Gene 480 (2011) 1–9

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j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / g e n e

Review

Genetically engineered bacteria: An emerging tool for environmental remediation

and future research perspectives
Jay Shankar Singh a,⁎, P.C. Abhilash b, H.B. Singh c, Rana P. Singh a, D.P. Singh a
a
Department of Environmental Science, BB Ambedkar (Central) University, Lucknow-226025, India
b
Agronomy and Soil Science Division, Central Institute of Medicinal and Aromatic Plants, Lucknow 226 015, Uttar Pradesh, India
c
Department of Mycology and Plant Pathology, Institute of Agricultural Sciences, Banaras Hindu University, Varanasi-221005, India

a r t i c l e i n f o a b s t r a c t

Article history: This minireview explores the environmental bioremediation mediated by genetically engineered (GE) bacteria
Accepted 3 March 2011 and it also highlights the limitations and challenges associated with the release of engineered bacteria in field
Available online 11 March 2011 conditions. Application of GE bacteria based remediation of various heavy metal pollutants is in the forefront
due to eco-friendly and lesser health hazards compared to physico-chemical based strategies, which are less
Received by A.J. van Wijnen
eco-friendly and hazardous to human health. A combination of microbiological and ecological knowledge,
Keywords:
biochemical mechanisms and field engineering designs would be an essential element for successful in situ
Bioremediation bioremediation of heavy metal contaminated sites using engineered bacteria. Critical research questions
Engineered bacteria pertaining to the development and implementation of GE bacteria for enhanced bioremediation have been
Environmental pollution identified and poised for possible future research. Genetic engineering of indigenous microflora, well adapted
Ecoogical risk to local environmental conditions, may offer more efficient bioremediation of contaminated sites and making
the bioremediation more viable and eco-friendly technology. However, many challenges are to be addressed
concerning the release of genetically engineered bacteria in field conditions. There are possible risks associated
with the use of GE bacteria in field condition, with particular emphasis on ways in which molecular genetics
could contribute to the risk mitigation. Both environmental as well as public health concerns need to be
addressed by the molecular biologists. Although bioremediation of heavy metals by using the genetically
engineered bacteria has been extensively reviewed in the past also, but the bio-safety assessment and factors of
genetic pollution have been never the less ignored.
© 2011 Elsevier B.V. All rights reserved.

1. Introduction
Soil and water are the most vital natural resources on earth.
However, in the past few decades, these have been contaminated with
several complex toxic compounds including heavy metals. The
problem of environmental pollution is escalating day by day due to
anthropogenic and natural sources because of increase in huge
population, industrialization and urbanization (Fulekar et al., 2009).
The contaminants causing environmental troubles leading to inequity
in environment is of worldwide apprehension. The model resolution
Abbreviations: arsenic, As; cadmium, Cd; cobalt, Co; deoxyribonucleic acid, DNA;
genetically engineered, GE; glutathione S-transferase fusion protein, GSM-MT; lead, Pb;
magnesium, Mg; mercury, Hg; metal binding peptide, MBP; metallothionein, MT;
nickel, Ni; nitrogen, N; phosphorus, P; phytochelatins, PCs; potassium, K; sodium, Na;
suicidal genetically engineered microorganisms, S-GEMS; trichloroethene, TCE;
trimethylarsine, TMA; uranium, U; zinc, Zn.
⁎ Corresponding author.
E-mail address: jayshankar_1@yahoo.co.in (J.S. Singh).
for toxic waste abatement could be bioremediation, which makes use
of microbial systems for treatment of contaminants. Although, this
novel technology involves multidisciplinary approach, but its vital
power may depend on the nature of microorganisms (Shukla et al.,
2010). Several microorganisms (Pseudomonas, Burkholderia, Sphingo-
monas, Ralstonia, Comamonas, Achromobacter, Alcaligenes, Rhodococ-
cus, Dehalococcoides) are known to degrade xenobiotics, or to
accumulate or detoxify heavy metal pollutants such as Cd, Hg, Pb,
Zn, U, etc., (Daly, 2000; Lloyd et al., 2003). An important difference
between bioremediation of toxic metals and bioremediation of
xenobiotics is the existence of heavy metals under different elemental
state (e.g., conversion of Hg2+ to the volatile Hg0, thus moving the
metal from the soil to the atmosphere). In contrast, the bioremedi-
ation of xenobiotics results in complete mineralization of the toxic
substances. In situ bioremediation uses naturally occurring non
engineered microorganisms and is often enhanced (biostimulation)
by the addition of nutrients, such as N and P, surfactants and oxygen
during the treatment (Watanabe, 2001). In such treatments, nature of
the microbial ecological niches is unpredictable. Another possible
method to improve the bioremediation efficiency is the bioaugmenta-
tion, where the indigenously isolated bacteria are injected into the

0378-1119/$ – see front matter © 2011 Elsevier B.V. All rights reserved.
doi:10.1016/j.gene.2011.03.001
2 J.S. Singh et al. / Gene 480 (2011) 1–9
contaminated site, which may include the genetically engineered
(GE) bacterial strains, but it is rarely done. A major difficulty with in
situ bioremediation by using transgenic bacteria is the unpredictable
end result on account of various environmental factors that may
interfere. However, bioremediation based on GE bacteria is an emerging
technology and has been receiving more attention as an eco-
friendly and efficient way of cleaning up toxic-metal contaminations
(Mejare and Bulow, 2001; Cases and de Lorenzo, 2005a,b; Shukla et al.,
2010; Liu et al., 2011).
