Review TRENDS in Biotechnology
3 March 2005
Accessing microbial diversity for
bioremediation and environmental
restoration
Debarati Paul, Gunjan Pandey, Janmejay Pandey and Rakesh K. Jain
Institute of Microbial Technology, Sector 39A, Chandigarh, 160036, India
Biological methods for decontamination promise an
improved substitute for ineffective and costly physico-
chemical remediation methods, although so far only a
fraction of the total microbial diversity (i.e. the cultur-
able fraction with metabolic potential) has been har-
nessed for this purpose. Exploring and exploiting the
overlooked genetic resource might ameliorate con-
cerns associated with the degradation of recalcitrant
and xenobiotic pollutants that are not degraded or only
poorly degraded by known culturable bacteria. Recent
advances in the molecular genetics of biodegradation
and in knowledge-based methods of rational protein
modification provide insight into the development of
designer biocatalysts for environmental restoration.
The application of such genetically engineered micro-
organisms (GEMs) in the environment has been limited,
however, owing to the risks associated with uncon-
trolled growth and proliferation of the introduced
biocatalyst and horizontal gene transfer. Programming
rapid death of the biocatalyst soon after the depletion of
the pollutant could minimize the risks in developing
these technologies for successful bioremediation.
Introduction
The variety of organic pollutants released by the ever-
increasing number of industries is the direct cause of
environmental and health-related problems that have
detrimental effects on living beings [1]. Conventional
approaches (e.g. land-filling, recycling, pyrolysis and
incineration) to the remediation of contaminated sites
are inefficient and costly and can also lead to the formation
of toxic intermediates [1,2]. Thus, biological decontamina-
tion methods are preferable to conventional approaches
because, in general, microorganisms degrade numerous
environmental pollutants without producing toxic inter-
mediates [3,4].
Some environmental pollutants, however, persist in the
environment. The mineralization of such pollutants by
cultivable bacteria has not been reported [2,5,6] because
the fraction of microbial diversity that is culturable does
not contain the metabolic potential for mineralizing all of
the different xenobiotic pollutants that are present in the
environment [7]. As such, it is possible and pragmatic to
Corresponding author: Jain, R.K. (rkj@imtech.res.in).
Available online 26 January 2005
www.sciencedirect.com 0167-7799/$ - see front matter Q 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.tibtech.2005.01.001
Vol.23 No.
envisage, to exploit and to apply the remaining uncultur-
able microorganisms (diversity) for bioremediation
applications.
In this review, we focus on the application of chemo-
taxis to bioremediation, the use of 16S rDNA to determine
the microbial diversity of polluted sites and the use of
unculturable microorganisms (unculturables) for bio-
remediation, We also discuss the containment of geneti-
cally engineered microorganisms (GEMs) that are
released into the environment.
Bioremediation: prospects and problems
The many different organic pollutants that are present in
the environment persist for varying periods of time.
Depending on their half-life, some pollutants either
disappear within a short period of time owing to
hydrolysis and microbial activities or are transformed
into non-toxic end products [2,5,6]. Many others, however,
such as polychlorinated dibenzodioxyfurans (PCDDF/Fs),
dioxins, dichlorodiphenyltrichloroethane (DDT), chlor-
dane and hexachlorocyclohexane (HCH), have long half-
lives in the environment and tend to enter the food web,
where they are subsequently biomagnified [2,5,8,9]. On
entering mammalian systems, most of these pollutants
are often metabolized in a way that generates more toxic
and harmful intermediates owing to the nonspecific
activity of different mammalian enzymes [10,11]. For
example, a study on a common fresh water fish, Clarius
batrachus, has suggested that the frequently used
fertilizer endosulfan causes impairment of metabolism in
fish by inhibiting transcription [12]. Similarly, commonly
used nitroaromatic compounds are readily reduced to
more reactive nitroso and hydroxylamino intermediates
that act as potentially more mutagenic or carcinogenic
agents on entering mammalian systems [1,11,12].
Bioremediation is the process by which living organ-
isms degrade or transform hazardous organic contamin-
ants. Microorganisms that can degrade various pollutants
(e.g. nitroaromatics, chloroaromatics, polycyclic aro-
matics, biphenyls, polychlorinated biphenyls and com-
ponents of oil) have been isolated with the eventual goal of
exploiting their metabolic potential for the bioremediation
of contaminated sites [1,13,14]. However, some of the more
recalcitrant and toxic xenobiotic compounds, such as
highly nitrated and halogenated aromatic compounds, as
well as some pesticides and explosives, are usually stable,
136 Review TRENDS in Biotechnology Vol.23 No.3 March 2005

