Das 2016

Appl Microbiol Biotechnol
DOI 10.1007/s00253-016-7364-4
MINI-REVIEW
Genetic basis and importance of metal resistant genes in bacteria

for bioremediation of contaminated environments with toxic
metal pollutants
Surajit Das 1 & Hirak R. Dash 1 & Jaya Chakraborty 1
Received: 5 October 2015 / Revised: 26 January 2016 / Accepted: 28 January 2016

# Springer-Verlag Berlin Heidelberg 2016
Abstract Metal pollution is one of the most persistent and Introduction

complex environmental issues, causing threat to the ecosys-
tem and human health. On exposure to several toxic metals Microorganisms are the oldest member of the living systems
such as arsenic, cadmium, chromium, copper, lead, and mer- and possess higher adaptability to thrive in adverse conditions.
cury, several bacteria has evolved with many metal-resistant They are the primary respondant to the changes in environment
genes as a means of their adaptation. These genes can be by altering their genetic system, transfer of genetic elements,
further exploited for bioremediation of the metal- and many other mechanisms for maintaining the ecosystem
contaminated environments. Many operon-clustered metal-re- structure and function (Ryan et al. 2009). They serve in many
sistant genes such as cadB, chrA, copAB, pbrA, merA, and ways for sustenance of the ecosystem such as nutrient cycling,
NiCoT have been reported in bacterial systems for cadmium, primary production, and catabolism of pollutants produced as
chromium, copper, lead, mercury, and nickel resistance and by-products of rapid urbanization and industrialization.
detoxification, respectively. The field of environmental biore- Environmental pollution caused by hazardous metals can
mediation has been ameliorated by exploiting diverse bacterial arise from natural as well as anthropogenic sources. Natural
detoxification genes. Genetic engineering integrated with bio- sources include seepage from rocks into water, forest fires, and
remediation assists in manipulation of bacterial genome which volcanic eruptions. However, vast industrial practices and con-
can enhance toxic metal detoxification that is not usually per- sumerist lifestyle add to the anthropogenic source of metal pol-
formed by normal bacteria. These techniques include genetic lution. The most common metal pollutants in the environment
engineering with single genes or operons, pathway construc- include cadmium, lead, mercury, chromium, arsenic, copper,
tion, and alternations of the sequences of existing genes. vanadium, nickel, molybdenum, and zinc. The toxicity of these
However, numerous facets of bacterial novel metal-resistant metal pollutants causes chronic and degenerative conditions.
genes are yet to be explored for application in microbial bio- The general symptoms of acute metal toxication include head-
remediation practices. This review describes the role of bac- ache, short-term memory loss, mental confusion, gastro-
teria and their adaptive mechanisms for toxic metal detoxifi- intestinal upsets, vision problems, and allergies in humans
cation and restoration of contaminated sites. (Das et al. 2014a). However, chronic exposure to toxic metals
impart many dreadful effects to humans, such as increased can-
Keywords Bioremediation . Metal resistant genes . Bacterial cer risk, disruption of gene expression, and deregulation of cell
diversity . Gene manipulation . Metal resistance growth and development. Continuous increase in the level of
metal pollution in the environment at a greater pace worsens the
situation seeking immediate action (Naser 2013). To combat this
* Surajit Das
surajit@nitrkl.ac.in; surajit@myself.com
persistent environmental problem, recent developments of novel
technologies involving multidisciplinary approaches utilizing
microorganisms for enhanced bioremediation capability have
1
Laboratory of Environmental Microbiology and Ecology (LEnME),
been proposed. This includes biostimulation, bioaugmentation,
Department of Life Science, National Institute of Technology, bioaccumulation, biosorption, phytoremediation, and
Rourkela, Odisha 769 008, India rhizoremediation (Niti et al. 2013).
In this present scenario of incessantly increasing pollution microbial diversity in the environment, there is no such gold
level, microbial bioremediation is an environment-friendly standard available for the assessment of environmental micro-
approach to treat noxious environmental conditions. This is flora for application in bioremediation practices.
way ahead of the conventional methods because it does not Molecular biology tools have been widely exploited for the
alter the natural microenvironment and maintain ecosystem study of microbial ecology. Moreover, there should be a clear
balance. Microorganisms are able to degrade the organic con- understanding of the role of metal-resistant genes from the
taminants present in the environment and subsequently release diverse microbial population prior to their application in con-
harmless products such as CO2, water, and cellular biomass taminated environments (Dash and Das 2012). The advanced
termed biomineralization. However, inorganic toxic metal re- techniques of culture-independent practices take advantage of
moval by the microorganisms is by biosorption to the cell sequencing and in silico approaches for both sequence and
surface with Van der Waals force, covalent binding, redox function-driven screening of genes for bioremediation appli-
interactions, extracellular precipitation, or combination of cation (Khan et al. 2013). In addition, knowing the physico-
these processes. Though many discoveries have taken place, chemical parameters of the environmental conditions, we can
many aspects of exploiting the enormous bioremediation po- manipulate the environmental DNA as well as functional
tential of microorganisms still remain unexplored. Their high RNA to model the species interaction in a community
surface area to volume ratio, presence of extra chromosomal structure.
genetic material, less complex genetic system, higher rate of The complexity and variability of biological organization
adaptation, rapid growth rate, high metabolic activity, super- at different levels is known as microbial diversity. This term
lative enzymatic, and nutritional versatility play a useful role includes the amount and distribution of genetic information
in their implication toward effective bioremediation practices within microbial species in microbial communities and their
(Smith 2005). Genetic system of bacteria provides every op- variation in community structure, complexity associated with
portunity for their survival, gaining energy for subsequent interactions, number of trophic levels, and number of guilds
degradation of the organic contaminants and bioremediation (Hinojosa et al. 2010). Microbes possess the de novo potential
of inorganic toxic metals from the contaminated environments of degrading all naturally occurring compounds through the
(Ronchel et al. 1995). Hence, huge diversity of genes present principle of microbial infallibility (Dash et al. 2013).
in bacterial genomes provides them the opportunity to sustain Microorganisms can degrade organic contaminants by oxidiz-
and subsequently contribute toward sustainable development. ing them to carbon dioxide, whereas in case of toxic metals,
This review aims to provide updated information regarding microorganisms can only change the speciation of metal con-
the exploitation of genetic potential of bacteria for bioremedi- taminants and mobility (Lovley and Coates 1997). So far, only
ation practices of most noxious toxic metals. As bacterial en- a fraction of the total microbial diversity with metabolic po-
tities are more prone to genetic changes due to many mecha- tential has been explored and further exploitation of the
nisms such as gene transfer by conjugation, transformation, overlooked genetic resources will accelerate the remediation
and transduction as well as transposon-mediated events, grad- practices of toxic metals (Naser 2013). In addition, recently
ually new genes of varied functions arise which are of prime developed molecular genetics of bioremediation and
interest for use in treating toxic environmental pollution. knowledge-based approaches of rational protein modification
provides greater insight for the development of designer
biocatalysts for environmental bioremediation (Pieper and
Microbial diversity and bioremediation Reineke 2000; Paul et al. 2005).
Bioremediation is a technique that utilizes microbes to re-