For delivering predictable GE bacteria for in situ bioremediation,
there is still enormous challenge regarding making the transgenic
bacteria to work as catalysts of bioremediation under the uncon-
trolled field conditions. In this respect, genetic engineering may
prove to be good tool that helps in optimization of bioremediation
process provided we focus on the large reservoirs of bio-catalytic
activities that have yet remained unexplored in the realms of the
anaerobic and the non-culturable bacteria by using the meta-
genomic technologies (Cases and de Lorenzo, 2002). In this way,
key genes essential for heavy metal bioremediation can be
scavenged from nature for constructing the engineered bacteria
without caring for the original host.
Recent developments in bioremediation technology, which
includes the utilization of protein engineering, metabolic engineering,
whole-transcriptome profiling, and proteomics are also considered
significant for removal of obstinate pollutants such as heavy metals
(Thomas, 2008). Cell surface appearance of precise proteins in the GE
bacteria is known to contribute in detoxification of heavy metals as
well as other recalcitrant compounds (Muhammad et al., 2008). Now,
it is widely considered that molecular biology has the potential
application in designing the bacteria for remediative tasks. The GE
bacteria have higher degradative capacity and have been demon-
strated successfully for the degradation of various pollutants under
defined conditions (Barac et al., 2004). Various strains of Ralstonia
eutropha, Pseudomonas putida, Mycobacterium marinum, Escherichia
coli, Sphingomonas desiccabilis, Bacillus idriensis, etc., have genes
inserted into their genomes, which empowers them to specifically
bioremediation of toxic metal compounds in contaminated environ-
ment (Valls et al., 2000; Ackerley et al., 2004; Kube et al., 2005; Parnell
et al., 2006; Schue et al., 2009; Liu et al., 2011).
Sriprang et al. (2003) reported the ability of the recombinant
Mesorhizobium huakuii subsp. rengei strain B3 to produce phytoche-
latins (PCs) and to accumulate Cd2+. Such bacterial strains might be
useful for the development of a novel plant–bacterium remediation
system for the removal of heavy metals from rice fields when
genetically engineered M. huakuii subsp. rengei strain B3 establishes a
symbiotic relationship with Astragalus sinicus (a nitrogen-fixing
legume). Recently Hasin et al. (2010) reported a well-characterized
model methanotroph Methylococcus capsulatus (Bath), able to
bioremediate chromium(VI) pollution over a wide range of concen-
trations (1.4–1000 mg L− 1 of Cr6+), thus extending the bioremedi-
ation potential via successful genetic manipulation of this major group
of microorganisms.
It has been reported that the metal regulatory genes are involved
in encoding the proteins that help in the conversion of more toxic
form of heavy metals to lesser toxic forms (Bondarenko et al., 2008;
Jan et al., 2009; Ng et al., 2009; Hasin et al., 2010). Sometimes,
complexity of the problems, arising due to presence of mixed
contaminants, presents a situation where survival of GE microbe is
in danger (Shukla et al., 2010). Thus, a combination of microbiological
and ecological knowledge of the site would be a precondition for
application of bioremediation technology by using the GE bacteria.
Besides the competitive situation and adverse environmental condi-
tions existing in the field conditions hampers the survival of GE
bacteria.
It has been observed that the biodegradative gene clusters in
microbes end up under the control of substrate responsive promoters
(Cases and de Lorenzo, 2005b; Tropel and Meer, 2004). The exotic
bacterial strains find it difficult to cope up with these stressful
conditions. However, pressure of natural selection can accomplish
all of these attributes in bacterial strains, but it takes a longer time.
Recreating these natural events in the laboratory is again a difficult
task (Cases and de Lorenzo, 2005b). The genetic engineering offers
the advantage of constructing the bacterial strains which can
withstand the adverse stressful conditions and can be deliberately
employed as bioremediators under complex environmental condi-
tions. Thus, identification and genetic engineering of indigenous
bacteria, which are fully adapted to such complex environmental
conditions, is largely expected to offer a most viable future
remediation technology.
Several significant research investigations in the mid and late
1980s raised immense prospect about manipulative microorganisms
for in situ bioremediation with the genetic engineering. As soon as the
hope of releasing recombinant microorganisms for bioremediation of
toxic compounds in natural environment became an authenticity, a
lot of the research attempt in the area was aimed at bio-safety and risk
evaluation (Cases and de Lorenzo, 2005a).
