway to facilitate acquisition of metals from theenvironment by identifying the mechanism of transport ma-chineries can provide a key direction for future research.

Application of GMOs for bioremediation is in forefrontdue to their efficiency and cost-benefit approaches.However, a complete information on the genes is requiredwhich can be achieved by microarray and fluorescent in situhybridization technologies. Another impediment regardingapplication of these GMOs is the regulatory affairs and thehazards associated with these organisms. Future of bacteria-based bioremediation relies on the development of suicidalgenetically engineered microorganisms (S-GEMs) which willenhance the application of these GMOs for on-site applica-tion. These S-GEMs are constructed by the use of a killer geneand regulatory circuit to control the expression of the killergene in response to presence or absence of environmentalsignals (Paul et al. 2005). Thus, the constructed S-GEMs willundergo programmed cell death due to the presence of killer–anti-killer genes after removal of toxic substances for theirsafe demise in the environment.

Future directions

Discovery of novel genes and proteins associated with theability of eco-friendly cleanup will be a great help to achieveenhanced bioremediation. Randommutations may be effectedin the genes known to provide heavy metal resistance to mi-crobes. Such mutations usually have harmful effects on theorganism, but sometimes may also have opposite effects andresult in the formation of strains with higher detoxificationT

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References

Gram-negativebacteria

Transposons

Introductionof

vectorsderivedfrom

Tn5

andTn10

Efficient

introductionof

genes

Lorenzo

etal.1998

E.coliJM109

endA

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Δ(lac-proAB)e14-

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lacZΔM15]hsdR

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nof

mt-1,ppkgenes

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Hgaccumulation/transformation

Ruizetal.2011

Deinococcus

radiodurans

merA

Introductio

nof

merAgene

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inpresence

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andHg,Hgvolatilization

Brim

etal.2000

Bacillus

cereus

BW-03(pP

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merAgene

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Hgvolatilizationandbiosorption

DashandDas

2015

Cupriavidus

metallid

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operon

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merB,m

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genes

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orange

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ability. This phenomenon is termed as Bgain-of-function^mu-tation (Arora et al. 2010). To identify new genes whichmay beexpressed in the presence of a particular pollutant, whole ge-nome assays may be performed using microarray technology.This enables one to study the expression of thousands of genesat once and thus may enable scientists to determine whether asingle gene or a gene cluster is responsible for the detoxifica-tion of a particular pollutant.

Another easy method that may be used to study the pres-ence of certain target genes is fluorescence in situhybridisation (FISH) (Pernthaler et al. 2002). FISH is gener-ally used in medical science to detect the presence of patho-gens in case of infections. However, its scope may be extend-ed to include in other studies as well. Metagenomic analysismay enable us to identify and study many unknown genomeswhich have vast potential in pollution control. Finally, oncespecific genes are identified, they may be transferred to otherpotent strains and stably expressed for the creation of mi-crobes that are highly efficient candidates for performingbioremediation.

Microbes possess many unique characteristic features suchas biofilm formation, biosurfactant production, secondary me-tabolites synthesis, and many more to withstand the stressconditions in the environment. These properties of metal-resistant bacteria may be harnessed for their enhanced utiliza-tion in bioremediation. Recently, multispecies biofilm com-munities have been explored for their metal tolerance andbio-mineralization properties (Golby et al. 2014). Thisshowed promising result of employing microbial Bvillage^for application in metal bioremediation which needs to beexplored further.

Conclusion

Bacteria-mediated bioremediation is an emerging field andshould be studied extensively in order to achieve results forthe betterment of the environment. Though bacteria developmetal-resistant genotypes as a mode of adaptation in the con-taminated environments, the gene pool of these resistant bac-teria can be studied extensively for a proper insight into thegenetic approaches for metal bioremediation. Many importantmetal detoxification genes have been discovered; however,owing to the vast majority of the microbial diversity and en-vironmental conditions, a complete understanding of the mi-crobial genetic machineries is required to develop a suitabletoxic metal bioremediation strategy.

Acknowledgments Authors would like to acknowledge the authoritiesof NIT, Rourkela, for providing facilities. Financial supports received byS.D. through the research projects from the Department ofBiotechnology, Ministry of Science and Technology, Government ofIndia, on various aspects of microbial bioremediation of organic andinorganic contaminants is gratefully acknowledged.

Compliance with ethical standards

Conflict of interest The authors declare no conflict of interest.

Ethical approval This article does not contain any studies with humanparticipants or animals performed by any of the authors.