chemically inert under natural conditions, and not known example, databases such as the University of Minnesota
to be degraded efficiently by many microorganisms. Biocatalysis/Biodegradation Database (http://umbbd.ahc.
Table 1 lists some compounds that are co-metabolized umn.edu) and the Biodegradative Strain Database (http://
by microorganisms only in the presence of an alternative bsd.cme.msu.edu/bsd/index.html) provide insight into the
carbon source; these compounds are only transformed and various pathways involved in the degradation of xeno-
not effectively mineralized by microorganisms. Such biotic compounds and provide scope for the in silico design
compounds cause problems for biodegradation and bio- of biocatalysts for in situ application [1719].
remediation for several reasons. First, the toxicity of these
organic pollutants to the existing microbial populations,
coupled with complications caused by mixtures of pollu- Enhancing biodegradation: optimizing biocatalysis
tants, is a major hindrance to successful biodegradation. Genetic engineering
Second, some pollutants, such as petroleum hydrocarbons, The construction of an optimized biocatalyst requires a
polycyclic aromatic hydrocarbons and some chlorinated bank of genetic modules that encode desired properties
pesticides among others, are not easily available to the that can be combined to generate novel, improved and
microorganisms because of their hydrophobicity and efficient degradation activities. So far, several micro-
persistence in soil due to aging [15,16]. Accessing the organisms have been modified to make them potent
fraction of pollutants that remains inaccessible to treat- biocatalysts. With the aim of treating a site contaminated
ment by microorganisms is a promising area for research. with various polychlorinated biphenyls (PCBs), for
Third, there is often insufficient knowledge of the example, genetic engineering has been used to alter the
degradation pathways for several recalcitrant compounds, substrate specificity of a biphenyl dioxygenase enzyme
such as those listed in Table 1. involved in PCB degradation in Pseudomonas sp. LB400
These limitations to bioremediation have paved the and Pseudomonas alcaligenes KF707 [20]. Variants of the
way for the development of GEMs or designer biocata- enzyme biphenyl dioxygenase were created by combining
lysts that contain artificially designed catabolic path- the substrate range of the enzyme obtained from both of
ways. Enhancing the performance of such biocatalysts these organisms so that the variants could hydroxylate
both double ortho- and double para-substituted PCBs.
through genetic engineering involves incorporating novel
Site-directed mutagenesis and gene shuffling, assisted
and efficient metabolic routes, widening the substrate
by computerized modeling of 3D protein structures, have
range of existing pathways, increasing the stability of
also facilitated the successful tailoring of enzymes for
catabolic activity, and so on. In addition, rational optim-
enhancing the process of bioremediation [2,21]. In one
ization of biocatalysis has been facilitated by the extensive
case study, a variant of haloalkane dehalogenase (which is
and rapidly growing databases of pathway genetics and by
involved in degradation of chloroaromatic compounds)
increasing knowledge of the biochemical and structure
was generated by replacing specific amino acids at the
function relationships of the component enzymes. For
active site of the enzyme, resulting in a several-fold
Table 1. Recalcitrant pollutants are co-metabolized in the increase in enzyme activity [22,23]. Genetic engineering
presence of an alternative carbon sourcea has also facilitated the construction of GEMs with
Pollutant Chemical class Use Refs multiple degradation pathways through the creation of a
Alachlor Acetamide Herbicide [57] bank of genetic modules encoding broad specificity
Ametryne Triazine Herbicide [57] enzymes or pathway segments that can be combined to
Benthiocarb Substituted Herbicide [58] generate new or improved transformations [24].
carbamate
Chlorpropham Halogenated Herbicide [58]
phenylamide
CL-20 Cyclic nitramine Explosive [32,59] Biosurfactants
Cyanazine Triazine Herbicide [57] The bioavailability of organic contaminants to degradative
DDT Chlorophenyl Insecticide [58]
bacteria is a chief limitation to efficient bioremediation of
alkane
Diuron Phenylamide Pesticide [57,58] sites contaminated with hydrophobic pollutants. This
HMX Cyclic nitramine Explosive [1,32] limitation can be overcome by using surfactants that
Lindane Organochlorine Pesticide [60] facilitate direct contact between the microbes and such
Linuron Halogenated Herbicide [57] pollutants. Because they are non-toxic, non-hazardous,
phenylamide
biodegradable and cheap, biosurfactants have been
Metachlor Acetamide Herbicide [57]
Picric acid Nitroaromatic Explosive [1] already used for bioremediation purposes [15,16,25]. For
Propanil Halogenated Pesticide [58] example, enhanced microbial degradation of the chlorin-
phenylamide ated pesticide endosulfan through the addition of a
RDX Cyclic nitramine Explosive [1,32] biosurfactant isolated from Bacillus subtilis MTCC1427
TCDD Dioxin Unintensional [6,24,
by-product 58]
has been reported: the biosurfactant enhanced the rate of
TNT Nitroaromatic Explosive [1,5] biodegradation by 3045%; it also mobilized the residual
a
Some recalcitrant pollutants do not serve as a sole source of carbon and energy to endosulfan, which otherwise would have remained un-
microorganisms but are co-metabolized in the presence of an alternative carbon degraded, towards biodegradation [26]. Oil bioremedia-
source or sources. Abbreviations: CL-20, 2,4,6,8,10,12-Hexanitro-2,4,6,8,10,12-
tion and the biodegradation of n-alkanes and polycyclic
hexaazaisowurtzitane; DDT, dichlorodiphenyl-trichloroethane; HMX, cyclotetra-
methylenetetranitramine; RDX, cyclotrimethylenetrinitramine; TCDD, 2,3,7,8 aromatic hydrocarbons have been also facilitated by
-tetrachlorodibenzo-para-dioxin; TNT, trinitrotoluene. biosurfactants [15,27].
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 Review TRENDS in Biotechnology
Chemotaxis