2. Engineered bacteria for enhanced bioremediation and sustainable
environment
With the aid of biotechnology and genetic engineering, the
symbiotic relationship between bacteria and plants has been exploited
for in situ bioremediation of a wide range of organic pollutants such as
trichloroethylene (Shim et al., 2000), toluene (Barac et al., 2004),
polychlorinated biphenyls (Villacieros et al., 2005), etc. Nevertheless,
very few reports have attempted to concentrate on the heavy metal
remediation using such symbiotic relationship. There is a report about
Pseudomonas putida 06909, a robust and versatile antifungal rhizo-
sphere bacterium was engineered to produce metal binding peptide
(MBP)-EC20, a metal-binding peptide that has high affinity for Cd. The
bacterium P. putida 06909 was modestly Cd resistant, due to the
presence of an efflux pump in the metalloregulatory cad operon (Lee
et al., 2001). Production of MBP-EC20 peptide in P. putida 06909 not
only enabled an enhanced Cd binding but also protected the
engineered strain from metal toxicity. These results demonstrated
that a combination of enhanced microbial biosorption and plant–
bacterium symbiosis can be a promising strategy for heavy metal
cleanup. An increased resistance to metal by the engineered rhizobac-
teria even at higher concentrations is particularly important, as it
provides a competitive advantage to the engineered bacterial strain in
the contaminated soil environment. This could be crucial factor for
sustaining the growth of the engineered strain in the presence of the
native bacterial population.
Unlike the approach reported by Valls et al. (2000), who have
shown expression of metallothionein (MT) genes in a heavy metal-
resistant soil bacterium, Ralstonia eutropha, resulting in a protective
effect on plant growth when inoculated directly into metal contam-
inated soil, The current strategy to utilize the recombinant rhizobac-
teria, which may colonize as a rhizosymbiont, offers the advantage of
removing the metal bound on the rhizobacteria while harvesting the
plants. This feature is particularly important because unlike the
organic pollutants, which are enzymatically degraded, the seques-
tered heavy metals must be physically removed to remediate the site.
This self-sustainable rhizobacterial population is likely to provide
both long-term growth plant protection and removal of the heavy
metals.
It is naturally assumed that free living GE bacteria may have a
decreased level of survival due to the additional stress conditions
imposed by both environmental conditions as well as introduced
foreign genes. Besides, another most important factor would be the
competition of GE bacteria with the other naturally occurring
 J.S. Singh et al. / Gene 480 (2011) 1–9
Fig. 1. Genetically engineered bacteria for enhanced bioremediation and eco-friendly and sustainable environment.
microorganisms for the nutrients and other resources. Considering
the huge selection pressure on the free living GE bacteria imposed by
the environmental factors, both biotic (antagonism, competition and
predation) and abiotic (temperature, pH, moisture, adsorption, etc.),
it is difficult and discouraging task to derive a competent modeling
scheme for survival of engineered bacteria. It is considered that the
indigenous microbial flora, if taken for constructing the recombinant
bacteria, will have natural advantage over the exotic strain of GE
bacteria. Since the rate of bioremediation is not the only function of
degradative genes but is also dependent on total population and
innate capabilities of the bacteria to withstand the existing complex
stressful environmental conditions. Therefore, choosing and engi-
neering the right bacterial strain with rapid growth (largest potential
population and greatest nutrient responses) and more efficient
bioremediation capabilities without environmental risk will be a
crucial step for achieving a safe and sustainable environment as
depicted in Fig. 1.
3. Remediation of contaminated sites with both heavy metal and
organic pollutants
Conventional approaches to the remediation of sites having a mix
of waste by physico-chemical or thermal methods may present a
complex situation and may also lead to the formation of unknown
toxic intermediates (Dua et al., 2002). Thus, decontamination of such
sites by GE bacteria may be preferred over the conventional
approaches because of the special attributes of microorganisms,
involving designed metabolic pathways, to bioremediate the numer-
ous mix environmental pollutants without producing toxic inter-
mediates (Pieper and Reineke, 2000; Furukawa, 2003).