References

Abdel-Monem MO, Al-Zubeiry AH, Al-Gheethi AA (2010) Biosorptionof nickel by Pseudomonas cepacia 120S and Bacillus subtilis 117S.Water Sci Technol 61(12):2994–3007

Ackerley DF, Gonzalez CF, Keyhan M, Blake R, Matin A (2004)Mechanism of chromate reduction by the Escherichia coli protein,NfsA, and the role of different chromate reductases in minimizingoxidative stress during chromate reduction. Environ Microbiol 6:851–860

Albarracin VH, Avila AL, Amoroso MJ, Abate CM (2008) Copper re-moval ability by Streptomyces strains with dissimilar growth pat-terns and endowed with cupric reductase activity. FEMSMicrobiol Lett 288:141–148

Arora PK, Srivastava A, Singh VP (2010) Application ofmonooxygenases in dehalogenation, desulphurization, denitrifica-tion and hydroxylation of aromatic compounds. J BioremedBiodegrad 1:112. doi:10.4172/2155-6199.1000112

Balasubramanian R, Kenney GE, Rosenzweig AC (2011) Dual pathwaysfor copper uptake by methanotrophic bacteria. J Biol Chem 286:37313–37319

Barkay T,Wagner-Dobler I (2005)Microbial transformations of mercury:potentials, challenges, and achievements in controlling mercury tox-icity in the environment. Adv Appl Microbiol 57(1):1–52

Barkay T, Miller SM, Summers AO (2003) Bacterial mercury resistancefrom atoms to ecosystems. FEMS Microbiol Rev 27:355–384

Batool R, Yrjala K, Hasnain S (2012) Hexavalent chromium reduction bybacteria from tannery effluent. J Microbiol Biotechnol 22:547–554

Bender CL, Cooksey DA (1986) Indigenous plasmids in Pseudomonassyringae pv. tomato: conjugative transfer and role in copper resis-tance. J Bacteriol 165:534–541

Bhaskar PV, Bhosle NB (2006) Bacterial extracellular polymeric sub-stances (EPS) a carrier of heavy metals in the marine food-chain.Environ Int 32:192–198

Blindauer CA, HarrisonMD, RobinsonAK, Parkinson JA, Bowness PW,Sadler PJ, Robinson NJ (2002) Multiple bacteria encodemetallothioneins and SmtA-like zinc fingers. Mol Microbiol 45(5):1421–1432

Bondarczuk K, Piotrowska-Seget Z (2013) Molecular basis of activecopper resistance mechanisms in Gram-negative bacteria. CellBiol Toxicol 29:397–405

Borremans B, Hobman JL, Provoost A, Brown NL, van Der Lelie D(2001) Cloning and functional analysis of the pbr lead resistancedeterminant of Ralstonia metallidurans CH34. J Bacteriol 183:5651–5658

Boyd ES, Barkay T (2012) Themercury resistance operon: from an originin a geothermal environment to an efficient detoxification machine.Front Microbiol 3. doi:10.3389/fmicb.2012.00349

Bramhachari PV, Kavi Kishor PB, Ramadevi R, Kumar R, Rao BR,Dubey SK (2007) Isolation and characterization of mucousexopolysaccharide produced by Vibrio furnissii VB0S3. JMicrobiol Biotechnol 17:44–51

Branco R, ChungAP, Johnston T, Gurel V,Morais P, Zhitkovich A (2008)The chromate-inducible chrBACF operon from the transposable


element TnOtChr confers resistance to chromium (VI) and superox-ide. J Bacteriol 190(21):6996–7003

BrimH,McFarlan SC, Fredrickson JK,MintonKW, ZhaiM,Wackett LP,Daly MJ (2000) Engineering Deinococcus radiodurans for metalremediation in radioactive mixed waste environments. NatBiotechnol 18:85–90

BrimH,VenkateswaranA,Kostandarithes HM, Fredrickson JK, DalyMJ(2003) Engineering Deinococcus geothermalis for bioremediationof high-temperature radioactive waste environments. Appl EnvironMicrobiol 69:4575–4582

Brunke M, Deckwer WD, Frischmuth A, Horn JM, Lünsdorf H, RhodeM, Röhricht M, Timmis KN, Weppen P (1993) Microbial retentionof mercury from waste streams in a laboratory column containingmerA gene bacteria. FEMS Microbiol Rev 11(1–3):145–152

Cervantes C, Campos-García J (2007) Reduction and efflux of chromateby bacteria. In: molecular microbiology of heavy metals. SpringerBerlin, Heidelberg, pp. 407–419

Chakraborty J, Das S (2014) Characterization and cadmium-resistantgene expression of biofilm-forming marine bacteriumPseudomonas aeruginosa JP-11. Environ Sci Pollut Res. doi:10.1007/s11356-014-3308-7

Chang FM, Coyne HJ, Cubillas C, Vinuesa P, Fang X, Ma Z, Ma D,Helmann JD, García-de los Santos A, Wang YX, Dann CE3rd,Giedroc DP (2014) Cu(I)-mediated allosteric switching in acopper-sensing operon repressor (CsoR). J Biol Chem 289(27):19204–19217

Chaouni LBA, Etienne J, Greenland T, Vandenesch F (1996) Nucleic acidsequence and affiliation of pLUG10, a novel cadmium resistanceplasmid from Staphylococcus lugdunensis. Plasmid 36:1–8

Chaturvedi R, Archana G (2014) Cytosolic expression of syntheticphytochelatin and bacterial metallothionein genes in Deinococcusradiodurans R1 for enhanced tolerance and bioaccumulation ofcadmium. Biometals 27(3):471–482