move, neutralize, or detoxify pollutants from the contaminat- Unique mechanisms in bacteria: natural
ed environment. There have been many studies regarding development of resistance toward toxic elements
characterization of bacterial communities, their response to
pollutants, identification of genes responsible for degradation, Bacteria are known to thrive in all types of environments,
and many more (Das 2014). Many reports suggested that the ranging from the frigid poles to the hot deserts, the wet
occurrence of huge varieties of unidentified microbes helping swamps to the aquatic systems. The unique features that they
in bioremediation in the contaminated environment can be possess are their small size, high surface area to volume ratio,
traced only by the culture-independent approaches efficient transfer of genetic traits, and adaptability. In the case
(Marzorati et al. 2010). 16S rRNA gene analysis has revolu- of organic pollutants, bacteria generally degrade them into
tionized the study of microbial diversity in the natural envi- non-toxic products by utilizing them as a source of carbon.
ronment both by culture-dependent as well as culture- However, in the case of inorganic metal pollution, microor-
independent approaches (Das et al. 2014b). Though polypha- ganisms have developed three methods of resistance, namely,
sic approaches have been widely used for the study of efflux of the irritant metals outside the cell by transporters,
transformation of the metals into non-toxic/less toxic forms, include the conversion of arsenate to arsenite, and chromium
and biosorption. Often, biosorption and enzymatic conversion (VI) to its less toxic counterpart chromium (III).
of metal into another form are coupled; i.e., once a metal has However, hazardous waste sites are usually co-
been absorbed into the cell, it is acted upon by enzymes, contaminated with organic compounds and metal pollutants.
which precipitate the metal as a salt (Williams et al. 2012). Bioavailability of metals is determined by medium
The first line of protection from heavy metals is efflux, which composition/soil type and pH and governs the extent to which
involves members of the heavy-metal efflux resistance- metals contradict biodegradation. In a mixed environment of
nodulation cell division (HME-RND) protein superfamily various pollutants, the non-biodegradable metal component is
with efflux pumps (Nies 2003). Another method by which removed or stabilized by mobilization, separation and collec-
bacteria are known to resist as well as remove the harmful tion, off-site transport, and disposal. Metals inhibit the organic
effects of toxic metals is biosorption (Vieira and Volesky pollutant degradation process by interacting with their specific
2010). degradation enzymes and enzymes involved in general metab-
Biosorption simply means the binding of certain sub- olism. The bioavailability of metals is dictated by their inter-
stances, especially contaminants, onto the cellular surface. action with organic compounds. In a report by Rosner and
This involves a solid phase (biosorbent, biological material) Aumercier (1990), a common intermediate of aromatic hydro-
and a liquid phase (solvent) with the dissolved species which carbons, salicylate increased uptake of cadmium and toxicity
is to be sorbed (sorbate, metal ions). The affinity of sorbent- in Escherichia coli. Presence of metals as soluble complexed
sorbate is maintained till equilibrium is established between species and ionic solutes in an environment extends acclima-
the amount of solid-bound sorbate species and its remaining tion periods and reduces the biodegradation rate of com-
part in solution, till the sorbate is removed. In bacteria, sorp- pounds (Sandrin and Maier 2003). In a co-contaminated en-
tion of metals is generally mediated by a family of proteins vironment, the energy requirements to maintain concurrent
called metallothioneins (MTs) which are 0.5–14-kDa proteins, metal resistance and organic degradation is too high; there-
rich in cysteine residues (Blindauer et al. 2002). In some cases, fore, simultaneous activity of bioremediation by microbes is
they may have histidine residues as well. It has already been needed to perform both activities under environmental condi-
reported that microbial metallothioneins act as a Bstorehouse^ tions. Thus, organic pollutant-degrading metal-resistant bac-
for zinc and also protect the cells from cadmium toxicity teria with the properties of biosorption and biotransformation
(Klaassen and Liu 1999). They may also act as a free radical are essential for removal of metal toxicity as well as organic
scavenger and combine with harmful molecules like superox- pollution.
ide and hydroxide ions. Cysteine undergoes oxidation and is
converted to cystine, and bound metals are released into the
environment. The process of biosorption is highly efficient Diversity of metal-resistant genes and biotechniques
with regeneration of biosorbent and metal recovery possibili- in metal-resistant bacteria for bioremediation
ties (Kratchovil and Volesky 1998). However, early saturation
of metal-interactive sites might sometimes be a problem. In the environments contaminated with compounds that are
Bacterial cell wall with different functional groups on the sur- naturally toxic, microorganisms have devised ways not only
face with variety of polysaccharides and proteins act as active to withstand such compounds but also to remediate them for
sites for binding different metal ions. This depends on the their own benefits (Guo et al. 2010). The ability of bacteria to
status of biomass (living or non-living), type of biomaterials, resist toxic metals comes from highly modified genetic sys-
properties of metal solution chemistry, and ambient environ- tems, by means of which they synthesize proteins enabling
mental conditions (Gee and Dudeney 1998). them to thrive in the presence of such elements. Bacteria sur-
Bacteria are known to have the ability to detoxify metals by vive by expressing several metal-resistant genes toward toxic
reducing them to a dissimilar oxidation state. Common reduc- metals such as cadmium, chromium, copper, lead, mercury,
tion mechanisms include the conversion of Hg2+ to Hg0, Cr6+ and nickel. They survive in the highly toxic environment with
to Cr3+, and AsO43− to AsO33−. Metal precipitation can also be the help of these resistant genes which are recruited further for
achieved by metallic phosphate precipitation which is a result bioremediation. Specific genes responsible for bioremediation
of dissimilarity reduction or secondary consequence of meta- of toxic metals discovered so far have been listed in Table 1.
bolic processes related to the transformed metals (Valls and de The focus of the metal selection is the relative abundance of
Lorenzo 2002). The chemical modification of compounds by these metal contaminants in the environments and their degree
biological agents is termed as biotransformation, and produc- of toxicity as indicated by USEPA and the US Department of
tion of CO2, NH4+, and H2O is termed as mineralization. An Labor (https://www.osha.gov/SLTC/metalsheavy/). Bacteria
example of this phenomena is the reduction of inorganic mer- develop resistance to the toxic metals as a part of their
cury (Hg2+) to elemental mercury (Hg0), mediated by mercu- defense mechanism which may be exploited for cleaning the
ric ion reductase (Dash and Das 2012). Other similar reactions contaminated environments (Fig. 1).
Table 1 Operon/Gene clusters involved in conferring toxic metal resistance/detoxification in bacterial system and their specific function
Toxic metal Gene Encoded protein/enzyme Biological function Harbored microorganisms References
Cadmium cadB Cadmium-binding protein It protects the bacterial cell by binding Staphylococcus sp. Perry and Silver 1982
cadmium at cell membrane
Metallothionein Binds cadmium under high cadmium Synechococcus Robinson et al. 1990
stress conditions
cadD Cadmium binding CadD confers a modest level of cadmium Staphylococcus aureus Crupper et al. 1999
resistance by similar action to that of CadB
Chromium chrA Chromate reductase Biotransforms Cr(VI) to the non-toxic Cr(III) Arthrobacter aurescens, Bacillus atrophaeus, Park et al. 2000;
with NADH or NADPH as co-factors Pseudomonas putida, Rhodococcus Patra et al. 2010
Sometimes Cr(VI) is used as an electron acceptor erythropolis Desulfotomaculum reducens Tebo and Obraztova 1998
in the electron transport chain under
anaerobic conditions
Copper cusF Copper accumulation Bind to copper in the periplasmic space E. coli Yu et al. 2014
and enhances copper accumulation
inside the cells
cueO Copper detoxification Oxidizes Cu(I) to a less toxic Cu(II) E. coli Djoko et al. 2010
Lead pbrD Lead binding protein Putative protein, binds with the Pb2+intracellularly Cupriavidus (Ralstonia) metallidurans Borremans et al. 2001
and thus reducing its toxic effect
Mercury merA Mercuric ion reductase Helps in the reduction of Hg2+ to volatile Hg0 Bacillus subtilis, Pseudomonas putida Hamlett et al. 1992;
Zhang et al. 2012;
Dash et al. 2014
merB Organomercurial lyase Responsible for reducing toxic organomercurial Desulfovibrio desulfuricans, Geobacter Osborn et al. 1997;
compounds (methylmercury and phenyl sulfurreducens, Streptomyces sp. Ravel et al. 2000;
mercuric acetate) into nontoxic volatile Schaefer et al. 2011
elemental mercury, i.e., lysis
of C–Hg+ bond
Nickel NiCoT Nickel cobalt transferase Nickel bioaccumulation Cloned in E.coli Deng et al. 2003;
Zhang et al. 2007
Fig. 1 A generalized illustration

of the genetic mechanism of
resistance to toxic metals by
bacteria. Resistance to toxic
metals in bacteria is attributed to
efflux systems, presence of metal-
resistant genes, detoxification
genes, biosorption, or
bioaccumulation
Cadmium genes together provide high level of resistance to cadmium.

