Accessing microbial diversity forbioremediation and environmentalrestorationDebarati 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 ofenvironmental and health-related problems that havedetrimental effects on living beings [1]. Conventionalapproaches (e.g. land-filling, recycling, pyrolysis andincineration) to the remediation of contaminated sitesare inefficient and costly and can also lead to the formationof toxic intermediates [1,2]. Thus, biological decontamina-tion methods are preferable to conventional approachesbecause, in general, microorganisms degrade numerousenvironmental pollutants without producing toxic inter-mediates [3,4].

Some environmental pollutants, however, persist in theenvironment. The mineralization of such pollutants bycultivable bacteria has not been reported [2,5,6] becausethe fraction of microbial diversity that is culturable doesnot contain the metabolic potential for mineralizing all ofthe different xenobiotic pollutants that are present in theenvironment [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

envisage, to exploit and to apply the remaining uncultur-able microorganisms (diversity) for bioremediationapplications.

In this review, we focus on the application of chemo-taxis to bioremediation, the use of 16S rDNA to determinethe microbial diversity of polluted sites and the use ofunculturable microorganisms (‘unculturables’) for bio-remediation, We also discuss the containment of geneti-cally engineered microorganisms (GEMs) that arereleased into the environment.

Bioremediation: prospects and problems

The many different organic pollutants that are present inthe environment persist for varying periods of time.Depending on their half-life, some pollutants eitherdisappear within a short period of time owing tohydrolysis and microbial activities or are transformedinto 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]. Onentering mammalian systems, most of these pollutantsare often metabolized in a way that generates more toxicand harmful intermediates owing to the nonspecificactivity of different mammalian enzymes [10,11]. Forexample, a study on a common fresh water fish, Clariusbatrachus, has suggested that the frequently usedfertilizer endosulfan causes impairment of metabolism infish by inhibiting transcription [12]. Similarly, commonlyused nitroaromatic compounds are readily reduced tomore reactive nitroso and hydroxylamino intermediatesthat act as potentially more mutagenic or carcinogenicagents 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 ofexploiting their metabolic potential for the bioremediationof contaminated sites [1,13,14]. However, some of themorerecalcitrant and toxic xenobiotic compounds, such ashighly nitrated and halogenated aromatic compounds, aswell as some pesticides and explosives, are usually stable,

Review TRENDS in Biotechnology Vol.23 No.3 March 2005

. doi:10.1016/j.tibtech.2005.01.001

Review TRENDS in Biotechnology Vol.23 No.3 March 2005136

chemically inert under natural conditions, and not knownto be degraded efficiently by many microorganisms.

Table 1 lists some compounds that are co-metabolizedby microorganisms only in the presence of an alternativecarbon source; these compounds are only transformed andnot effectively mineralized by microorganisms. Suchcompounds cause problems for biodegradation and bio-remediation for several reasons. First, the toxicity of theseorganic pollutants to the existing microbial populations,coupled with complications caused by mixtures of pollu-tants, is a major hindrance to successful biodegradation.Second, some pollutants, such as petroleum hydrocarbons,polycyclic aromatic hydrocarbons and some chlorinatedpesticides among others, are not easily available to themicroorganisms because of their hydrophobicity andpersistence in soil due to ‘aging’ [15,16]. Accessing thefraction of pollutants that remains inaccessible to treat-ment by microorganisms is a promising area for research.Third, there is often insufficient knowledge of thedegradation pathways for several recalcitrant compounds,such as those listed in Table 1.

These limitations to bioremediation have paved theway for the development of GEMs or ‘designer biocata-lysts’ that contain artificially designed catabolic path-ways. Enhancing the performance of such biocatalyststhrough genetic engineering involves incorporating noveland efficient metabolic routes, widening the substraterange of existing pathways, increasing the stability ofcatabolic activity, and so on. In addition, rational optim-ization of biocatalysis has been facilitated by the extensiveand rapidly growing databases of pathway genetics and byincreasing knowledge of the biochemical and structure–function relationships of the component enzymes. For

Table 1. Recalcitrant pollutants are co-metabolized in the

presence of an alternative carbon sourcea

Pollutant Chemical class Use Refs

Alachlor Acetamide Herbicide [57]

Ametryne Triazine Herbicide [57]

Benthiocarb Substituted

carbamate

Herbicide [58]

Chlorpropham Halogenated

phenylamide

Herbicide [58]