Chaturvedi KS, Hung CS, Crowley JR, Stapleton AE, Henderson JP(2012) The siderophore yersiniabactin binds copper to protect path-ogens during infection. Nat Chem Biol 8:731–736

Chaturvedi KS, Hung CS, Giblin DE, Urushidani S, Austin AM, DinauerMC, Henderson JP (2014) Cupric yersiniabactin is a virulence-associated superoxide dismutase mimic. ACS Chem Biol 9:551–561

Chihomvu P, Stegmann P, Pillay M (2015) Characterization and structureprediction of partial length protein sequences of pcoA, pcoR andchrB genes from heavy metal resistant bacteria from the KlipRiver, South Africa. Int J Mol Sci 16:7352–7374

Chillappagari S, Miethke M, Trip H, Kuipers OP, Marahiel MA (2009)Copper acquisition is mediated by YcnJ and regulated by YcnK andCsoR in Bacillus subtilis. J Bacteriol 191:2362–2370

Cooksey DA, Azad HR, Cha JS, Lim CK (1990) Copper resistance genehomologs in pathogenic and saprophytic bacterial species from to-mato. Appl Environ Microbiol 56:431–435

Crupper SS, Worrell V, Stewart GC, Iandolo JJ (1999) Cloning and ex-pression of cadD, a new cadmium resistance gene of Staphylococcusaureus. J Bacteriol 181:4071–4075

Das S (2014) Microbial biodegradation and bioremediation. Elsevier,USA, p. 634

Das S, Raj R, Mangwani N, Dash HR, Chakraborty J (2014a) Heavymetals and hydrocarbons: adverse effects and mechanism of toxic-ity. In: Das S (ed) Microbial Biodegradation and Bioremediation.Elsevier, USA, pp. 23–54

Das S, Dash HR, Mangwani N, Chakraborty J, Kumari S (2014b)Understanding molecular identification and polyphasic taxonomicapproaches for genetic relatedness and phylogenetic relationships ofmicroorganisms. J Microbiol Meth 103:80–100

Das P, Sinha S,Mukherjee SK (2014c) Nickel bioremediation potential ofBacillus thuringiensis KUNi1 and some environmental factors innickel removal. Bioremediation J 18(2):169–177

DashHR, Das S (2012) Bioremediation ofmercury and the importance ofbacterial mer genes. Int Biodet Biodeg 75:207–213

Dash HR, Das S (2015) Enhanced bioremediation of inorganic mercurythrough simultaneous volatilization and biosorption by transgenicmarine bacterium Bacillus cereus BW-03(pPW-05). Int BiodeteriorBiodegrad 103:179–185

Dash HR, Mangwani N, Chakraborty J, Kumari S, Das S (2013) Marinebacteria: potential candidates for enhanced bioremediation. ApplMicrobiol Biotechnol 97:561–571

Dash HR, Mangwani N, Das S (2014) Characterization and potentialapplication in mercury bioremediation of highly mercury-resistantmarine bacterium Bacillus thuringiensis PW-05. Environ Sci PollutRes 21(4):2642–2653

De J, Ramaiah N, Bhosle NB, Garg A, Vardanyan L, Nagle VL, FukamiK (2007) Potential of mercury resistant marine bacteria for detoxi-fication of chemicals of environmental concern. Microbes Environ22:336–345

De J, Ramaiah N, Vardanyan L (2008) Detoxification of toxic heavymetals by marine bacteria highly resistant to mercury. MarBiotechnol 10:471–477

Deng X, Li QB, Lu YH, Sun DH, Huang YL, Chen XR (2003)Bioaccumulation of nickel from aqueous solutions by geneticallyengineered Escherichia coli. Water Res 37(10):2505–2511

Diaz-Magana A, Aguilar-Barajas E, Moreno-Sánchez R, Ramírez-Díaz MI, Riveros-Rosas H, Vargas E, Cervantes C (2009)Short-chain chromate ion transporter proteins from Bacillussubtilis confer chromate resistance in Escherichia coli. JBacteriol 191:5441–5445

Dixit R, Malaviya D, Pandiyan K, Singh UB, Sahu A, Shukla R, SinghBP, Rai JP, Sharma PK, Lade H, Paul D (2015) Bioremediation ofheavy metals from soil and aquatic environment: an overview ofprinciples and criteria of fundamental processes. Sustainability7(2):2189–2212

Djoko KY, Chong LX, Wedd AG, Xiao Z (2010) Reaction mechanismsof the multicopper oxidase CueO from Escherichia coli support itsfunctional role as a cuprous oxidase. J Am Chem Soc 132:2005–2015

Duprey A, Chansavang V, Frémion F, Gonthier C, Louis Y, Lejeune P,Dorel C (2014) BNiCo Buster^: engineering E. coli for fast andefficient capture of cobalt and nickel. J Biol Eng 8(1):1–11

Eriksson PO, Sahlman L (1993) 1H NMR studies of the mercuric ionbinding protein MerP: sequential assignment, secondary structureand global fold of oxidized MerP. J Biomol NMR 3:613–626