cadX was found to be 40 % similar in sequence to cadC. In
Cadmium is a highly toxic non-essential metal, and its effect can turn, the cadX* gene from the pRW001 plasmid is similar in
be disastrous even when present in trace amounts. First report sequence to the cadX gene, but only over the first 78 nucleo-
on cadmium genetics suggested that there are no special mech- tides. Beyond this, it is truncated and hence loses its transcrip-
anisms for cadmium resistance in bacteria (Silver and Misra tion regulation ability. The cadX protein also displays about
1984). However, this was widely disproven by Trevors et al. 30 % homology to the ArsR protein of the ars operon (Yoon
(1986), who studied cadmium transport, resistance, and toxicity and Silver 1991). These are the genetic adaptations by which
in bacteria, algae, and fungi. Cadmium resistance is conferred bacteria resist the cadmium-rich environment.
by plasmid-borne operon named the cad operon, generally In the practical sense, metals cannot be degraded; therefore,
found in Staphylococcus spp., and czc operon in most biological metal remediation approaches are based on
Pseudomonas aeruginosa located on a 3972-bp element detoxification and immobilization of metal which can reduce
(Crupper et al. 1999; Chakraborty and Das 2014). The cad its biological toxicity and also hinder metal transportation.
operon has been found to be a two-component operon Cadmium remediation by bacteria initially involves metal
consisting of the cadA and cadB operons (Zhang et al. 2015). binding with the bacterial cell wall. In vicinity with cadmium,
The cadA operon is carried by the plasmid pI258 (Nucifora et al. calcium ions and protons are released which is an indication of
1989). It carries two genes, cadA and cadC. CadA protein is the competitive binding nature of the cell wall. On surround-
analogous to the ArsB protein of the ars operon and provides ing with bivalent ions, the cell wall becomes positively
protection by forming an energy-dependent ATPase which ef- charged based on pH-dependent charging and metal binding
fluxes cadmium from the bacterial cell. The CadC protein is the data as reported by Plette et al. (1996). Carboxylic and phos-
transcriptional regulator of the operon (Hsieh et al. 2010). phatic sites present on the cell wall of bacteria are used for
The cadB operon encodes a 204-residue polypeptide; the strong coordination with the cadmium ions helping in their
mechanism of action is not yet well elucidated completely. It is site remediation. Gram-negative bacterial cell wall is com-
hypothesized that cadB, located on the plasmid pII147, causes posed of peptidoglycan, phospholipids, and lipopolysaccha-
cellular binding of cadmium, most probably at the plasma rides, of which the highly anionic character and charged na-
membrane (Smith and Novick 1972). Crupper et al. (1999) ture of lipopolysaccharides render metal binding on the cell
reported the presence of another new mechanism of cadmium envelope. Mechanisms include a combination of ion ex-
resistance through the cadD system in the Staphylococcus change, complexation, coordination, adsorption, electrostatic
aureus plasmid pRW001 containing two genes, cadD and interaction, chelation, and microprecipitation (Vijayaraghavan
cadX*. cadD has been found to be similar to the gene cadB. and Yun 2008). In a report by Roane et al. (2001),
cadX* encodes an inactive transcription regulator. However, Pseudomonas strain H1 and Bacillus strain H9 showed an
this operon provides only low-level resistance to cadmium. intracellular mechanism of cadmium sequestration (36 %)
Chaouni et al. (1996) reported the presence of another cadmi- for reducing cadmium toxicity. These strains showed the pro-
um resistance operon in the Staphylococcus lugdunensis plas- duction of exopolymers (EPS) which accumulated cadmium
mid pLUG10. This comprised a cadB-like resistance gene and and reduced soluble cadmium levels by 22 and 11 %,
another transcription regulatory gene named cadX. These two respectively.
Another alternative is the usage of microbial biomass for sequence LFVTPEYNXXXXXX-LKNAIDXXS, which could al-
cadmium bioremediation which is cost effective and environ- so provide additional protection against H2O2. The general chro-
ment friendly. This mechanism is due to independent extra- mate transport reaction involves the family of chromate ion
cellular adsorption by surface complexation, ion exchange, or transporters. Three other genes, chrJ, chrK, and chrL, were
electrostatic interaction leading to intracellular accumulation identified in Arthrobacter sp. strain FB24 (Henne et al. 2009).
by the cell surface (Vargas-García et al. 2012). A study by Viti et al. (2013) proposed that chrJ encodes a putative malate/
Khan et al. (2015) showed the removal of 18.8, 37, and quinone reductase protein, chrK, which specifies a YVTN beta-
56 % Cd+2 from aqueous medium after 48, 96, and 144 h, propeller repeat-containing protein, and chrL forms a probable
respectively. But an increase in Cd2+ concentration in the me- conserved lipoprotein of the LppY/LpqO family.
dium was observed after 192 h, with a decrease in intracellular Another method of chromate detoxification is the enzymat-
accumulation of Cd2+. It is due to the activation of efflux ic reduction of Cr6+ to Cr3+ (Batool et al. 2012). Presence of
system in E.coli for its survival. This illustrates the interde- chromate-reducing enzyme and fast reduction of Cr6+ in
pendence of metal accumulation and efflux system in bacteria. chromium-resistant Lactobacillus strain have proven to be
Another strategy of cadmium remediation is the use of CdS useful for bioremediation of Cr6+ from contaminated environ-
nanoparticles synthesized by functionalized EPS of ment (Mishra et al. 2012). Cervantes and Campos-García
Pseudomonas aeruginosa JP-11, which removed 88.66 % of (2007) broadly classified Cr6+ reduction by three mecha-
cadmium from aqueous solutions (Raj et al. 2016). nisms—aerobic reduction by reductases in presence of
NADH or NADPH, usage of Cr6+ as an electron acceptor in
Chromium the electron transport system, or by reaction of Cr6+ with or-
ganic compounds. During anaerobic reduction, when Cr6+ is
Chromium is the seventh most abundant metal on earth and made to act as an electron acceptor, the catalyzing enzymes
exists in two stable states in the environment, trivalent Cr3+ generally show a flavin oxidoreductase activity. Examples of
and hexavalent Cr6+. Chromium causes oxidative damage and such enzymes include ferric reductase from Paracoccus
inhibits sulfate membrane transport in bacteria. To fight chro- denitrificans, which reduces both Fe3+ and Cr6+. Other en-
mium toxicity, microbes have developed two mechanisms of zymes include YieF Cr6+ reductase from E. coli which is sim-
chromium resistance. The first is a method of chromate efflux ilar in structure with the ChrR of Pseudomonas putida
from the cells, and the second method involves enzymatic (Ackerley et al. 2004). Many bacterial strains such as
reduction of toxic Cr6+ to less toxic Cr3+. The chromate efflux Enterobacter sp. and Pseudomonas sp. have also been isolated
protein is encoded by a chrA gene, which has homologs in and utilized in Cr6+ reduction under anaerobic conditions
eubacteria, archaea, and even eukaryotes. Nies et al. (1998) using chromate reductase (Kamaludeen et al. 2003). The
described two chromate efflux pumps with six transmembrane YieF reaction involves transfer of four electrons. Three elec-
segments. However, Diaz-Magana et al. (2009) showed that in trons are used to reduce Cr6+ to Cr3+, while the single electron
E. coli, these two pumps are not separate entities, but together combines with O2 and generates reactive oxygen species
form a heterodimer of 12 segments. (ROS) (Viti et al. 2013). Since these ROS are harmful to the
In another study, the presence of a chromium resistance op- cell, superoxide dismutases act and convert these ROS to ox-
eron in Ochrobactrum tritici was described which conferred ygen or water. Similarly, ArsH from Synechocystis sp. PCC
resistance up to 50 mM of Cr6+ (Branco et al. 2008). This op- 6803 has also been found to be capable of reducing Cr6+ (Xue
eron, present on the 7189-bp transposable element TnOtChr, et al. 2014). These are the genetic adaptation mechanisms in
comprised four genes in chrBACF. These genes were not found bacteria for chromium toxicity.
to be continuous, but interspaced with other genes. chrB was
found to be the regulator of the operon, which induces its ex- Copper
pression in the presence of chromium (Chihomvu et al. 2015).
chrA encodes a chromate ion transporter, which is sensitive to Copper is the third oldest Bhuman used^ metal having wide