CL-20 Cyclic nitramine Explosive [32,59]

Cyanazine Triazine Herbicide [57]

DDT Chlorophenyl

alkane

Insecticide [58]

Diuron Phenylamide Pesticide [57,58]

HMX Cyclic nitramine Explosive [1,32]

Lindane Organochlorine Pesticide [60]

Linuron Halogenated

phenylamide

Herbicide [57]

Metachlor Acetamide Herbicide [57]

Picric acid Nitroaromatic Explosive [1]

Propanil Halogenated

phenylamide

Pesticide [58]

RDX Cyclic nitramine Explosive [1,32]

TCDD Dioxin Unintensional

by-product

[6,24,

58]

TNT Nitroaromatic Explosive [1,5]aSome recalcitrant pollutants do not serve as a sole source of carbon and energy to

microorganisms but are co-metabolized in the presence of an alternative carbon

source or sources. Abbreviations: CL-20, 2,4,6,8,10,12-Hexanitro-2,4,6,8,10,12-

hexaazaisowurtzitane; DDT, dichlorodiphenyl-trichloroethane; HMX, cyclotetra-

methylenetetranitramine; RDX, cyclotrimethylenetrinitramine; TCDD, 2,3,7,8

-tetrachlorodibenzo-para-dioxin; TNT, trinitrotoluene.

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example, databases such as the University of MinnesotaBiocatalysis/Biodegradation Database (http://umbbd.ahc.umn.edu) and the Biodegradative Strain Database (http://bsd.cme.msu.edu/bsd/index.html) provide insight into thevarious pathways involved in the degradation of xeno-biotic compounds and provide scope for the in silico designof biocatalysts for in situ application [17–19].

Enhancing biodegradation: optimizing biocatalysis

Genetic engineering

The construction of an optimized ‘biocatalyst’ requires abank of genetic modules that encode desired propertiesthat can be combined to generate novel, improved andefficient degradation activities. So far, several micro-organisms have been modified to make them potentbiocatalysts. With the aim of treating a site contaminatedwith various polychlorinated biphenyls (PCBs), forexample, genetic engineering has been used to alter thesubstrate specificity of a biphenyl dioxygenase enzymeinvolved in PCB degradation in Pseudomonas sp. LB400and Pseudomonas alcaligenes KF707 [20]. Variants of theenzyme ‘biphenyl dioxygenase’ were created by combiningthe substrate range of the enzyme obtained from both ofthese organisms so that the variants could hydroxylateboth double ortho- and double para-substituted PCBs.

Site-directed mutagenesis and gene shuffling, assistedby computerized modeling of 3D protein structures, havealso facilitated the successful tailoring of enzymes forenhancing the process of bioremediation [2,21]. In onecase study, a variant of haloalkane dehalogenase (which isinvolved in degradation of chloroaromatic compounds)was generated by replacing specific amino acids at theactive site of the enzyme, resulting in a several-foldincrease in enzyme activity [22,23]. Genetic engineeringhas also facilitated the construction of GEMs withmultiple degradation pathways through the creation of abank of genetic modules encoding broad specificityenzymes or pathway segments that can be combined togenerate new or improved transformations [24].

Biosurfactants

The bioavailability of organic contaminants to degradativebacteria is a chief limitation to efficient bioremediation ofsites contaminated with hydrophobic pollutants. Thislimitation can be overcome by using surfactants thatfacilitate direct contact between the microbes and suchpollutants. Because they are non-toxic, non-hazardous,biodegradable and cheap, biosurfactants have beenalready used for bioremediation purposes [15,16,25]. Forexample, enhanced microbial degradation of the chlorin-ated pesticide endosulfan through the addition of abiosurfactant isolated from Bacillus subtilis MTCC1427has been reported: the biosurfactant enhanced the rate ofbiodegradation by 30–45%; it also mobilized the residualendosulfan, which otherwise would have remained un-degraded, towards biodegradation [26]. Oil bioremedia-tion and the biodegradation of n-alkanes and polycyclicaromatic hydrocarbons have been also facilitated bybiosurfactants [15,27].

Review TRENDS in Biotechnology Vol.23 No.3 March 2005 137

Chemotaxis

Another characteristic feature of some motile bacteriathat has far-reaching applications in biodegradation ischemotaxis. This process is the movement of microorgan-isms under the influence of a chemical gradient that helpsthem to find optimum conditions for growth and survival.In past few years, several microbes have been reported tobe chemotactic towards different environmental pollu-tants (Table 2). It has been argued that bacterialchemotaxis might have evolved as a selective advantagefor microbes, enabling them to sense and to locateenvironmental pollutants, and that this property can besuccessfully used for bioremediation [28].