Franke S, Grass G, Rensing C, Nies DH (2003) Molecular analysis of thecopper-transporting efflux system CusCFBA of Escherichia coli. JBacteriol 185(13):3804–3812

Ge Z, Taylor DE (1996) Helicobacter pylori genes hpcopA and hpcopPconstitute a cop operon involved in copper export. FEMSMicrobiolLett 145:181–188

Gee AR, Dudeney AWL (1998) Adsorption and crystallization of gold atbiological surfaces. In PR Norris, DP Kelly (Eds). Proceedings ofthe international symposium on Biohydrometallurgy (pp 437–451)

Golby S, Ceri H, Marques LLR, Turner RJ (2014) Mixed-speciesbiofilms cultured from an oil sand tailings pond can biomineralizemetals. Microb Ecol 68:70–80

Gonzalez CF, Ackerley DF, Lynch SV, Matin A (2005) ChrR, a solublequinone reductase of Pseudomonas putida that defends againstH2O2. J Biol Chem 280:22590–22595

Grass G, Große C, Nies DH (2000) Regulation of the cnr cobalt andnickel resistance determinant from Ralstonia sp. strain CH34. JBacteriol 182:1390–1398

Grass G, Thakali K, Klebba PE, Thieme D, Muller A, Wildner GF,Rensing C (2004) Linkage between catecholate siderophores andthe multicopper oxidase CueO in Escherichia coli. J Bacteriol186:5826–5833


Grass G, Fricke B, Nies DH (2005) Control of expression of a periplasmicnickel efflux pump by periplasmic nickel concentrations. Biometals18:437–448

Guo H, Luoa S, Chen L, Xiao X, Xi Q, Wei W, Zeng G, Liu C, Wan Y,Chen J, He Y (2010) Bioremediation of heavy metals by growinghyperaccumulaor endophytic bacterium Bacillus sp. L14. BioresourTechnol 101:8599–8606

Hakansson T, Suer P, Mattiasson B, Allard B (2008) Sulphate reducingbacteria to precipitate mercury after electrokinetic soil remediation.Int J Environ Sci Technol 5(2):267–274

Hamlett NV, Landale EC, Davis BH, Summers AO (1992) Roles of theTn21 merT, merP, and merC gene products in mercury resistanceand mercury binding. J Bacteriol 174(20):6377–6385

Henne KL, Nakatsu CH, Thompson DK, Konopka AE (2009) High-levelchromate resistance in Arthrobacter sp. strain FB24 requires previ-ously uncharacterized accessory genes. BMC Microbiol 9:199

Hinojosa BM, Garcia-Ruiz R, Carreira JA (2010) Utilizing microbialcommunity structure and function to evaluate the health of heavymetal polluted soils. In Soil HeavyMetals by Sherameti I, Varma A.Chapter 9

Hsieh PF, Lin HH, Lin TL, Wang JT (2010) CadC regulates cad and tdcoperons in response to gastrointestinal stresses and enhances intes-tinal colonization of Klebsiella pneumoniae. J Infect Dis 202:52–64

Hu YH, Wang HL, Zhang M, Sun L (2009) Molecular analysis of thecopper-responsive CopRSCD of a pathogenic Pseudomonasfluorescens strain. J Microbiol 47:277–286

Hynninen A, Touze T, Pitkänen L, Mengin-Lecreulx D, Virta M (2009)An efflux transporter PbrA and a phosphatase PbrB cooperate in alead-resistance mechanism in bacteria. Mol Microbiol 74:384–394

Jarosławiecka A, Piotrowska-Seget Z (2014) Lead resistance in micro-organisms. Microbiology 160:12–25

Johnsen AR, Wick LY, Harms H (2005) Principles of microbial PAH-degradation in soil. Environ Pollut 133:71–84

Juhnke S, Peitzsch N, Hübener N, Grobe C, Nies DH (2002) New genesinvolved in chromate resistance in Ralstonia metallidurans strainCH34. Arch Microbiol 179:15–25

Kalyaeva ES, Kholodii GY, Bass IA, Gorlenko ZM, Yurieva OV,Nikiforov VG (2001) Tn5037, a Tn21-like mercury resistance trans-poson from Thiobacillus ferrooxidans. Rus J Gen 37:972–975

Kamaludeen SPB, Arunkumar KR, Avudainayagam SA, Ramasamy K(2003) Bioremediation of chromium contaminated environments.Indian J Exp Biol 41:972–985

Kanehisa M, Goto S (2000) KEGG: kyoto encyclopedia of genes andgenomes. Nucleic Acids Res 28:27–30

Kang SH, Singh S, Kim JY, Lee W, Mulchandani A, Chen W (2007)Bacteria metabolically engineered for enhanced phytochelatin pro-duction and cadmium accumulation. Appl Environ Microbiol 73:6317–6320

Karp PD, Ouzounis CA, Moore-Kochlacs C, Goldovsky L, Kaipa P,Ahrén D, Ópez-Bigas N (2005) Expansion of the BioCyc collectionof pathway/genome databases to 160 genomes. Nucleic Acids Res33:6083–6089