Cr6+ but not Cr3+. ChrA contains a motif application in wires, motors, architecture, and medicines.
GGX12VX4WX16PGPX9/8G (X = any amino acid) homologous Excess copper in the body causes copperedius, leading to
to many species. ChrC is a 202-amino acid protein, similar to production of ROS, which have the potential to damage pro-
iron/manganese superoxide dismutase; however, the exact func- tein, lipids, and DNA. Copper cycles between two oxidation
tion is yet not well elucidated (Morais et al. 2011). Though ChrF states, Cu(I) and Cu(II), and can displace iron (Fe) from ac-
showed some similarity to putative superoxide dismutase pro- cessible Fe–S clusters in dehydratases and other iron–sulfur
teins (Branco et al. 2008), the exact function of ChrF is not well proteins (Macomber and Imlay 2009). Tetaz and Luke (1983)
understood. Deletion of chrF2 also did not affect chromate re- first reported a plasmid pRJ1004-mediated copper resistance
sistance levels (Juhnke et al. 2002). Gonzalez et al. (2005) iden- in bacteria. Later, Bender and Cooksey (1986) identified in-
tified ChrR as chromate reductase bearing the signature digenous pPT23D plasmids in Pseudomonas syringae pv.
tomato helping from copper toxicity. Subsequently, Mellano A Streptococcus strain was seen to have a copper transport
and Cooksey (1988) established the presence of a copper- operon named copYAZ in which copY and copZ were
inducible cop operon in the 35-kb pPT23D plasmid. It showed established as heavy metal-binding proteins (Vats and Lee
considerable similarity to the copper resistance genes of 2001). Pseudomonas fluorescens has been reported to possess
P. cichorii and P. fluorescens (Cooksey et al. 1990). a copRSCD operon (Hu et al. 2009). In contrast, Helicobacter
Wunderli-Ye and Solioz (1999) described the copper resis- pylori contain two separate operons for copper export and
tance mechanism in the bacterium Enterococcus hirae, which import, hpcopA and hpcopP (Ge and Taylor 1996). Bacillus
contained four genes: copYABZ. copY and copZ encode a subtilis has another copper regulatory system, mediated by
copper-responsive repressor and a chaperone protein, respec- YcnJ and regulated by YcnK and CsoR (Chillappagari et al.
tively. copA was postulated to encode a protein which helps 2009). Together, these genes maintain a state of copper
copper uptake in limiting conditions, and CopA and CopB are homoeostasis in the cell. However, ATPase-driven copper ef-
P-type ATPases. In this regard, Streptomyces sp. AB2A flux system is the main mechanism responsible for cytoplas-
showed early copper retention followed by drastic metal ef- mic copper removal. Periplasmic copper handling,
flux process suggesting the extracellular cupric reductase ac- multicopper oxidases, metallochaperones, and RND systems
tivity in the organism (Albarracin et al. 2008). are involved in this process (Bondarczuk and Piotrowska-
Copper resistance in E. coli has also been extensively Seget 2013).
studied. E. coli has been reported to have a double regula- Grass et al. (2004) reported the multicopper oxidase CueO
tory mechanism of copper resistance. The first is a two- in E.coli which expressed in presence of copper and oxidizes
component system called the cus (copper sensing) locus Cu(I) to Cu(II) in the periplasm and also catecholate-
(Munson et al. 2000). It has two regulator genes cusR and containing ligands such as enterobactin. As the siderophore,
cusS, which forms a regulator-sensor pair and regulates the enetrobactin is able to reduce Cu(II), it was proposed that
expression of cusCFBA. The CusCBA proteins are similar enterobactin oxidation by CueO plays a supplementary func-
to cation/proton antiporter complexes which export metal tion in Cu resistance not generating more toxic Cu(I) ions. In
ions outside the cell in exchange of influx of H+. CusF is addition, 2,3-dihydroxybenzoic acid (DHB), an intermediate
known to bind to copper in the periplasmic space enhancing in enterobactin biosynthesis, stably binds Cu ions, acting as a
copper accumulation inside the cells (Yu et al. 2014). CusF Cu sink. In another uropathogenic E.coli, yersiniabactin was
is a small 10-kDa protein located in perplasmic space and reported to sequester Cu(II) outside bacterial cells and pre-
binds one copper per polypeptide. It is involved in copper vents its catechol-mediated reduction to Cu(I) (Chaturvedi
resistance and contains several methionine, aspartate, and et al. 2012), thus protecting the bacteria from intracellular
histidine residues required for copper binding. However, killing. Furthermore, the Cu(II)–yersiniabactin complex has
site-directed mutagenesis deduced the involvement of me- superoxide dismutase activity protecting bacteria from oxida-
thionine residues for CusF binding with copper. Prediction tive stress inside phagocytic vesicles (Chaturvedi et al. 2014).
of sequence analysis of CusF revealed an N-terminal leader
sequence and a potential signal peptidase cleavage site sig- Lead
nifying the periplasmic location of CusF (Franke et al.
2003). Lead (Pb) is a non-essential, persistent, and hazardous toxic
The second copper homoeostasis system is called cue, or metal pollutant. Several bacteria, such as Arthrobacter spp.,
copper efflux system. The regulatory gene cueR regulates two Bacillus megaterium, Pseudomonas marginalis, Citrobacter
genes, copA and cueO. CopA, as described, is a P-type freundii, Staphylococcus aureus, and E. coli have been found
ATPase, and cueO encodes a multicopper oxidase, which ox- to be resistant to lead. Perhaps the most studied lead resistance
idizes Cu+. CueO oxidizes Cu(I) to a less toxic Cu(II) and operon is found in the endogeneous pMOL30 megaplasmid of
reduces dioxygen to water through four single-electron trans- the bacterium Cupriavidus (Ralstonia) metallidurans CH34
fer steps (Djoko et al. 2010). In the case of multicopper oxi- (Borremans et al. 2001). This operon, named pbr operon,
dases, three types of copper atoms, type 1 (T1), type 2 (T2), was found to contain many structural genes and one regulato-
and two type 3 (T3), are present, of which T1 is buried in the ry gene (pbrR), of which pbrT encodes Pb(II) uptake protein,
interior of the protein and catalyzes oxidation of the substrate pbrA encodes P-type Pb(II) efflux ATPase, pbrB encodes pre-
whereas, T2 and two T3 form a trinuclear center (TNC) where dicted integral membrane protein of unknown function, and
dioxygen is reduced. However, CueO has an extra pbrC encodes predicted prolipoprotein signal peptidase. pbrA
methionine-rich helix that blocks the solvent access to the was proposed to export Pb(II) from the cytoplasm, which
T1 site and forms an additional copper-binding site accumu- would then be converted to a phosphate salt by the inorganic
lating copper (Singh et al. 2004). CsoR is another regulatory phosphate produced by PbrB. Hynninen et al. (2009) showed
gene, which in the presence of Cu+ derepresses copper resis- that pbrB encoded an undecaprenyl pyrophosphate phospha-
tance genes (Chang et al. 2014). tase. A Pb(II)-binding protein encoded by pbrD is located
downstream of pbrC vital for lead sequestration. In the pres- resistant bacterial strains may serve as potential
ence of lead, the regulator pbrR induces the transcription of bioremediative agent (lead biosorbent) in lead-contaminated
pbrABCD operon. environmental sites.
Regulation of the pbr operon was done by pbrR, which was Bioprecipitation of toxic metals to insoluble complex for-
found to be similar to the merR family of heavy metal ion- mation is another strategy which reduces metal bioavailability
sensing regulatory genes. pbrR induced the expression of and toxicity. Bacillus iodinium GP13 and Bacillus pumilus S3
pbrABCD as a single transcriptional unit in the presence of were reported to precipitate lead as lead sulfide (PbS) (De
Pb2+. pbrABC combination was believed to be involved in et al. 2008). A phosphate-solubilizing bacterium E. cloacae
lead efflux, and pbrD is thought to be responsible for Pb2+ was found to resist lead by immobilizing lead as insoluble lead
accumulation (Borremans et al. 2001). PbrD possesses a po- phosphate mineral named pyromorphite (Park et al. 2011).
tential metal-binding motif, rich in cysteine residues (Cys-7X- This method of lead reclamation is an effective, eco-friendly
Cys-Cys-7X-Cys-7X-His-14X-Cys), and also bears a large approach for lead bioremediation. Siderophore induction in
number of proline and serine residues (Jarosławiecka and response to lead stress is also another detoxification strategy.