For example, Marx and Aitken [29] have shown thatnaphthalene is degraded more rapidly by a wild-typebacterial strain than by its non-motile or non-chemotacticmutant in a heterogeneous aqueous system. Recently, ithas been demonstrated that chemotaxis can substantiallyincrease the rate of mass transfer and degradation ofhydrophobic pollutants associated with the ‘non-aqueousphase liquid’ [30]. Hydrophobic organic contaminants thatremain adsorbed to non-aqueous phase liquids in con-taminated soils are easily accessed by bacteria that formbiofilms on such surfaces to derive nutrition from theadsorbed compounds. Chemotaxis aids in biofilm formationby guiding bacteria towards adsorbed nutrients and byproviding attachment to abiotic surfaces through theflagella [28]. In turn, this increases the bioavailability ofcontaminants to bacteria, thereby enhancing degradation.

In addition, chemotaxis has been shown to promotebioavailability 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) insuggesting that the genes involved in chemotaxis andbiodegradation might share a common pathway ofregulation.Furthermore, chemotaxis-mediatedbiodegrada-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) Refs

Agrobacterium

radiobacter J14a

Atrazine [61]

Clostridium

sp. EDB2

RDX, CL-20, HMX [32]

Pseudomonas

sp. ADP

Atrazine [61]

P. putida PRS2000 Aminobenzoate, nitrobenzoate,

3,4-chlorobenzoate

[62]

P. putida F1 Benzene, toluene, trichloroethylene,

ethylbenzene, isopropylbenzene,

perchloroethylene, dichloroethylene,

trifluorene, naphthalene

[63]

P. putida G7 Naphthalene [64]

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

[65]

R. eutropha JMP134 2,4-Dichlorophenoxyacetate [66]

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(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 strainClostridium sp. EDB2 initiates the biotransformation ofcyclic nitramines, which releases nitrite ions that attractother distantly located bacteria that help to accelerate thebiodegradation process [32]. These studies suggest thatchemotactic 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 beused to improvise successfully the process of bioremedia-tion. There is, however, insufficient information on thedegradation of several compounds such as lindane (b-HCH),dibenzofurans, polychlorophenols and high molecularweight polycyclic aromatic hydrocarbons, among others.Because molecular cloning and protein engineering arelabor- and cost-intensive processes, it seems reasonablethat new natural resources should be exploited fortreating such pollutants.

New microbial processes for biodegradation: infiltrating

microbial diversity

In the quest to explore other resources, it is important toassess the unexplored ‘unculturable’ microbial wealth. Ithas been estimated that 1 g of soil might contain between1000 and 10 000 species of unknown prokaryotes, andthere is likely to be further diversity within each species[7,33]. It has been suggested that, first, ‘unculturable’microorganisms are phylogenetically similar or identicalto the culturable minority but remain in a physiologicalstate that makes them recalcitrant to standard culturingmethods [34]; and second, the remaining microbesrepresent novel lineages that are phylogenetically distinctfrom the culturables and cannot be cultivated in standardmedia [34].

Because cultivation techniques underestimate thegreat microbial diversity of environmental samples, thetremendous chemical virtuosity and use of unculturables(expected to be a vast repertoire of new genetic andmolecular information) have not been harnessed to theirmaximum. Thus, a new science has evolved that aims tocharacterize the ‘unculturable’ organisms through the useof phylogenetic analysis based on DNA sequence infor-mation. For microbial identification, genes encoding thesmall subunit (SSU) rRNA molecule (16S rRNA or SSUrRNA) have been compared because of their high degree ofsequence conservation. 16S rDNA sequences obtainedfrom soil feature prominently in several newly proposedbacterial divisions and have contributed greatly to ourexpanding picture of bacterial diversity [34,35].