Kermani AJN, GhasemiMF, Khosravan A, Farahmand A, ShakibaieMR(2010) Cadmium bioremediation bymetal-resistantmutated bacteriaisolated from active sludge of industrial effluent. Ira J EnvironHealth Sci Eng 7(4):279–286

Khan F, Sajid M, Cameotra SS (2013) In silico approach for the biore-mediation of toxic pollutants. J Pet Environ Biotechnol 4:2

Khan Z, Nisar MA, Hussain SZ, Arshad MN, Rehman A (2015)Cadmium resistance mechanism in Escherichia coli P4 and its po-tential use to bioremediate environmental cadmium. ApplMicrobiolBiotechnol 99(24):10745–10757

Kiyono M, Pan-Hou H (1999) The merG gene product is involved inphenylmercury resistance in Pseudomonas strain K-62. J Bacteriol181:726–730

KiyonoM,OkaY, Sone Y, Nakamura R, SatoMH, SakabeK, Pan-HouH(2013) Bacterial heavy metal transporter MerC increases mercuryaccumulation in Arabidopsis thaliana. Biochem Engin J 71:19–24

Klaassen CD, Liu SCJ (1999) Metallothionein: an intracellular protein toprotect against cadmium toxicity. Ann Rev Pharmacol Toxicol 39:267–294

Kornberg A (1995) Inorganic polyphosphate: toward making a forgottenpolymer unforgettable. J Bacteriol 177:491–496

Kratchovil D, Volesky B (1998) Advances in the biosorption of heavymetals. Trends Biotechnol 16:291–300

Letunic I, Copley RR, Pils B, Pinkert S, Schultz J, Bork P (2006) SMART5: domains in the context of genomes and networks. Nucleic AcidsRes 34:D257–D260

Lorenzo V, Herrero M, Sánchez JM, Timmis KN (1998) Mini-transposons in microbial ecology and environmental biotechnology.FEMS Microbiol Ecol 27:211–224

Lovley DR, Coates JD (1997) Bioremediation of metal contamination.Curr Opin Biotechnol 8:285–289

Macomber L, Imlay JA (2009) The iron-sulfur clusters of dehydratasesare primary intracellular targets of copper toxicity. Proc Natl AcadSci U S A 106:8344–8349

Marzorati M, Balloi A, De Ferra F, Daffonchio D (2010) Identification ofmolecular markers to follow up the bioremediation of sites contam-inated with chlorinated compounds. Methods Mol Biol 668:219–134

Mellano MA, Cooksey DA (1988) Induction of the copper resistanceoperon from Pseudomonas syringae. J Bacteriol 170:4399–4401

Mindlin S, Kholodii G, Gorlenko Z, Minakhina S, Minakhin L, KalyaevaE, Nikiforov V (2001) Mercury resistance transposons of gram-negative environmental bacteria and their classification. ResMicrobiol 152:811–822

Mishra R, Sinha V, Kannan A, Upreti RK (2012) Reduction ofchromium-VI by chromium resistant Lactobacilli: a prospectivebacterium for bioremediation. Toxicol Int 19:25–30

Monchy S, Benotmane MA, Janssen P, Vallaeys T, Taghavi S, van derLelie D, Mergeay M (2007) Plasmids pMOL28 and pMOL30 ofCupriavidus metallidurans are specialized in the maximal viableresponse to heavy metals. J Bacteriol 189(20):7417–7425

Moore MJ, Distefano MD, Zydowsky LD, Cummings RT, Walsh CT(1990) Organomercurial lyase and mercuric ion reductase: nature’smercury detoxification catalysts. Acc Chem Res 23:301–308

Morais PV, Branco R, Francisco R (2011) Chromium resistance strategiesand toxicity: what makes Ochrobactrum tritici 5bvl1 a strain highlyresistant. Biometals 24:401–410

Morillo JA, Garcia-Ribera R, Quesada T, Aguilera M, Ramos-Cormenzana A, Monteoliva-Sanchez M (2008) Biosorption ofheavy metals by the exopolysaccharide produced by Paenibacillusjamilae. World J Microbiol Biotechnol 24:2699–2704

Munson GP, Lam DL, Outten FW, O’Halloran TV (2000) Identificationof a copper-responsive two-component system on the chromosomeof Escherichia coli K-12. J Bacteriol 182:5864–5871

Naik MM, Dubey SK (2011) Lead-enhanced siderophore production andalteration in cell morphology in a Pb-resistant Pseudomonasaeruginosa strain 4EA. Curr Microbiol 62:409–414

NaikMM, PandeyA,Dubey SK (2012a)Pseudomonas aeruginosa strainWI-1 from Mandovi estuary possesses metallothionein to alleviatelead toxicity and promotes plant growth. Ecotoxicol Environ Safety79:129–133

Naik MM, Shamim K, Dubey SK (2012b) Biological characterization oflead resistant bacteria to explore role of bacterial metallothioneininlead resistance. Curr Sci 103:1–3