Another characteristic feature of some motile bacteria
that has far-reaching applications in biodegradation is
chemotaxis. This process is the movement of microorgan-
isms under the influence of a chemical gradient that helps
them to find optimum conditions for growth and survival.
In past few years, several microbes have been reported to
be chemotactic towards different environmental pollu-
tants (Table 2). It has been argued that bacterial
chemotaxis might have evolved as a selective advantage
for microbes, enabling them to sense and to locate
environmental pollutants, and that this property can be
successfully used for bioremediation [28].
For example, Marx and Aitken [29] have shown that
naphthalene is degraded more rapidly by a wild-type
bacterial strain than by its non-motile or non-chemotactic
mutant in a heterogeneous aqueous system. Recently, it
has been demonstrated that chemotaxis can substantially
increase the rate of mass transfer and degradation of
hydrophobic pollutants associated with the non-aqueous
phase liquid [30]. Hydrophobic organic contaminants that
remain adsorbed to non-aqueous phase liquids in con-
taminated soils are easily accessed by bacteria that form
biofilms on such surfaces to derive nutrition from the
adsorbed compounds. Chemotaxis aids in biofilm formation
by guiding bacteria towards adsorbed nutrients and by
providing attachment to abiotic surfaces through the
flagella [28]. In turn, this increases the bioavailability of
contaminants to bacteria, thereby enhancing degradation.
In addition, chemotaxis has been shown to promote
bioavailability in bacteria isolated from a polluted rhizo-
sphere that degrade polycyclic aromatic hydrocarbons
[31]; Ortego-Calvo et al. [31] agreed with previous reports
(on chemotaxis towards naphthalene and toluene) in
suggesting that the genes involved in chemotaxis and
biodegradation might share a common pathway of
regulation. Furthermore, chemotaxis-mediated biodegrada-
tion of cyclic nitramine explosives such as cyclotrimethyl-
enetrinitramine (RDX), cyclotetramethylenetetranitramine
Table 2. Pollutants that act as chemoattractants to hydro-
carbon-utilizing bacteria
Bacterial strain Pollutant(s)
Agrobacterium Atrazine
radiobacter J14a
Clostridium RDX, CL-20, HMX
sp. EDB2
Pseudomonas Atrazine
sp. ADP
P. putida PRS2000 Aminobenzoate, nitrobenzoate,
3,4-chlorobenzoate
P. putida F1 Benzene, toluene, trichloroethylene,
ethylbenzene, isopropylbenzene,
perchloroethylene, dichloroethylene,
trifluorene, naphthalene
P. putida G7 Naphthalene
Ralstonia sp. SJ98 p-Nitrophenol, 4-nitrocatechol,
o-nitrobenzoate, p-nitrobenzoate,
3-methyl-4-nitrophenol, m-nitro-
phenol, 2,4-dinitrophenol,
2,5-dinitrophenol, 2,6-dinitrophenol,
3,5-dinitrobenzoate
R. eutropha JMP134 2,4-Dichlorophenoxyacetate
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Vol.23 No.3 March 2005 137
(HMX) and 2,4,6,8,10,12-hexanitro-2,4,6,8,10,12-hexa-
azaisowurtzitane (CL-20) has been recently reported [32].
Bhushan et al. [32] suggested that the degradative strain
Clostridium sp. EDB2 initiates the biotransformation of
cyclic nitramines, which releases nitrite ions that attract
other distantly located bacteria that help to accelerate the
biodegradation process [32]. These studies suggest that
chemotactic bacteria might be more competent for bio-
remediation than their non-chemotactic counterparts.
Taken together, the above studies suggest that knowl-
edge of molecular biology and bacterial physiology can be
used to improvise successfully the process of bioremedia-
tion. There is, however, insufficient information on the
degradation of several compounds such as lindane (b-HCH),
dibenzofurans, polychlorophenols and high molecular
weight polycyclic aromatic hydrocarbons, among others.
Because molecular cloning and protein engineering are
labor- and cost-intensive processes, it seems reasonable
that new natural resources should be exploited for
treating such pollutants.
New microbial processes for biodegradation: infiltrating
microbial diversity
In the quest to explore other resources, it is important to
assess the unexplored unculturable microbial wealth. It
has been estimated that 1 g of soil might contain between
1000 and 10 000 species of unknown prokaryotes, and
there is likely to be further diversity within each species
[7,33]. It has been suggested that, first, unculturable
microorganisms are phylogenetically similar or identical
to the culturable minority but remain in a physiological
state that makes them recalcitrant to standard culturing
methods [34]; and second, the remaining microbes
represent novel lineages that are phylogenetically distinct
from the culturables and cannot be cultivated in standard
media [34].
Because cultivation techniques underestimate the
great microbial diversity of environmental samples, the
tremendous chemical virtuosity and use of unculturables
(expected to be a vast repertoire of new genetic and
molecular information) have not been harnessed to their
maximum. Thus, a new science has evolved that aims to
characterize the unculturable organisms through the use
Refs of phylogenetic analysis based on DNA sequence infor-
[61] mation. For microbial identification, genes encoding the
small subunit (SSU) rRNA molecule (16S rRNA or SSU
[32]
rRNA) have been compared because of their high degree of
[61] sequence conservation. 16S rDNA sequences obtained
from soil feature prominently in several newly proposed
[62] bacterial divisions and have contributed greatly to our
expanding picture of bacterial diversity [34,35].
[63]
Molecular methods for assessing microbial diversity
have overcome the culturing bias and have been success-
fully used to elucidate the taxonomical diversity of various
[64] environments including contaminated sites (Table 3).
[65]
Recently, molecular techniques have been developed to
characterize and to monitor the functional diversity in
various environments, some of which are shown in
Figure 1. Analyses of the microbial diversity of contamin-
ated ecosystems have suggested that such systems contain
[66]
significant diversity of unknown and uncultured
138 Review TRENDS in Biotechnology Vol.23 No.3 March 2005