Many heavy metal contaminated sites are co-contaminated with
organic pollutants such as trichloroethene (TCE) and other toxic
compounds (Lee et al., 2006). Brim et al. (2006) have reported that
the engineered Deinococcus radiodurans strain, cloned with tod and
xyl genes of Pseudomonas putida, is capable of complete degradation
of organic contaminants. They also suggested that as such, the Tod/
Xyl strain could provide a model for examining the reduction of
metals coupled to organic contaminant. Similarly, Lee et al. (2006)
have reported a gene coding for the metal-binding peptide, EC20,
3
was incorporated into rhizobacteria engineered for TCE mineraliza-
tion, resulting in strains with TCE degradation potentials. The
characteristic feature of rhizobacteria offers the flexibility of
utilizing different engineered rhizobacteria to remediate mixed-
waste contaminated soil, as many superfund sites are co-contam-
inated with a myriad of organics and heavy metals (Sandrin and
Maier, 2003). Since most organic-degrading microorganisms (e.g.,
Pseudomonas sp.) are sensitive to the toxic effects of heavy metals. A
successful strategy to address this mixed-waste situation requires
the use of microorganisms that will survive and thrive in the
presence of toxic level of heavy metals. Thus, the success of
introducing the EC20 peptides into different root-colonizing bacteria
would depend upon both metal resistance and metal remediation
capabilities of the strains Thus, the rhizosphere bacterial community
can be specifically engineered to target various pollutants at co-
contaminated sites to provide a customized rhizoremediation
system. The flexibility of bacterial strain like P. putida 06909 in
adapting to different plant hosts and the easy molecular manipula-
tion makes it invaluable for designing plant-microbe remediation
systems. However, selection of specific biodegradative genes and
plant species should be made in accordance to the pollutants
present and plant growth conditions at the contaminated sites. The
strategy of simultaneous rhizoremediation of trichloroethylene and
cadmium has been shown by Wu et al. (2006).
4. Bioremediation of heavy metals
A number of pollutants and waste materials containing heavy
metals are released into the environment due to rapid industrializa-
tion (Shukla et al., 2010). Heavy metals such as mercury (Hg),
cadmium (Cd), arsenic (As), nickel (Ni), cobalt (Co), lead (Pb), etc., are
known to cause damage to vital organs in living organisms including
human beings. Therefore, the heavy metal contamination due to
natural and anthropogenic sources is a global environmental concern.
Usually the contaminated sites are treated with traditional methods
like physical, chemical and thermal processes similar to excavation
and transportation. By this process, the expenditure of taking away of
1 m3 soil from a 1-acre polluted spot is expected as US$0.6–2.5 million
(McIntyre, 2003).The bioremediation technology is more eco-
4 J.S. Singh et al. / Gene 480 (2011) 1–9

Table 1
Engineered bacteria involved in remediation of heavy metals.

Bacteria Modified gene expression Heavy metals References

Ralstonia eutropha CH34
Deinococcus radiodurans strains
Escherichia coli and Moraxella sp.
E. coli strian
P. fluorescens 4F39
Mesorhizobium huakuii B3
E. coli strain
P. putida strain
E. coli SE5000
E. coli JM109
E .coli strain
Acidithiobacillus ferrooxidans strain
P. putida 06909
Pseudomonas K-62
E .coli strain
E. coli JM109
E. coli strain
E. coli strain
Pseudomonas fluorescens OS8;
Escherichia coli MC1061;
Bacillus subtilis BR151;
Staphylococcus aureus RN4220
Pseudomonas strain K-62
Achromobacter sp AO22
Methylococcus capsulatus (Bath)
Caulobacter crescentus JS4022/p723–6H
Sphingomonas desiccabilis and
Bacillus Idriensis strains
B. subtilis BR151 (pTOO24)
Metallothionein (MT) Cd2+ (In laboratory) Valls et al. (2000)
Hg (II) resistance gene (merA) Hg (Radioactive waste sites Brim et al. (2000)
from nuclear weapons)
Expressing EC20 Cd and Hg (In laboratory) Bae et al. (2001, 2003)
(with 20 cysteines)
Organomurcurial lyase Hg (Geographically distinct Murtaza et al. (2002)
gene expression regions of India)
Ni transport system Ni (In laboratory) Lopez et al. (2002)
Phytochelatin synthase (PCS) Cd2+ (From rice fields) Sriprang et al. (2003)
gene expression
Metalloregulatory protein ArsR As (Contaminated drinking Kostal et al. (2004)
(overexpressing ELP153AR) and ground water)
Chromate reductase (ChrR) Cr (Bacterial cultures, as well Ackerley et al. (2004)
as cell suspensions)
Ni transport system and MT Ni (Accumulation of Ni2+ Deng et al. (2005)
from aqueous solution)
Hg2+ transporter and MT Hg (In laboratory) Zhao et al. (2005)
Overexpression of serine Ni and Co (In laboratory) Freeman et al. (2005)
acetyltransferase
Hg ion transporter gene expression Hg (In laboratory) Sasaki et al. (2005)
Expression of a metal-binding Cd (Mixed organic-metal- Wu et al. (2006)
peptide (EC20) contaminated sites)
Express Hg transport system and Hg (Mercurials from Kiyono and Pan-Hou (2006)
organomercurial lyase contaminated sites)
PCS gene expression (SpPCS) Cd2+ (Microbial sorbents Kang et al. (2007)
for Cd removal)
Cd transport system and MT Cd (Removal of Cd from Deng et al. (2007)
(namely M4) aqueous solution)
MT As (Whole-cell binding experiments Singh et al. (2008)
in laboratory conditions)
AsIII S-adenosylmethionine As (From bacteria cultures) Yuan et al. (2008)
methyltransferase gene
MerR/CadC/ZntR/Pmer/PcadA/PzntA Cd, Zn, Hg and Pb (water-suspensions Bondarenko et al. (2008)
(expression of luxCDABE genes) and extracts of soils)
MerE protein encoded by transposon Hg (Across the bacterial membrane) Kiyono et al. (2009)
Tn21 (broad Hg transporter)
Hg reductase expressing mer gene Hg (In situ bioremediation Ng et al. (2009)
of contaminated sites)
CrR genes for Cr (VI) Cr (VI) (Cell-associated Cr Hasin et al. (2010)
reductase activity removal in laboratory conditions)
RsaA-6His fusion protein Cd (II) (From the bacterial growth medium) Patel et al. (2010)
Overexpression of arsM gene As (Laboratory conditions) Liu et al. (2011)
luminescent Cd sensors Cd (Naturally polluted soils) Ivask et al. (2011)

friendly, a good substitute to conventional treatments, which rely on
incinerations, volatilization or immobilization of the toxic metals. The
conventional treatment technologies simply transfer the pollutants,
creating a new waste such as incineration residues and not get rid of
the problem (Shukla et al., 2010).