Nascimento AM, Chartone-Souza E (2003) Operon mer: bacterial resis-tance to mercury and potential for bioremediation of contaminatedenvironments. Gen Mol Res 2(1):92–101


Naser HA (2013) Assessment and management of heavy metal pollutionin the marine environment of the Arabian Gulf: a review. Mar PollutBull 72:6–13

Nies DH (2003) Efflux-mediated heavy metal resistance in prokaryotes.FEMS Microbiol Rev 27:313–339

Nies DH, Koch S, Wachi S, Peitzsch N, Saier MH (1998) CHR, a novelfamily of prokaryotic proton motive force-driven transporters prob-ably containing chromate/sulfate antiporters. J Bacteriol 180:5799–5802

Niti C, Sunita S, Kamlesh K, Rakesh K (2013) Bioremediation: Anemerging technology for remediation of pesticides. Res J ChemEnviron 17:88–105

Nucifora G, Chu L, Misra TK, Silver S (1989) Cadmium resistance fromStaphylococcus aureus plasmid pI258 cadA gene results from acadmium-efflux ATPase. Proc Nat Acad Sci 86:3544–3548

Obenauer JC, Cantley LC, Yaffe MB (2003) Scansite 2.0: proteome-wideprediction of cell signalling interactions using short sequence motifs.Nucleic Acids Res 31:3635–3641

Osborn AM, Bruce KD, Strike P, Ritchie DA (1997) Distribution, diver-sity and evolution of the bacterial mercury resistance (mer) operon.FEMS Microbiol Rev 19:239–262

Pal A, Paul AK (2008) Microbial extracellular polymeric substances:central elements in heavy metal bioremediation. Indian JMicrobiol 48:49–64

Pan-Hou HS, Imura N (1981) Role of hydrogen sulfide in mercury resis-tance determined by plasmid of Clostridium cochlearium T-2. ArchMicrobiol 129(1):49–52

Pan-Hou H, Kiyono M, Omura T, Endo G (2002) Polyphosphate pro-duced in recombinant Escherichia coli confers mercury resistance.FEMS Microbiol Lett 207:159–164

Park CH, KeyhanM,Wielinga B, Fendorf S,Matin A (2000) Purificationto homogeneity and characterization of a novelPseudomonas putidachromate reductase. Appl Environ Microbiol 66:1788–1795

Park JH, Bolan N, Meghraj M, Naidu N (2011) Concomitant rock phos-phate dissolution and lead immobilization by phosphate solubilisingbacteria (Enterobacter sp.). J Environ Manag 92:1115–1120

Patra RC, Malik S, Beer M, Megharaj M, Naidu R (2010) Molecularcharacterization of chromium (VI) reducing potential in Gram pos-itive bacteria isolated from contaminated sites. Soil Biol Biochem42(10):1857–1863

Paul D, Pandey G, Pandey J, Jain RK (2005) Accessing microbial diver-sity for bioremediation and environmental restoration. TrendsBiotechnol 23:135–142

Pernthaler A, Pernthaler J, Amann R (2002) Fluorescence in situ hybrid-ization and catalyzed reporter deposition for the identification ofmarine bacteria. Appl Environ Microbiol 68:3094–3101

Perry RD, Silver S (1982) Cadmium and manganese transport inStaphylococcus aureus membrane vesicles. J Bacteriol 150:973–976

Pieper DH, Reineke W (2000) Engineering bacteria for bioremediation.Curr Opin Biotechnol 11:262–270

Plette AC, Benedetti MF, van RiemsdijkWH (1996) Competitive bindingof protons, calcium, cadmium, and zinc to isolated cell walls of agram-positive soil bacterium. Environ Sci Technol 30(6):1902–1910

Raj R, Dalei K, Chakraborty J, Das S (2016) Extracellular polymericsubstances of a marine bacterium mediated synthesis of CdS nano-particles for removal of cadmium from aqueous solution. J ColloidInterf Sci 462:166–175

Ravel J, Diruggiero J, Robb FT, Hill RT (2000) Cloning and sequenceanalysis of the mercury resistance operon of Streptomyces sp. strainCHR28 reveals a novel putative second regulatory gene. J Bacteriol182:2345–2349

Rebello RCL, Gomes KM, Duarte RS, Rachid CTCC, Rosado AS,Regua-Mangia AH (2013) Diversity of mercury resistant

Escherichia coli strains isolated from aquatic systems in Rio deJaneiro. Brazil Int J Biodiv. doi:10.1155/2013/265356

Roane TM (1999) Lead resistance in two bacterial isolates from heavymetal-contaminated soils. Microbial Ecol 37:218–224

Roane TM, Josephson KL, Pepper IL (2001) Dual-bioaugmentation strat-egy to enhance remediation of cocontaminated soil. Appl EnvironMicrobiol 67(7):3208–3215

Robinson NJ, Gupta A, Fordham-Skelton AP, Croy RRD, Whitton BA,Huckle JW (1990) Prokaryotic metallothionein gene characteriza-tion and expression: chromosome crawling by ligation-mediatedPCR. Proc R Soc London B 242:241–247