Table 3. Effect of pollutants on the community structure of contaminated sites as indicated by 16S rDNA studies
Contaminant Technique Effects Refs
Acid from mine drainage 16S rDNA Microbial communities affected by sulfuric-acid-rich solutions present in mine [67]
sequencing drainage
4-Chlorophenol T-RFLP Marked changes observed in indigenous soil bacterial communities [68]
Fractions of petroleum DGGE Phylogenetically diverse and novel microbial tolerant to high levels of pollutants [69]
detected
Methidathion (insecticide) Domain-specific Impacts on soil microbial population observed [70]
PCR and RFLP
Petroleum compounds DGGE Both cyanobacterial and bacterial communities showed noticeable changes [71]
during degradation
Phenylurea DGGE Structure and metabolic potentials of microbial communities affected by long- [72]
term application of phenylurea
Phenanthrene TGGE Root exudates and phenanthrene induced modifications in bacterial communities [73]
in polluted environments spatially
Polychlorinated biphenyl RTPCR and 16S Representatives of two novel lineages detected [74]
rRNA sequencing
Abbreviations: DGGE, denaturing gradient gel electrophoresis; RFLP, restriction fragment length polymorphism; RTPCR, polymerase chain reaction with reverse
transcription; TGGE, thermal gradient gel electrophoresis; T-RFLP, terminal restriction fragment length polymorphism.