Application of GE bacteria for bioremediation of heavy metals
has come in the forefront due to its selective nature of transforming
the toxicants and thus, posing lesser health hazards as compared to
physico-chemical methods. Bacterial metal detoxification and
removal can be an efficient strategy due to its low cost, high
efficiency and eco-friendly nature. Recent advances in the field of
molecular biology have enabled us to understand the metal-microbe
interaction and their application for bioremediation of metal in the
environment (Rajendran et al., 2003). The detoxification machiner-
ies in GE bacteria have been considered useful for metal
bioremediation, and a comparison of its efficiency of remediation
with the previously isolated metal-resistant bacteria may yield
interesting results. Heavy metal-tolerant Ralstonia eutropha was
genetically engineered to express mouse metallothionein on the cell
surface (Valls et al., 2000). It has been demonstrated that the
inoculation of Cd2+-polluted soil with the GE Ralstonia significantly
decreased the toxic effects of heavy metal on the growth of tobacco
plants. An enhanced ability to sequester heavy metals (e.g.,
cadmium) has been achieved by transfer of a mouse gene, encoding
metallothionein as in Ralstonia eutropha (a natural soil inhabitant)
(Valls et al., 2000). There are several reports on the bioremediation
of heavy metals by different microorganisms. However, only
potential bioremediation of heavy metals by using the GE bacteria
is the subject of review, and the GE bacteria particularly involved in
heavy metal remediation are listed in Table 1.
4.1. Mercury (Hg) remediation
Hg has been reported as one of the most potent toxic heavy metals
in the environment and has been released into the various ecosystems
in substantial amounts through natural events and human activities
(Kiyono and Pan-Hou, 2006). However, various bioremediation
technologies such as biosorption, biotransformation and bioprecipita-
tion of Hg have been invented, but their applications are limited for
removal of Hg from the environment. In response to Hg toxicity,
bacteria have developed a surprising array of resistance mechanisms
 J.S. Singh et al. / Gene 480 (2011) 1–9
to overcome the effect of Hg toxicity. An extensively studied
resistance system based on clustered genes in an operon (mer),
allows bacteria to detoxify Hg2+ into volatile Hg by enzymatic (Hg
reductase) reduction (Barkay et al., 2003). A mechanism for mercury
detoxification (Hg resistance mer operon) based on intracellular
reduction of Hg2+ to non-toxic Hg0 by the Hg reductase enzyme and
subsequent loss of Hg0 from the cell by diffusion (Wagner-Dobler,
2003).The narrow Hg resistance mer operon comprises three major
functions: transport of Hg2+ into the cell, enzymatic NADPH-
dependent conversion of the ionic mercury into relatively less toxic
elemental mercury (Hg0) and regulation of the functional genes.
Organic Hg detoxification requires a fourth function, namely the
cleavage of Hg from the organic residue and the resistance is termed
as broad spectrum. The mer operon genes are under regulatory control
at transcriptional level both positively as well as negatively (Gupta
and Ali, 2004).The mer operon usually consists of structural genes
encoding functional proteins associated with various functions such
as transport (mer T and mer P), regulation (mer R and mer D) and
reduction (mer A and mer B) (Ruiz and Daniell, 2009). Recently, two
other mer genes, mer E and mer H (membrane bound) assisting in the
membrane transport of mercury has been reported in bacteria
(Kiyono et al., 2009; Schue et al., 2009). Various approaches have
been proposed for the GE of bacteria as a device for enhanced
bioremediation of Hg. One particular among them is to make use of
proteins expressed by the genes of mer operon as mer genes of Hg
resistant bacteria serve as important factor involved in the biocycling
of Hg in the environment (Jan et al., 2009). Thus, advance genetic
engineering approaches have opened up new ways to shift the focus
towards the GE bacteria to clean up the Hg contaminated
environment.