Rodrigue A, Effantin G, Mandrand-Berthelot MA (2005) Identificationof rcnA (yohM), a nickel and cobalt resistance gene in Escherichiacoli. J Bacteriol 187:2912–2916

Rojas LA, Yáñez C, González M, Lobos S, Smalla K, Seeger M (2011)Characterization of themetabolically modified heavymetal-resistantCupriavidus metallidurans strain MSR33 generated for mercurybioremediation. PLoS one 6:e17555

Ronchel MC, Ramos C, Jensen LB, Molin S, Ramos JL (1995)Construction and behaviour of biologically contained bacteria forenvironmental adaptations in bioremediation. Appl EnvironMicrobiol 61:2990–2994

Rosner JL, Aumercier M (1990) Potentiation by salicylate and salicylalcohol of cadmium toxicity and accumulation in Escherichia coli.Appl Environ Microbiol 34:2402–2406

Ruiz ON, Alvarez D, Gonzalez-Ruiz G, Torres C (2011) Characterizationof mercury bioremediation by transgenic bacteria expressing metal-lothionein and polyphosphate kinase. BMC Biotechnol 11:82

Ryan RP, Monchy S, Cardinale M, Taghavi S, Crossman L, Avison MB,Berg G, van der Lelie D, Dow JM (2009) The versatility and adap-tation of bacteria from the genus Stenotrophomonas. Nat RevMicrobiol 7:514–525

Sandaa RA, Torsvik V, Enger O, Daae FL, Castberg T, Hahn D (1999)Analysis of bacterial communities in heavy metal-contaminatedsoils at different levels of resolution. FEMS Microbiol Ecol 30:237–251

Sandrin TR, Maier RM (2003) Impact of metals on the biodegradation oforganic pollutants. Environ Health Perspect 111(8):1093

Sasaki Y, Hayakawa T, Inoue C, Miyazaki A, Silver S, Kusano T (2006)Generation of mercury-hyperaccumulating plants through transgen-ic expression of the bacterial mercury membrane transport proteinMerC. Transgen Res 15:615–625

Sathyavathi S, Manjula A, Rajendhran J, Gunasekaran P (2014)Extracellular synthesis and characterization of nickel oxide nanopar-ticles from Microbacterium sp. MRS-1 towards bioremediation ofnickel electroplating industrial effluent. Bioresour Technol 165:270–273

Schaefer JK, Rocks SS, Zheng W, Liang L, Gu B, Morel FMM (2011)Active transport, substrate specificity, and methylation of Hg(II) inanaerobic bacteria. Proc Nat Acad Sci USA 108:8714–8719

Schelert J, Drozda M, Dixit V, Dillman A, Blum P (2006) Regulation ofmercury resistance in the crenarchaeote Sulfolobus solfataricus. JBacteriol 188:7141–7150

Schelert J, Rudrappa D, Johnson T, Blum P (2013) Role of MerH inmercury resistance in the archaeon Sulfolobus solfataricus.Microbiol 159:1198–1208

Schmidt T, Schlegel HG (1994) Combined nickel-cobalt-cadmium resis-tance encoded by the ncc locus of Alcaligenes xylosoxidans 31A. JBacteriol 176:7045-7054

Schneiker S, Keller M, Dröge M, Lanka E, Pühler A, Selbitschka W(2001) The genetic organization and evolution of the broad hostrange mercury resistance plasmid pSB102 isolated from a microbialpopulation residing in the rhizosphere of alfalfa. Nuc Acids Res 29:5169–5181

Schue M, Dover LG, Besra GS, Parkhill J, Brown NL (2009) Sequenceand analysis of a plasmid-encoded mercury resistance operon from


Mycobacteriummarinum identifiesMerH, a newmercuric ion trans-porter. J Bacteriol 191:439–444

Silver S, Misra TK (1984) Bacterial transformations of and resistances toheavy metals. In: Genetic control of environmental pollutants.Springer USA, pp. 23–46

Silver S, Phung LT (2013) Bacterial mercury resistance proteins.Encyclopedia of Metalloproteins 209-217

Singh SK, Grass G, Rensing C, Montfort WR (2004) Cuprous oxidaseactivity of CueO from Escherichia coli. J Bacteriol 86:7815–7817

Smith TC (2005) Economy, speed and size matter: evolutionary forcesdriving nuclear genomeminiaturization and expansion. Ann Bot 95:147–175

Smith K, Novick RP (1972) Genetic studies on plasmid-linked cadmiumresistance in Staphylococcus aureus. J Bacteriol 112:761–772

Stahler FN, Odenbreit S, Haas R, Wilrich J, Van Vliet AH, Kusters JG,Kist M, Bereswill S (2006) The novelHelicobacter pylori CznABCmetal efflux pump is required for cadmium, zinc, and nickel resis-tance, ureasemodulation, and gastric colonization. Infect Immun 74:3845–3852