microorganisms that contribute to biodegradation [36].
For example, comparisons of the gene pool between
cultivated and uncultivated bacteria that degrade
a-halocarboxylic acid have shown that Gram-positive
bacteria account for a significant fraction of the total
diversity. Owing to culturing bias, however, no Gram-
positive a-halocarboxylic acid degraders have been
reported from studies based on enrichment techniques
since 1964 [37].
In another study, significant differences were found
between the community structure of experimental plots in
which oil was deliberately spilt and that of control plots by
using phospholipids fatty acid analysis and denaturing
gradient gel electrophoresis analysis [38]. The control plots
were devoid of a-proteobacteria, whereas the experimental
plots contained a wide range of a-proteobacteria and other
phyla. Similarly, the possible involvement of unculturable
soil microorganisms has been predicted in the degradation
of a phosphonate herbicide, glyphosate [39]. These studies
indicate that culture-based methods elucidate only a small
fraction of the whole microbial diversity of soil; thus, it can
be argued that the soil metagenome remains an unexplored
resource for novel genes and/or gene clusters that might be
exploited for bioremediation.
To analyze the diversity of the pollutant-degrading
population in the environment, fragments of genes for
catabolic enzymes in degradation pathways and/or 16S
rDNA have been amplified from environmental DNA
samples by PCR [34,40]. This approach requires prior
information of the gene sequences, however, for the design
of suitable primers. Metagenomics (the genomics of total
microbial DNA extracted directly from environments), by
contrast, circumvents this limitation because it involves
cloning DNA directly from soil [33,40]. Large genomic
fragments are cloned into bacterial artificial chromo-
somes, fosmids and/or cosmid vectors such that the
whole metagenome is represented by a finite number of
screenable clones [41].
Metagenomics has been successfully used to access the
biosynthetic diversity of microorganisms from varied
environments by acquiring operons or genes involved in
synthesis of commercially or biotechnologically important
compounds such as lipases, cellulases, amylases,
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chitinases, esterases and enzymes involved in biotin
synthesis among others [4145]. Moreover, through
metagenomic analysis the genomes of uncultured organ-
isms have been reconstructed by identifying overlapping
fragments in metagenomic libraries and by walking,
clone to clone, to assemble each chromosome [45,46]. For
example, by using metagenomic profiling Henne et al. [47]
have characterized the enzyme p-hydroxybutyrate dehy-
drogenase, which is involved in the degradation of
p-hydroxybutyratem; genes encoding this enzyme had
been reported from only two other organisms before this
study. This result indicates that metagenomic profiling
promises enormous potential for identifying novel
enzymes or pathways involved in the biodegradation of
poorly and inefficiently degraded pollutants.
The scope of metagenomics can be further broadened by
introducing the desired genes into other hosts such as
pseudomonads, which have active roles in the degradation
of several pollutants. Efficient bacterial artificial chromo-
some shuttle vectors are now available for the construc-
tion of environmental libraries in hosts other than
Escherichia coli [40,41,48]. Recent advances in screening
for biological activity have made the screening process of
up to 1 000 000 clones (representing a metagenomic
library) simpler, quicker and more sensitive [33]. Large-
scale sequencing projects, such as that led by The Institute
for Genomic Research and Monterey Bay Coastal Ocean
Microbial Observatory (http://www.tigr.org/tdb/MBMO/),
are attempting to generate vast databases that, in turn,
will expand the available metagenomic information on
some of the more obscure microbial metabolic potentials.
These approaches will facilitate the construction of
efficient designer biocatalysts for bioremediation and
other biotechnological applications.
Genetically engineered organisms: in situ
bioremediation
The rational combination of catabolic segments from
different organisms in one recipient strain is a useful
strategy for developing bacteria with enhanced degra-
dation capabilities [2,24]. In the present era of functional
genomics, in which complete microbial genomes and
thousands of catabolic operons are known, it is easy to
 Review TRENDS in Biotechnology Vol.23 No.3 March 2005 139