Bacterial remediation of Hg is aimed at altering its toxic ionic form
into a less toxic species in the presence of proteins expressed by mer
operon genes (Jan et al., 2009). Acidithiobacillus ferrooxidans strain
with its mercuric ion transporter gene merC expression has been
reported by Sasaki et al. (2005). A new mercuric ion transporter gene
merH present on mercury resistance operon from Mycobacterium
marinum was reported by Schue et al. (2009). Brim et al. (2000) have
developed a radiation resistant bacterium Deinococcus radiodurans for
the treatment of mixed radioactive wastes containing ionic mercury.
It has also been shown that a genetically engineered Escherichia coli,
which simultaneously harbored merT–merP and MT genes, is capable
of removing Hg2+ effectively from electrolytic wastewater taken
directly from a contaminated site containing Na, K, Mg and other ions
besides Hg (Deng and Wilson, 2001). Various mer operon systems
(encodes mer genes responsible for Hg remediation) on the basis of
DNA sequence analysis among different species of bacteria has been
Table 2
Various mer operon systems (encodes mer genes responsible for Hg remediation) on
the basis of DNA sequence analysis among different species of bacteria.
Source: Jan et al. (2009).
Bacteria Type of mer operon system
Shigella Flexneri transposon Tn21
Pseudomonas aeruginosa GD86 transposon Tn501
Klebsiella pneumoniae M426 Tn5073
Morganella morgani M567 Tn5074
Xanthomonas sp W17 Tn5053
Achromobacter sp AO22 Tn5051
Acinetobacter calcoaceticus JM83 pKHL2
Serratia marcescens J53 pDU1358
Pseudomonas sp K-62 pMR26
P. fluorescens pMER419
Mycobacterium marinum pMM23
P. stutzeri pPB
Staphylococcus aureus p1258
5
presented in Table 2. To overcome the above mentioned Hg pollution
problems and to keep check of Hg pollution, new strategies based on
the different arrangement and expression of genes of mer operon are
needed to be designed in order to combat Hg pollution of the
environment completely(Jan et al., 2009).
The high cost of remediating radioactive waste sites from nuclear
weapons production site has stimulated the development of biore-
mediation strategies using D. radiodurans, the most radiation resistant
organism known (Brim et al. 2006). They have engineered D.
radiodurans strains expressing the cloned Hg (II) resistance gene
(merA) from E. coli strain BL308. Their results have demonstrated that
the engineered strains of D. radiodurans can grow in the presence of
both radiation and ionic mercury at concentrations well above those
found in radioactive waste sites and to effectively reduce Hg (II) to the
less toxic volatile elemental mercury. They also suggested that these
expression systems could provide models for future maneuvering of
D. radiodurans strain aimed at integrating several remediation
functions into a single bacterium.
In laboratory experiments, it has been observed that the bioreactor
inoculated with several mercury resistant isolates was invaded by
other Hg-resistant bacteria and a new consortium of mercury-
transforming bacteria evolved that dynamically changed the popula-
tion over a period of time (von Canstein et al., 2001). In such
laboratory-scale experiments, the presence of a consortium was
shown to be a reliable, beneficial, disturbance-independent mercury
removal system (von Canstein et al., 2002). Obviously, this demon-
strated that continuous selection pressure in the bioreactor caused the
mercury-resistant bacteria to adapt to prevailing conditions, best
suited for the stable population.
4.2. Remediation of cadmium (Cd)
A large-scale application of metals like Cd in various industries
and a greater awareness about its ecological effects has led to
numerous reports relating to accumulation and toxicity of Cd. The
application of GE bacteria with higher efficiency has demonstrated
greater need for removal of this metal from the contaminated sites.
Metal-binding peptides such as metallothioneins and phytochela-
tins (PCs) have been shown to contribute enhanced heavy metal-
binding capabilities (Bae et al., 2001). PCs are known to bind
heavy metals such as Cd, Hg, As, and Pb, especially cadmium, with
high affinity through formation of thiolate complexes (Schmoger et
al., 2000). Recently, the genes coding for PCs have been cloned
from plants and fungi, and are functionally expressed in E. coli
(Sauge-Merle et al., 2003). Deng et al. (2003) reported that GE
bacteria with both high bioaccumulation capacity and high affinity
for the target metal have shown that the new recombinant
bacterial strains selectively accumulate the metal ions from
multi-component pollutants. Deng et al. (2007) demonstrated
that capability of the GE bacteria for specifically accumulating
Cd2+ from multi-component metal polluted site was due to
specific cadmium transport system and metallothionein (MT)
protein.