Taghavi S, Lesaulnier C,Monchy S,Wattiez R,MergeayM, van der LelieD (2009) Lead(II) resistance in Cupriavidus metallidurans CH34:interplay between plasmid and chromosomally-located functions.Antonie Van Leeuwenhoek 96:171–182

Tebo BM, Obraztova AY (1998) Sulfate-reducing bacterium grows withCr(VI), U(VI), Mn(IV), and Fe(III) as electron acceptors. FEMSMicrobiol Lett 162:193–198

Tetaz TJ, Luke RK (1983) Plasmid-controlled resistance to copper inEscherichia coli. J Bacteriol 154:1263–1268

Tibazarwa C, Wuertz S, Mergeay M, Wyns L, van Der Lelie D (2000)Regulation of the cnr cobalt and nickel resistance determinant ofRalstonia eutropha (Alcaligenes eutrophus) CH34. J Bacteriol182(5):1399–1409

Timmis KN, Pieper DH (1999) Bacteria designed for bioremediation.Trends Biotechnol 17:201–204

Trepreau J, de Rosny E, Duboc C, Sarret G, Petit-Hartlein I, Maillard AP,Covès J (2011) Spectroscopic characterization of the metal-bindingsites in the periplasmic metal-sensor domain of CnrX fromCupriavidus metallidurans CH34. Biochem 50:9036–9045

Trevors JT, Stratton GW, Gadd GM (1986) Cadmium transport, resis-tance, and toxicity in bacteria, algae, and fungi. Can J Microbiol32:447–464

Valls M, de Lorenzo V (2002) Exploiting the genetic and biochemicalcapacities of bacteria for the remediation of heavy metal pollution.FEMS Microbiol Rev 26:327–338

Vargas-García MC, López MJ, Suárez-Estrella F, Moreno J (2012)Compost as a source of microbial isolates for the bioremediationof heavy metals: In vitro selection. Sci Total Environ 431:62–67

Vats N, Lee SF (2001) Characterization of a copper-transport operon,copYAZ, from Streptococcus mutans. Microbiol 147:653–662

Vieira RH, Volesky B (2010) Biosorption: a solution to pollution? IntMicrobiol 3:17–24

Vijayaraghavan K, Yun YS (2008) Bacterial biosorbents and biosorption.Biotechnol Adv 26:266–291

Viti C, Marchi E, Decorosi F, Giovannetti L (2013) Molecular mecha-nisms of Cr (VI) resistance in bacteria and fungi. FEMS MicrobiolRev 38:633–659

Von Mering C, Jensen CJ, Kuhn M, Chaffron S, Doerks M, Krüger B,Bork P (2007) STRING 7-recent developments in the integrationand prediction of protein interactions. Nucleic Acids Res 35:D358–D362

von Rozycki T, Nies DH (2009) Cupriavidus metallidurans: evolution ofa metal-resistant bacterium. Antonie Van Leeuwenhoek 96(2):115–139

Wagner-Dobler I (2003) Pilot plant for bioremediation of mercury-containing industrial wastewater. Appl Microbiol Biotechnol62(2–3):124–133

Williams GP, GnanadesiganM, Ravikumar S (2012) Biosorption and bio-kinetic studies of halobacterial strains against Ni2+, Al3+ and Hg2+

metal ions. Bioresour Technol 107:526–529Wu CH, Wood TK, Mulchandani A, Chen W (2006) Engineering plant-

microbe symbiosis for rhizoremediation of heavy metals. ApplEnvironmental Microbiol 72:1129–1134

Wunderli-Ye H, Solioz M (1999) Copper homeostasis in Enterococcushirae. In: Copper Transport and Its Disorders. Springer USA. pp.255–264

Xue XM, Yan Y, Xu HJ, Wang N, Zhang X, Ye J (2014) ArsH fromSynechocystis sp. PCC 6803 reduces chromate and ferric iron.FEMS Microbiol Lett 356:105–112

Yoon KP, Silver S (1991) A second gene in the Staphylococcus aureuscadA cadmium resistance determinant of plasmid pI258. J Bacteriol173:7636–7642

Yu P, Yuan J, Deng X, Ma M, Zhang H (2014) Subcellular targeting ofbacterial CusF enhances Cu accumulation and alters root to shootCu translocation in Arabidopsis. Plant Cell Physiol 55(9):1568–1581

Zhang YM, Yin H, Ye JS, Peng H, Zhang N, Qin HM, Yang F, He BY(2007) Cloning and expression of the nickel/cobalt transferase genein E. coli BL21 and bioaccumulation of nickel ion by geneticallyengineered strain. Huan Jing Ke Xue 28(4):918–923

Zhang W, Chen L, Liu D (2012) Characterization of a marine-isolatedmercury resistant Pseudomonas putida strain SP1 and its potentialapplication in marine mercury reduction. Appl MicrobiolBiotechnol 93:1305–1314

Zhang H, Zhou Y, Bao H, Zhang L, Wang R, Zhou X (2015) Plasmid-borne cadmium resistant determinants are associated with the sus-ceptibility of Listeria monocytogenes to bacteriophage. MicrobiolRes 172:1–6


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