Piotrowska-Seget 2014). Monchy et al. (2007) showed the Lead-resistant P. aeruginosa strain 4EA showed lead-induced
presence of another gene pbrU, which is induced in the pres- siderophore (pyochelin and pyoverdine) production (Naik and
ence of Pb2+. However, pbrU codes for a permease belonging Dubey 2011). Therefore, genetic adaptations rendering lead
to the major facilitator superfamily (MFS) which is present in resistance followed by various biotransformation techniques
the inner membrane of C. metallidurans (Taghavi et al. 2009). for lead remediation may be employed for microbial remedi-
Hither to this, C. metallidurans was identified as Ralstonia ation of lead.
metallidurans. However, the name of some of the genes in
the Deutsche Sammlung von Mikroorganismen und Mercury
Zellkulturen (DSMZ) database are still starting with BRme^
which stands for R. metallidurans (von Rozycki and Nies Mercury is one of the most toxic elements in the universe and
2009). is found to have severe health concerns in comparison to other
Roane (1999) investigated the intracellular sequestration of toxic metal pollutants. It is concentrated in sediments, soils,
0.6 mM of lead by B. megaterium by metallothionein-like atmosphere, and water. Consumption of fish has been shown
proteins. P. aeruginosa strain WI-1 isolated from Mandovi to be the most potent source of mercury ingestion. Mercury
estuary also possesses bacterial metallothionein (BmtA) which also enters the environment as industrial wastes. Two different
bioaccumulated 26.5 mg lead/g dry weight of cells intracellu- operons are present in bacteria for mercury resistance. One is a
larly to reduce the toxic effect of lead (Naik et al. 2012a). narrow-spectrum mer operon, while the other is the broad-
Metallothionein protein from the smtAB gene was amplified spectrum mer operon (Silver and Phung 2013). The simplest
from Salmonella choleraesuis and Proteus penneri mer operons are those found on the transposons, Tn5037
bioaccumulating 19 and 22 mg of lead, respectively (Naik (Kalyaeva et al. 2001) and Tn5070 (Mindlin et al. 2001),
et al. 2012b). Therefore, presence of metallothioneins in bac- whereas the most complex is the mer operon of Tn5718
teria may be employed for lead bioremedaition in contaminat- (Schneiker et al. 2001).
ed environmental sites. The narrow-spectrum mer operon consists of the genes
Metal immobilization by the process of extracellular se- merR, merT, merC, merF, merP, and merD (Dash and Das
questration is important for regulating metal toxicity. 2012). This operon is inducible by inorganic mercury (Hg2+)
Extracellular polymeric substances (EPS) consisting of poly- and provides resistance to inorganic mercury salts only. merR
saccharides, proteins, nucleic acids, humic substances, and acts as the positive transcriptional regulator of the operon and
lipids with diverse functional groups of hydroxyl, carboxyl, is transcribed separately. In the presence of extracellular mer-
amides, and phosphoryl exhibit high affinity toward heavy cury, or absence of intracellular mercury, it binds to the
metals with high specificity and affinity (Bhaskar and promoter/operator region of the operon to positively and neg-
Bhosle 2006; Bramhachari et al. 2007). Lead binding by the atively regulate the expression of the functional genes. The
negatively charged components of EPS of P. aeruginosa outermost protein of the operon is MerP, located in the peri-
CH07 was also investigated by De et al. (2007). plasmic space. This is a 72-amino acid protein with a
Pseudomonas marginalis was able to resist 2.5 mM lead by β-α-β-β-α-β fold. The two α helices are found to overlay
sequestering lead in an exopolymer (Roane 1999). Similarly, the four-strand antiparallel β sheet (Eriksson and Sahlman
EPS of Paenibacillus jamilae biosorbed 303.03 mg lead/g 1993). The mercury-binding site contains the GMTCAAC
EPS from lead solution (Morillo et al. 2008). There are many consensus sequence. MerP scavenges inorganic mercury ions
enzymatic activities in the bacterial EPS which assist in toxic and transports them to the MerT protein (Hamlett et al. 1992).
metal transformation by chemical reaction, precipation, or en- MerT, encoded by the transposon Tn501, is a 116-amino acid
trapment (Paul 2008). Therefore, EPS producing lead- protein, which receives the inorganic mercury from MerP at
the plasma membrane and transports it inside the cell by bind- reductase (Dash and Das 2012). Hg0, due to its high vapor
ing to cysteine residues on its transmembrane helices. pressure, is volatilized out of the bacterial cell.
Removal or mutation of these residues results in loss of mer- Mercury bioremediation by microbes is mediated by vari-
curic resistance. ous enzymatic transformations like reduction of Hg2+ to Hg0,
The exact mechanism of MerC is obscure. However, MerC breakdown of organomercury compounds, methylation of
has been shown to play a part in mercury transportation and Hg2+, and oxidation of Hg0 to Hg2+. In a study by Dash and
accumulation (Kiyono et al. 2013). merC expression in Das (2015), a transgenic bacterium Bacillus cereus BW-
Arabidopsis thaliana and Nicotiana tabacum has led to their 03(pPW-05) was constructed by transforming a plasmid-
doubling ability to accumulate mercury (Sasaki et al. 2006). harboring mer operon of the marine bacterium Bacillus
MerF, on the other hand, is a 8.7-kDa protein with two trans- thuringiensis PW-05 isolated from Bay of Bengal (Dash
membrane helices. It also functions as a broad-spectrum mer- et al. 2014) into another mercury-resistant marine bacterium
cury transporter. The ∼1600-nucleotide-long merA gene codes B. cereus BW-03 with mercury biosorption ability. This could
for a protein called mercuric ion reductase (Moore et al. 1990). remove >99 % of mercury supplement in vitro by simulta-
This dimeric enzyme functions to reduce Hg2+ in the cell to neous volatilization (>53 %) and biosorption (∼40 %). Many
form volatile Hg0, which is then easily released from the cell. mercury-resistant mer gene-incorporating bacterial isolates
MerA protein is a flavoprotein, which requires NADPH as an were isolated which could volatilize and reduce Hg2+ to Hg0
electron donor to perform the reaction. MerD forms a second- (De et al. 2008). Reduction of mercury and breakdown of
ary regulator protein, which binds, albeit weakly, to the same organomercury compounds are performed by proteins of the
promoter/operator region as MerR and negatively regulates mer operon. In addition to that, the electrochemical potential
the operon (Nascimento and Chartone-Souza 2003). In a re- of Hg2+/Hg0 at pH 7 is +430 mV, and this indicates that the
cent study, merH has been identified as a mercuric ion trans- living cells reduce Hg2+ to the elemental form, which is non-
porter (Schue et al. 2009). Located upstream of the merA gene, toxic to bacteria. The melting point/boiling point of mercury is
it has been found to transport Hg2+ ions by cysteine residues low (−39/357 °C); therefore, metallic mercury leaves the cell
and is co-expressed with merA itself. merH putatively func- by passive diffusion and is volatilized into the air or precipi-
tions as a metal-trafficking protein to merR, which in turn tates due to its low solubility in water removing toxic Hg2+
induces the mer operon for merA-mediated volatilisation of (Wagner-Dobler 2003). Methymercury production in the en-
Hg2+ (Schelert et al. 2013). merI has also been identified im- vironment is controlled by both microbial and abiotic trans-
mediately downstream of the merA gene (Schelert et al. 2006). formations. Direct transformation includes Hg2+ methylation
However, its exact function has not yet been deciphered, and and MeHg degradation. Hg2+ reduction to Hg0 and its cyclic
the huge diversity of arrangement and sequence of mer operon oxidation affect MeHg formation indirectly by controlling
can be explored further for bioremediation application Hg 2+ levels, the substate for methylation (Barkay and
(Rebello et al. 2013). Wagner-Dobler 2005). Moreover, large-scale cleanup of
The broad-spectrum mer operon contains similar genes as mercury-containing wastewater by mercury-resistant mi-
the narrow-spectrum operon. In addition to the genes already crobes is an environment friendly and cost-effective treatment
present, additional genes like merE, merG, and merB are pres- technology practiced nowadays. This was evidenced by the
ent. The broad-spectrum operon provides protection to organ- construction of pilot plants for mercury removal which treated
ic mercury (Barkay et al. 2003). These compounds are more 100 m3 of 50 % mercury-containing wastewater per day
toxic as they can easily enter the cell without transporter mol- (Wagner-Dobler 2003).