Sriprang et al. (2003) reported an enhanced accumulation of Cd2+
by a Mesorhizobium huakuii, which was transformed with a gene from
Arabidopsis thaliana coding for PCs. Kang et al. (2007) reported that
the level of Cd accumulation in recombinant E. coli strain was about
25-fold higher than that in the control strain. Wu et al. (2006; 2010
demonstrated that expression of a metal-binding peptide (EC20) in a
rhizobacterium Pseudomonas putida 06909, which alleviated the
cellular toxicity of Cd. Very recently, the recombinant Caulobacter
crescentus strain JS4022/p723-6H, expressing RsaA-6His fusion
protein was able to remove 94.3–99.9% of the Cd(II), whereas the
control strain could remove only 11.4–37.0% (Patel et al., 2010).
Without questioning the authenticity of the results, it is assumed that
6 J.S. Singh et al. / Gene 480 (2011) 1–9
there is great potential of utilizing the recombinant technology in
efficient removal of the target heavy metals.
4.3. Arsenic (As) remediation
Arsenic (As) is an extremely toxic metalloid found in nature
(Jackson et al., 2006; Chen et al., 2008; Williams et al., 2009).
Arsenic gets accumulated in edible parts of plants and subsequently
finds its way into the food chain, which poses a serious health risk
to humans (Abedin et al., 2002). Thus, there is an urgent need to
efficiently remove arsenic from contaminated water and soil.
Bioremediation of As from contaminated sites by using GE bacteria
could be a better option as the transformation of As to non-toxic
form requires the involvement of enzyme mediated redox reactions
(Valls and de Lorenzo, 2002). Recently E. coli expressing arsenite S-
adenosylmethionine methyltransferase gene (arsM) coloned from
Rhodopseudomonas palustris has been found to methylate more toxic
inorganic As to less toxic volatile trimethylarsine (TMA) (Qin et al.,
2006; Yuan et al., 2008). Therefore, practical application of GE
bacteria for As bio-volatilization from As contaminated soils is
becoming possible. Very recently, Liu et al. (2011) demonstrates
that As could be removed through volatilization from the contam-
inated soil by GE bacteria, which show expression of arsM gene.
These findings suggested that over expression of arsM genes in
Sphingomonas desiccabilis and Bacillus idriensis, resulted into many
fold increase in methyled As gas, when compared with activity of
wild type (WT) strain. Thus, it is now accepted that the
volatilization of arsenic gas by using the GE bacteria with multiple
copies of the arsM gene may be highly useful.
The metalloregulatory protein (ArsR) also offers immense poten-
tial of bioremediation by GE bacteria due to high-affinity transport
system and selectivity toward the substrate arsenite. Kostal et al.
(2004) have demonstrated that overexpression of ArsR genes in E. coli
could increase the bioaccumulation of As metal. Thus, over expression
of ArsR in bacteria by genetic manipulation of the strain could be a
promising strategy to increase the cellular accumulation of As and
provide a highly selective ligand for As binding, which may ultimately
enable the bacteria for selective removal of As.
4.4. Nickel (Ni) remediation
The Ni ion is a more recalcitrant pollutant as compared to other
heavy metal ions (Deng et al., 2003, 2005). To construct strains that
are capable of specifically accumulating Ni2+ from dilute solution, a E.
coli SE5000 strain was genetically engineered to express a Ni2+
transport system (the products of nixA gene) which enabled the
bacterium to specifically accumulate the Ni2+ from aqueous (Fulk-
erson et al., 1998). It has been demonstrated that over expressed
metallothionein (MT) and glutathione S-transferase fusion protein
(GSM-MT) (Chen and Wilson, 1997) as well as nixA gene, encoding a
37 kDa integral membrane protein consisting of eight transmembrane
domains, exhibit very high affinity for Ni2+. It is presumed that over
expression of Ni2+ transport proteins along with intracellular over
expressed MTs would accumulate large amount of Ni2+.
Freeman et al. (2005) reported that the over expression of serine
acetyltransferase from the Ni-hyper-accumulating plant Thlaspi
goesingense causes enhanced Ni resistance in E. coli. Enhanced Ni
resistance is directly related to constitutive over activation of sulfur
assimilation and glutathione biosynthesis, driven by the overproduc-
tion of O-acetyl-L-serin- a product of serine acetyltransferase and a
positive regulator of the cysteine regulon (Freeman et al., 2005). Thus,
genetic engineering techniques have the potential to improve or
redesign bacteria so that biological metal binding systems will have
higher intrinsic capability and specificity, and they are more tolerant
to ambient conditions.