Contaminated environment

Culture-dependent Culture-independent

Enrichment culture Direct isolation of nucleic

acids (metagenome)
Efficient degradative strain

RNA DNA

cDNA

rRNA
sequence
RNASIP and FISH Microarray analysis
rDNA Whole-genome Library construction
sequencing sequencing (plasmid, cosmid and BAC vectors)
Strain
identification
Sequence-based Expression based
Strain screening screening
Influence of pollutants on gene expression
identification

DNA sequence data for constructing Isolation of catabolic

microarrays and oligo probes operon(s) for pollutant(s)

TRENDS in Biotechnology

Figure 1. Flow diagram showing the various genome-based techniques that are currently being used to characterize, to monitor and to harness the whole microbial diversity
from any contaminated site. Cells or nucleic acids isolated from environmental samples can be used to study both the taxonomic and the catabolic diversity of polluted
environments by these recently developed techniques. Abbreviations: BAC, bacterial artificial chromosome; FISH, fluorescence in situ hybridization; RNASIP, RNA-based
stable isotopic probing.

construct GEMs by shuffling genes and promoters to
enhance their performance in situ. Several GEMs have
been successfully constructed and experimentally proved
to have higher degradative capability and utility for
bioremediation under laboratory conditions [3,4,49]. In
spite of such studies, applying GEMs in situ has been
limited by the risks associated with horizontal gene
transfer and uncontrolled proliferation of the introduced
GEMs [50,51].
On release, GEMs either get eliminated from the
environment after performing their desired task or escape
elimination and subsequently evolve and proliferate. In the
past decade, various attempts have been made to restrict the
survival of the GEMs to the environment in which they
perform the desired function in such a way that any cell
escaping this environment will be killed by the induction of
a controlled suicide system. Such constructs, termed
bacterial containment systems, have been designed by
exploring the knowledge of various killer genes and
numerous catabolic regulatory genes [52,53].
www.sciencedirect.com
Several killer genes and killeranti-killer gene
pairs have been shown to be associated with various
bacterial plasmids, chromosomes and bacteriophages.
Cells harbor such systems to eliminate plasmid-free
cells from the population of plasmid-bearing ones. The
killinganti-killing mechanism essentially involves the
expression of two different genes the toxin or poison
and a respective antitoxin or antidote whereby the
killer toxin is stable for hours but its anti-killing
counterpart is very labile. The antidotes (either an
antisense RNA or a protein) either neutralize their
cognate toxins (protein) or prevent their synthesis [54].
The molecular mechanisms of toxin inactivation in
plasmid-free cells are controlled mainly by the differ-
ential decay rates of the toxin and the antidote.
Although the origin of such addiction systems
remains open to speculation, their utility in plasmid
maintenance is evident and promotes their application
in the construction of GEMs for bioremediation. For
example, hok, gef, sac, nuc, relF and colE3 are some of
140 Review TRENDS in Biotechnology Vol.23 No.3 March 2005