ecules. Organic mercury (R-Hg) enters the cell by passive Besides enzymatic transformation, mercury can also be
diffusion or is transported inside by MerE or MerG. merE bioremediated by using metallothioneins and polyphosphates
was originally located in the transposon Tn21 and can mediate by sequestering mercury ions in a biologically inactive form.
the transport of both methyl-mercury (CH3Hg+) and inorganic ppk gene encodes polyphosphate kinase which is involved in
mercury (Boyd and Barkay 2012). Phenylmercury resistance polyphosphate biosynthesis. These negatively charged ortho-
is conferred by the MerG (∼20 kDa) protein, which is the phosphate polymers are capable of binding mercury ions
product of a 654-bp gene (Kiyono and Pan-Hou 1999). (Kornberg 1995). ppk genes expressed in many transgenic bac-
Located between merA and merB on the operon, it protects teria were shown to withstand and accumulate up to 16 μM of
the cell from phenylmercury by preventing it from entering mercury from solutions (Pan-Hou et al. 2002). Another ap-
the cell (Schneiker et al. 2001). The other enzymatic molecule proach is the mercuric sulfide (HgS) formation by the direct
of the mer operon is merB, coding for the enzyme reaction of Hg 2+ with H 2 S produced anaerobically by
organmercurial lyase. MerB protein catalyzes the protonolysis Clostridium cochlearium (Pan-Hou and Imura 1981). Similar
of the carbon–mercury bond resulting in the formation of ionic work reported that Klebsiella aerogenes NCTC418 produced
mercury and a reduced hydrocarbon. The ionic mercury is HgS when grown in continuous aerobic culture with mercury
then reduced to the elemental form Hg0 by the mercuric ion chloride. Mercury was also biologically removed by a mercury-
reducing biofilm which had both the natural and engineered rcnA, the first nickel efflux system discovered in E. coli. Another
mercuric reductase (Brunke et al. 1993). Use of sulfate- efflux pump was identified in Helicobacter pylori and named
reducing bacteria is also used as source of H2S for precipitating cznABC, for cadmium–zinc–nickel (Stahler et al. 2006). Out of
metal as sulfides. The high-solubility product of HgS renders the three genes, cznA and cznC were found to confer nickel
mercury removal by H2S (Hakansson et al. 2008). homoeostasis.
E.coli JM109 was genetically engineered for the simulta-
Nickel neous expression of nickel transport system and metallothio-
nein gene to remove as well as recover Ni2+ from aqueous
Nickel exists as five stable isotopes 58Ni, 60Ni, 61Ni, 62Ni, and solution. It showed a sixfold increase in nickel-binding capac-
64
Ni with 58Ni being the most abundant metal in the environ- ity than the host cells following the linearized Langmuir iso-
ment. Nickel has an important role in the biochemistry of therm (Deng et al. 2003). In another study, the nickel/cobalt
microbes and plants. The enzyme urease contains nickel. transferase gene, NiCoT, from Staphylococcus aureus
Other enzymes like hydrogenases, superoxide dismutase, ATCC6538 was amplified and ligated into vector pET-3c
and glyoxalase enzyme contain Ni-Fe clusters or use nickel which established high bioaccumulation of nickel of
as a co-factor. However, nickel is highly toxic to animals and 11.33 mg/g which was three times more than that of the orig-
humans due to its potential to cross the placenta and affect the inal E. coli BL21 strain (Zhang et al. 2007). Genetic engineer-
fetus. Nickel resistance in bacteria is generally mediated by ing of bacterium E. coli by genomic suppression of the RcnA
efflux pumps. One such resistance mechanism has been stud- nickel (Ni) and cobalt (Co) efflux system was combined with
ied in Cupriavidus (Ralstonia) metallidurans CH34 by Grass the plasmid-controlled expression of a specific metallic trans-
et al. (2000). They reported the presence of a cnrCBA efflux porter, NiCoT from Novosphingobium aromaticivorans. This
pump encoded by the cnrYHXCBAT gene system. Once nickel Ni/Co buster strain resulted in enhanced nickel (II) and cobalt
enters the periplasm, transcription is initiated at the cnr pro- (II) uptake with metal accumulation of 6 mg/g bacterial dry
moter by cnrY and cnrC. The products of the three genes weight in the first 10 min of treatment. Additionally, a syn-
cnrYXH regulate the expression of the whole gene cluster. thetic adherence operon was introduced into the plasmid able
cnrH is an extracytoplasmic function (ECF) sigma factor to form thick biofilm structures in the presence of nickel (II)
(Grass et al. 2000) which constitutively activates cnrCBA ex- and cobalt (II). Therefore, genetic engineering demonstrated
pression. cnrX and cnrY located in the periplasm are increased metal sequestration and biofilm formation by E.coli
membrane-bound proteins, which putatively function as anti- which can be used for biofiltration of nickel as well as cobalt
sigma factors. The cnrCBA encodes a highly efficient pump in an industrial scale by the immobilized cells (Duprey et al.
which is activated only in micromolar concentrations of nick- 2014).
el. These gene products form an efflux pump to efflux excess Nickel metallochaperones are responsible for shuttling
nickel outside the cell. nickel and transferring it to enzyme precursors through pro-
Grass et al. (2005) also characterized the nre (nickel resis- tein–protein interactions in a complex stepwise process. These
tance) operon in Achromobacter xylosoxidans 31A. They found metallochaperones can be engineered for nickel binding
the nre locus on the plasmid pTOM9, and only one gene, nreB, and removal. Infact, nickel removal was reported to be
was responsible for conferring the entire nickel resistance. 234.4 μg/ml by Pseudomonas cepacia 120S (living and dead
However, similar to the cnr operon, this operon did not detoxify biomass) and 117.2 and 351.6 μg/ml by living and dead bio-
Ni2+, instead effluxed it outside the cell. Earlier, Schmidt and mass, respectively, by B. subtilis 117S (Abdel-Monem et al.
Schlegel (1994) reported another operon on the same pTOM9 2010). Soil-inhabiting Ni-resistant Bacillus thuringiensis was
plasmid termed ncc operon, which provided combined nickel, reported to tolerate up to 10 mM Ni and found to remove 82 %
cobalt, and cadmium resistance. However, these nickel resis- of Ni from the medium by the process of biosorption (Das
tance mechanisms do not contribute much to nickel bioremedi- et al. 2014c). A recent approach incorporated the synthesis
ation. Seven open reading frames (ORFs) were studied and des- of nickel oxide nanoparticles from Microbacterium sp.
ignated nccYXHCBAN. Nucleotide sequence revealed signifi- MRS-1 utilized for the treatment of nickel electroplating in-
cant similarity to the cnr and czc operons of Alcaligenes dustrial effluent which showed nickel removal efficiency of
eutrophus CH34 (Tibazarwa et al. 2000). The NccX protein is 95 % (Sathyavathi et al. 2014).
composed of 76 amino acids and has many His residues, indi-
cating that it may serve as a metal-binding protein utilized in
nickel remediation (Trepreau et al. 2011). In E. coli, Rodrigue Discovery of novel metal-resistant genes involved
et al. (2005) performed studies on the yohM gene and found that in bioremediation
it encoded a membrane-bound polypeptide which had the ability
to confer resistance to nickel and cobalt. Higher copies of yohM In response to toxic metal stress, bacteria have developed
led to reduced intracellular nickel. yohM was, thus, renamed as some amazing survival mechanisms incorporated into their
genome. They produce diverse group of enzymes and proteins be tested by means of 15 programs. Two of the most efficient
which help them to overcome these adversities (Johnsen et al. programs described were Biodegradability Evaluation and
2005). Bacteria may also be genetically modified or metabol- Simulation System (BESS) and Biochemical Network
ically engineered to yield products that confer special features Integrated Computational Explorer (BNICE). With this knowl-
to the host cells. Currently, scientists are examining the possi- edge in hand, and with the help of biological pathway prediction
bility of finding Bnew^ adaptation strategies in microorgan- softwares like Scansite 2.0 (Obenauer et al. 2003), BioCyc
isms for removal and subsequent detoxification of pollutants. (Karp et al. 2005), SMART5 (Letunic et al. 2006), STRING7
Introduction of genes encoding enzymes is required that can (Von Mering et al. 2007), and KEGG (Kanehisa and Goto
change the oxidation state of heavy metal from highly toxic to 2000), it is now possible to study the detailed interactions be-