5. Release of engineered bacteria in field condition
Use of GE bacteria for bioremediation has seen limited due to
very little development in the field over the past decade (Sayler and
Ripp, 2000). The recent advances in the area of recombinant DNA
technologies have paved the way for conceptualizing “suicidal
genetically engineered microorganisms” (S-GEMS) to minimize such
anticipated hazards and to achieve efficient and safer decontami-
nation of polluted sites (Pandey et al., 2005). A field release of
Pseudomonas fluorescens HK44 for bioremediation application has
been successfully conducted on moderately large-scale and con-
trolled field condition (Ripp et al., 2000). However, the future
application of GE bacteria for pollution remediation will not be free
from the risks associated with their release in the environment. The
future risk regarding use of other engineered bacteria is still
unclear. Therefore, the future perspectives of engineered bacterial
strains under the field conditions will be the focus of review, which
may help us to assess the obstacles related with application of GE
bacteria in environmental bioremediation.
During the 1980s, an increasing interest in development of
transgenic microorganisms for various environmental applications
ahs ignited an ethical argument related to possible risks of using the
genetically modified microorganisms. Such questions have genuinely
enthused the expansion of research in the field of Environmental
Microbiology and molecular biology. But the importance of recombi-
nant bacteria for environmental applications should not be overlooked
because of certain ethical questions. The variety of problems
associated with application of GE bacteria for addressing the
environmental issues need to be addressed with an integrated and
holistic approach. The major problem encountered in successful
bioremediation technology pertains to hostile field conditions for the
engineered microbes. Besides, the molecular applications are mainly
confined to only few well-characterized bacteria such as Escherichia
coli, Pseudomonas putida, Bacillus subtilis, etc. Other bacterial strains
need to be tried for developing the engineered microbes The specific
characteristic of open biotechnological applications has clearly
necessitated the development of engineered bacterial strains to meet
the new challenges. The main concern is to construct GE bacteria for
field release in bioremediation with an adequate degree of environ-
mental certainty. Efforts should be made to examine the performance
of engineered bacteria in terms of their survival, potential of horizontal
gene transfer, which may affect the indigenous microflora within a
complex environmental situation. Often the novel scientific researches
always give rise to still more fascinating questions pertaining to public
concern. In the majority cases, the bacteria designed for bioremedi-
ation processes have been designed for specific purpose under the
laboratory conditions, ignoring the field requirement and other
complex situations. However, there is no evidence that the deliberate
release of GE bacteria for bioremediation has caused a measurable
adverse impact on the natural microbial community. At least the
repeatedly overstated idea of risk appraisal has fuelled so much debate
and triggered so many research efforts, which have immensely
contributed a lot in the field of Environmental Microbiology. However,
survival of the GE bacteria in complex environmental situations is still
a big question, needs to be addressed in the light of latest findings as
mentioned in the given flow chart (Fig. 2).
6. Conclusions and future research perspectives
Investigations on bioremediation of environmental toxicants are
entering in a new era with the application of GE bacteria (Singh et al.
2008). However, majority of the studies related to bioremediation of
heavy metals have been conducted under the laboratory conditions. It
is too difficult to study the decontamination of the polluted sites in
natural environments because of the implication of various factors in
 J.S. Singh et al. / Gene 480 (2011) 1–9
Fig. 2. A conceptual model showing various steps that may be applied for the analyses of environmental risk assessment of engineered bacteria during their field release for
environmental bioremediation.
detoxification process. Therefore, future researches would be more
focused on identifying the factors that enhance the in situ bioreme-
diation by engineered bacteria. Regardless of several advantages and
disadvantages associated with bioremediation technology, bioreme-
diation of metal contaminated sites using the native rhizosphere
bacteria gives a ray of hope for successful application of bioremedi-
ation technology keeping an eye on the local factors prevailing at the
contaminated site. A slow development in the field of application of
GE bacteria is mainly ascribed to the possible risks, low public
acceptability. Perhaps the selection of indigenous microflora in the
recombinant technology will shed the public apprehension also.
Although, in a review article Davidson (2005) have described the
possible risks associated with the use of engineered bacteria, with
particular importance on ways in which molecular genetics could
contribute to risk mitigation. However, ecological and environmental
concerns and regulatory constraints are major obstacles for testing of
the GE bacteria in the field (Shukla et al., 2010). Since most of the
scientific researches on bioremediation by engineered bacteria are of
a basic nature, there is growing need for a regulatory, safety, or costs
benefit-driving systems, which may translate this potential biotech-
nology into a reality. Fig. 2 demonstrated various steps that may help
in the analyses of environmental risk assessment of GE bacteria during
their field release for bioremediation.
Acknowledgments
The authors are thankful to Dr. Andre van Wijnen, Reviews Editor–
GENE, for invitation to contribute this article. We also wish to thank
the reviewers for their brilliant comments and suggestions during
revision of this article. We thank the Head, Professor M. Yunus,
Department of Environmental Science, Babasaheb Bhimrao Ambedkar
7
(Central) University, Lucknow for providing infrastructural facilities.
This work was possible through grants given to Dr. Jay Shankar Singh
as Senior Research Associate (Scientist's Pool Scheme) by Council of
Scientific and Industrial Research, Human Resource Development
Group, Government of India, New Delhi.
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