the lethal genes that have been used to construct
various containment systems [52,54].
Molin et al. [55] proposed the first containment system
in 1987, after which the concept of a suicidal system was
modified by researchers to create a fail-safe system for
introducing GEMs into the environment for bioremedia-
tion purposes. A generalized scheme for constructing such
containment systems is shown in Figure 2a. In these
constructs, the killer gene is negatively regulated; that is,
it is switched off by default and is activated only when the
concentration of the pollutant falls below detectable
limits. Streptavidin-based systems have been sub-
sequently constructed in which the gene encoding strep-
tavidin (stv) is controlled by a promoter that is negatively
regulated by a repressor protein synthesized in
response to the presence of environmental contami-
nants. In the absence of the pollutant the cells produce
streptavidin, which can bind D-biotin (a prosthetic
group for some essential cellular enzymes) thereby
causing cell death [56]. The main disadvantage of this
type of system is that random mutations in the suicide
genes or control elements can enable GEMs to escape
from programmed killing.
Very recently, a dual lethal system has been con-
structed in which colicin E3 RNase and EcoRI endo-
nuclease (lethal genes) simultaneously control cell death
by regulating different cellular targets, namely, RNA
and DNA, respectively. This system has been developed
in an attempt to reduce the chances of escape because
simultaneous mutations in both enzymes seem improb-
able [53].
The construction strategy of containment systems for
bioremediation purposes requires further refinement to
decrease the probability of cells escaping programmed cell
death. We therefore envisage a novel construct in which
the killer gene product is produced constitutively and
expression of its cognate anti-killer gene is controlled by
environmental signals (Figure 2b). Thus, only in the
presence of the pollutant will the anti-killer be produced
and counteract the suicidal effects of the killer gene. In the
absence of the pollutant, cells will be rapidly killed,
thereby avoiding any chance of proliferation and uncon-
trolled growth of the introduced GEMs. In addition, the
use of a dual lethal system (two killerantikiller pairs), in
which one of the killer genes is placed on a plasmid and the
other toxin and the antidote genes are places on the

(a) Regulator Repressor Killer

Survival
Pollutant
Regulator Repressor Killer

Death

(b) Regulator Anti-killer Killer

Survival
Pollutant

Regulator Anti-killer Killer

Death
TRENDS in Biotechnology

Figure 2. Representation of bacterial containment systems designed for the safe introduction of GEMs into the environment. (a) Model containment system showing negative
regulation of the killer gene by environmental signals. In the presence of the pollutant, the killer gene is repressed; however, depletion of pollutant results in derepression of
the killer gene followed by cell death. (b) Proposed containment system in which the killer gene is constitutively expressed and its cognate anti-killer gene is positively
regulated by environmental signals. In the absence of the pollutant, synthesis of anti-killer ceases, which immediately leads to cell death. Block arrows represent promoters:
black arrows represent positive regulation, blue arrows represent constitutive regulation and yellow arrows represent negative regulation.

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 Review TRENDS in Biotechnology
chromosome, should result in a tighter regulation of
suicidal functions, thereby stimulating immediate cell
death in the case of plasmid-mediated horizontal gene
transfer. In this type of construct, the presence of more
than one promoterantidote fusion (i.e. promoters induc-
ible by different pollutants) would facilitate its use for
sites that are contaminated with a heterogeneous mixture
of pollutants.
Exploiting biodiversity for enhanced bioremediation
therefore involves the elucidation of (i) efficient or novel
catabolic pathways for improved detoxification, (ii) suit-
able chemotactic or biosurfactant-producing strains to
access insoluble or aged pollutants easily for microbial
degradation, and (iii) regulatory circuits and genes for
constructing suicidal systems. Biotechnologists should,
however, always emphasize the ethical responsibilities
before using such novel strategies for bioremediation. In
addition, before releasing such an organism, its genetic
modifications should be understood in full and it should be
possible to confine the organism to the site of introduction.
Concluding remarks
Growing awareness of the harmful effects of environ-
mental pollution has led to a marked increase in research
into various strategies that might be used to clean up the
contaminated environment. Microbial metabolism is now
accepted as a safer and efficient tool for the removal of
many such organic pollutants; however, some of the
more toxic and recalcitrant pollutants remain a challenge
for environmental biotechnologists because they are not
efficiently degraded by culturable microorganisms.
The remaining unculturable microbial diversity can
therefore be exploited to identify desirable degradative
genes and/or properties via the construction of meta-
genomic libraries and to transfer them subsequently to
culturable bacteria for enhanced bioremediation.
Although the resulting GEMs are expected to have
increased efficiency for bioremediation, they pose a threat
to the environment because of the possibility of horizontal
gene transfer and uncontrolled proliferation. The con-
struction of containment systems is thus a prerequisite for
the release of GEMs in situ in order to resolve these
concerns.
Acknowledgements
The authors are supported, in part, by the Council of Scientific and
Industrial Research (CSIR), India, and the Department of Biotechnology
(DBT), India. This is IMTECH communication no. 046/2004.
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