less toxic forms; e.g., for bacterial merA encoding, mercuric tween various biomolecules in silico and to deduce novel pro-
reductase was introduced into other bacterium for better bio- teins and genes which may be used for bioremediation purposes.
remediation (Dash and Das 2015). Thus, introduction of mod-
ified genes may be helpful in obtaining the new mechanisms
of detoxification of heavy metals (Arora et al. 2010). Manipulation of bacterial genetic system
Another approach which can be undertaken is the use of in for enhanced bioremediation
silico methods. Nowadays, with the advent of computers and
softwares, it is possible to get information on any object from a Most pollutants have high persistence in the environment,
single source. Various databases exist which provide informa- mainly due to sub-optimal degradation pathways (Timmis
tion on the toxicity of compounds and their occurrence, proper- and Pieper 1999). Many organic pollutants are degraded by
ties, and pathways by which they may be degraded. Some of the biological pathways, whereas inorganic toxic metals are trans-
important databases include USEPA (http://www.epa.gov/), formed by various enzymes. There exist many intracellular
ATSDR (http://www.atsdr.cdc.gov/), and KEGG PATHWAY and extracellular events in bacteria involving microbial biore-
Database (http://www.genome.jp/). An interesting comparison mediation of toxic pollutants from the environment. In re-
of utilizing such computational sources for carrying out sponse to the presence of toxic metals in the environment,
bioremediation virtually before testing it on site has been resistant bacteria synthesize many intracellular and extracellu-
reviewed by Khan et al. (2013). They listed 11 software tools lar enzymes to remove/degrade the toxic form of metals to
for predicting the toxicity of compounds. They also described 10 non-toxic/less toxic forms (Fig. 2). Each enzymatic pathway
databases with toxicity information on several compounds. In has a rate limiting step. Manipulation of this rate limiting step
addition, environmental degradability of compounds could also could be a solution to increase their bioremediation potential.
Fig. 2 Intracellular and extracellular events involving microbial oxidation-reduction of metals, B metal sequestration by metallothioneins,
bioremediation of toxic metals from the environment. In response to the C conjugate formation with organic compounds/precipitation, D metal
presence of toxic metals in the environment, resistant bacteria synthesize efflux by metal transporters followed by bioremoval by microbial
many intracellular and extracellular enzymes to remove/detoxify the toxic products, and E bioremoval of metals by microbial products
form of metals to non-toxic/less toxic forms. These processes include A (biosurfactants or EPS)
There are many techniques, involving the manipulation of mercury from the contaminated environments. The heavy
bacterial systems, which have been discussed below in details. metal-resistant bacterium C. metallidurans strain MSR33
The modern technology of genetic engineering allows design- was genetically modified to resist mercury (Rojas et al.
ing of microorganisms capable of transforming specific metal 2011). This bacterium was found to be tolerant to both inor-
contaminants. The opportunity of creating artificial combina- ganic mercury and methylmercury, in addition to copper and
tion of genes that are not present in nature offers huge out- chromate.
come for these GMOs to be used for in situ removal of metal In a study by Chaturvedi and Archana (2014), the expres-
pollutants. The most common techniques include engineering sion of two metal-binding peptides was expressed in
with a single gene or operon, alteration of existing gene se- Deinococcus radiodurans R1 which became an attractive
quences, and pathway switching. strategy for developing metal tolerance. A synthetic gene
(EC20) encoding a phytochelatin analog and cyanobacterial
Engineering single gene or gene cluster/operon metallothionein (MT) gene, smtA, was constructed by overlap
extension and expressed in DR1 under the native groESL
Microorganisms prevalent in the polluted sites with high con- promoter. This recombinant strain demonstrated 2.5-fold
centration of heavy metals adapt to resist the harmful effects higher tolerance to Cd2+ and accumulated 1.21-fold greater
by modulating various genetic mechanisms. These strains Cd2+. Therefore, engineering of desirous genes into compati-
with their intrinsic ability to survive in polluted sites are pre- ble bacteria bestows the environment with better approaches
ferred for bioremediation application. Thus, construction of a for remediation.
bacterium by introduction of a single gene or gene cluster into
the intrinsic bacterium will be suitable for bioremediation ap- Alteration of intrinsic genes
plication due to the de novo adaptation of these strains. Sandaa
et al. (1999) found that mostly Gram-positive and α- There are many disadvantages of using a genetically
proteobacteria is found in heavy metal-contaminated soils. engineered microorganism in environmental conditions.
Since majority of the indigenous bacteria are incapable of Some of the introduced genes may not be stable in various
surviving in such adverse conditions, genetically modified environmental conditions to carry out the desired function
recombinant strains may be used to enhance bioremediation. (Dixit et al. 2015). In certain instances, the expression level
It is preferred to alter the indigenous bacterial population ge- of the foreign genes is highly affected in the presence of con-
netically, but the major problem that arises is the instability of taminants and adversely affects the bioremediation practice
the cloned genes and subsequent transfer onto the future (Kiyono and Pan-Hou 1999). Thus, instead of using the for-
generations. Lorenzo et al. (1998) suggested the use of mini- eign genes, the intrinsic genes may be targeted or manipulat-
transposons, derived from the naturally occurring Tn5 and ed. This technique allows the growth of the microorganisms in
Tn10 transposons. These mini-transposons have the speciality their natural environment in addition to carrying out the de-
that only the functional segments of a DNA can be isolated sired function in terms of metal bioremediation.
and cloned into the Tn5 vector, following which it may be There are many studies on targeting the alteration/
inserted into the chromosome of Gram-negative bacteria. modification of the existing gene clusters of indigenous
Ruiz et al. (2011) transformed E. coli JM109 with vectors microflora for bioremediation application. Kermani et al.
resulting in enhanced expression of metallothionein (mt1) and (2010) isolated 3 cadmium-resistant Pseudomonas aeruginosa
polyphosphate kinase (ppk) genes. Metallothioneins are from industrial sludge. Mutation in these strains by exposure
membrane-bound proteins involved in binding of metals and to the dyes acridine orange and acriflavine increased the toler-
their subsequent reduction. This engineered bacterium was ance of these strains upto 7 mM of Cd2+. Phytochelatin syn-
found to accumulate more than 100 μM Hg. Brim et al. thase from Schizosaccharomyces pombe was cloned and
(2000) transformed the radiation-resistant Deinococcus overexpressed in E. coli. As a result, the genetically modified
radiodurans with the mercuric ion reductase gene (merA) E. coli strain was found to accumulate 25-fold higher concen-
from E. coli BL308 host. The resulting mutant strain was tration of Cd2+ as compared to the control strain (Kang et al.
found to grow both in the presence of high radiation and 2007). Wu et al. (2006) found that overexpressing a synthetic
mercury. They were also able to volatilize inorganic Hg phytochelatin EC20 in the soil bacterium Pseudomonas putida
(Hg2+) to elemental Hg (Hg0). In a similar approach, Dash 06909 resulted in triple cadmium binding capability and also
and Das (2015) developed a transgenic strain B. cereus BW- improved cell growth by 2-fold. The close relative of the
03(pPW-05) that harbors the mer operon-containing plasmid radiation-resistant Deinococcus radiodurans and
from a wild strain of B. thuringiensis PW-05. The transgenic Deinococcus geothermalis was engineered by Brim et al.
strain was reported to possess both the mechanisms of mercu- (2003). With the help of plasmids, D. geothermalis was found
ry resistance, i.e., Hg volatilization as well as Hg biosorption to reduce not only Hg2+ but also Fe3+, U6+, and even Cr6+.
which can be widely used in situ for efficient removal of Ackerley et al. (2004) overexpressed nfsA in E. coli and found
Gonzalez et al. 2005

Ackerley et al. 2004
Kermani et al. 2010
Dash and Das 2015
Lorenzo et al. 1998
increased chromate reduction upto 1.5-fold. Overproduction
Duprey et al. 2014

Rojas et al. 2011
Brim et al. 2000
Brim et al. 2003

Ruiz et al. 2011
References of ChrR in Pseudomonas putida increased Cr6+ reduction by
2.4 times (Gonzalez et al. 2005). A detailed list of the bacterial
strains which have effectively been engineered for bioremedi-
ation purpose has been summarized in Table 2.
Efficient Hg volatilization and biosorption

Pathway switching
Efficient Hg accumulation/transformation
Reduction of Hg2+, Fe3+, U6+, and Cr6+

Tolerance to inorganic and organic Hg,
Increased Ni2+ and Co2+ sequestration

Increased Cr6+ reduction by 2.4 times
Increase in Cd2+ tolerance to 7 mM
1.5-fold increase in Cr6+ reduction

Pathway switching involves the construction, extension, and
Growth in presence of radiation
Efficient introduction of genes
regulation of certain novel genetic mechanisms for bioreme-

and Hg, Hg volatilization
along with Cu and Cr diation applications. In the case of toxic metals constructing a
used for bio filtration

microbial consortium each performing a specific path, it can
be an effective measure for achieving complete bioremedia-
tion. Limited studies have been carried out so far for the im-
Applications
provement of the bacterial strains for metal removal by con-

struction of novel consortium. Pathway switching approach
may be employed for the metal removal using GMOs as well.
As the uptake of Cu–Mb (copper–methanobactin) by
aromaticivorans, synthetic adherence
methanotrophic bacteria is quite evident, the mechanism can

Introduction of vectors derived from
Mutation of cad operon by acridine
be developed for simultaneous uptake of Cu–Mb complexes

Introduction of plasmid pMD66
Introduction of mt-1, ppk genes
rather than Cu dissociating from Mb prior to uptake

NiCoT from Novosphingobium
(Balasubramanian et al. 2011). Hence, the identification of a

Introduction of merA gene
Modification of chrR gene

Introduction of merA gene
Alteration of merB, merG,
orange and acriflavine
Overexpression of nfsA
suitable pathway to facilitate acquisition of metals from the

and other mer genes
operon from E. coli

Genetic alterations
environment by identifying the mechanism of transport ma-

Tn5 and Tn10
chineries can provide a key direction for future research.

Application of GMOs for bioremediation is in forefront
List of genetically engineered bacteria with the potential of bioremediation of toxic metals
due to their efficiency and cost-benefit approaches.

However, a complete information on the genes is required
which can be achieved by microarray and fluorescent in situ
hybridization technologies. Another impediment regarding
endA1 glnV44 thi-1 relA1 gyrA96 recA1 mcrB+
Δ(lac-proAB) e14- [F′ traD36 proAB+ lacIq
application of these GMOs is the regulatory affairs and the

lacZΔM15] hsdR17(rK−mK+)
hazards associated with these organisms. Future of bacteria-

based bioremediation relies on the development of suicidal
genetically engineered microorganisms (S-GEMs) which will
enhance the application of these GMOs for on-site applica-
tion. These S-GEMs are constructed by the use of a killer gene
Genetic machinery
and regulatory circuit to control the expression of the killer

gene in response to presence or absence of environmental
Transposons
Consortium
signals (Paul et al. 2005). Thus, the constructed S-GEMs will

mer operon
cad operon
chr operon
Plasmid
Plasmid
undergo programmed cell death due to the presence of killer–

merA
merA
anti-killer genes after removal of toxic substances for their

safe demise in the environment.
Cupriavidus metallidurans strain MSR33
Future directions
Bacillus cereus BW-03(pPW-05)
Deinococcus geothermalis
Discovery of novel genes and proteins associated with the

Deinococcus radiodurans
Pseudomonas aeruginosa
Gram-negative bacteria
ability of eco-friendly cleanup will be a great help to achieve

Pseudomonas putida
enhanced bioremediation. Random mutations may be effected

in the genes known to provide heavy metal resistance to mi-
E. coli JM109
crobes. Such mutations usually have harmful effects on the

Bacteria
Table 2
E. coli
E. coli
organism, but sometimes may also have opposite effects and

result in the formation of strains with higher detoxification
ability. This phenomenon is termed as Bgain-of-function^ mu- Compliance with ethical standards
tation (Arora et al. 2010). To identify new genes which may be
Conflict of interest The authors declare no conflict of interest.
expressed in the presence of a particular pollutant, whole ge-
nome assays may be performed using microarray technology. Ethical approval This article does not contain any studies with human
This enables one to study the expression of thousands of genes participants or animals performed by any of the authors.
at once and thus may enable scientists to determine whether a
single 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-
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Appl Microbiol Biotechnol

Genetic basis and importance of metal resistant genes in bacteria

Received: 5 October 2015 / Revised: 26 January 2016 / Accepted: 28 January 2016

Abstract Metal pollution is one of the most persistent and Introduction

Bioremediation is a technique that utilizes microbes to re-

Fig. 1 A generalized illustration

Cadmium genes together provide high level of resistance to cadmium.

Gonzalez et al. 2005

Duprey et al. 2014

Brim et al. 2003

Efficient Hg volatilization and biosorption

Reduction of Hg2+, Fe3+, U6+, and Cr6+

Increased Ni2+ and Co2+ sequestration

1.5-fold increase in Cr6+ reduction

regulation of certain novel genetic mechanisms for bioreme-

used for bio filtration

provement of the bacterial strains for metal removal by con-

methanotrophic bacteria is quite evident, the mechanism can

Mutation of cad operon by acridine

be developed for simultaneous uptake of Cu–Mb complexes

rather than Cu dissociating from Mb prior to uptake

(Balasubramanian et al. 2011). Hence, the identification of a

Modification of chrR gene

Alteration of merB, merG,

orange and acriflavine

suitable pathway to facilitate acquisition of metals from the

operon from E. coli

environment by identifying the mechanism of transport ma-

chineries can provide a key direction for future research.

due to their efficiency and cost-benefit approaches.

application of these GMOs is the regulatory affairs and the

hazards associated with these organisms. Future of bacteria-

and regulatory circuit to control the expression of the killer

signals (Paul et al. 2005). Thus, the constructed S-GEMs will

undergo programmed cell death due to the presence of killer–

anti-killer genes after removal of toxic substances for their

Discovery of novel genes and proteins associated with the

ability of eco-friendly cleanup will be a great help to achieve

enhanced bioremediation. Random mutations may be effected

crobes. Such mutations usually have harmful effects on the

organism, but sometimes may also have opposite effects and

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