Molecular methods for assessing microbial diversityhave overcome the culturing bias and have been success-fully used to elucidate the taxonomical diversity of variousenvironments including contaminated sites (Table 3).Recently, molecular techniques have been developed tocharacterize and to monitor the functional diversity invarious environments, some of which are shown inFigure 1. Analyses of the microbial diversity of contamin-ated ecosystems have suggested that such systems containsignificant diversity of unknown and uncultured

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

sequencing

Microbial communities affected by sulfuric-acid-rich solutions present in mine

drainage

[67]

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

detected

[69]

Methidathion (insecticide) Domain-specific

PCR and RFLP

Impacts on soil microbial population observed [70]

Petroleum compounds DGGE Both cyanobacterial and bacterial communities showed noticeable changes

during degradation

[71]

Phenylurea DGGE Structure and metabolic potentials of microbial communities affected by long-

term application of phenylurea

[72]

Phenanthrene TGGE Root exudates and phenanthrene inducedmodifications in bacterial communities

in polluted environments spatially

[73]

Polychlorinated biphenyl RT–PCR and 16S

rRNA sequencing

Representatives of two novel lineages detected [74]

Abbreviations: DGGE, denaturing gradient gel electrophoresis; RFLP, restriction fragment length polymorphism; RT–PCR, polymerase chain reaction with reverse

transcription; TGGE, thermal gradient gel electrophoresis; T-RFLP, terminal restriction fragment length polymorphism.

Review TRENDS in Biotechnology Vol.23 No.3 March 2005138

microorganisms that contribute to biodegradation [36].For example, comparisons of the gene pool betweencultivated and uncultivated bacteria that degradea-halocarboxylic acid have shown that Gram-positivebacteria account for a significant fraction of the totaldiversity. Owing to culturing bias, however, no Gram-positive a-halocarboxylic acid degraders have beenreported from studies based on enrichment techniquessince 1964 [37].

In another study, significant differences were foundbetween the community structure of experimental plots inwhich oil was deliberately spilt and that of control plots byusing phospholipids fatty acid analysis and denaturinggradient gel electrophoresis analysis [38]. The control plotswere devoid of a-proteobacteria, whereas the experimentalplots contained a wide range of a-proteobacteria and otherphyla. Similarly, the possible involvement of unculturablesoil microorganisms has been predicted in the degradationof a phosphonate herbicide, glyphosate [39]. These studiesindicate that culture-based methods elucidate only a smallfraction of the whole microbial diversity of soil; thus, it canbe argued that the soil metagenome remains an unexploredresource for novel genes and/or gene clusters that might beexploited for bioremediation.

To analyze the diversity of the pollutant-degradingpopulation in the environment, fragments of genes forcatabolic enzymes in degradation pathways and/or 16SrDNA have been amplified from environmental DNAsamples by PCR [34,40]. This approach requires priorinformation of the gene sequences, however, for the designof suitable primers. Metagenomics (the genomics of totalmicrobial DNA extracted directly from environments), bycontrast, circumvents this limitation because it involvescloning DNA directly from soil [33,40]. Large genomicfragments are cloned into bacterial artificial chromo-somes, fosmids and/or cosmid vectors such that thewhole metagenome is represented by a finite number ofscreenable clones [41].

Metagenomics has been successfully used to access thebiosynthetic diversity of microorganisms from variedenvironments by acquiring operons or genes involved insynthesis of commercially or biotechnologically importantcompounds such as lipases, cellulases, amylases,

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chitinases, esterases and enzymes involved in biotinsynthesis among others [41–45]. Moreover, throughmetagenomic analysis the genomes of uncultured organ-isms have been reconstructed by identifying overlappingfragments in metagenomic libraries and by ‘walking’,clone to clone, to assemble each chromosome [45,46]. Forexample, by using metagenomic profiling Henne et al. [47]have characterized the enzyme p-hydroxybutyrate dehy-drogenase, which is involved in the degradation ofp-hydroxybutyratem; genes encoding this enzyme hadbeen reported from only two other organisms before thisstudy. This result indicates that metagenomic profilingpromises enormous potential for identifying novelenzymes or pathways involved in the biodegradation ofpoorly and inefficiently degraded pollutants.

The scope of metagenomics can be further broadened byintroducing the desired genes into other hosts such aspseudomonads, which have active roles in the degradationof several pollutants. Efficient bacterial artificial chromo-some shuttle vectors are now available for the construc-tion of environmental libraries in hosts other thanEscherichia coli [40,41,48]. Recent advances in screeningfor biological activity have made the screening process ofup to 1 000 000 clones (representing a metagenomiclibrary) simpler, quicker and more sensitive [33]. Large-scale sequencing projects, such as that led by The Institutefor Genomic Research and Monterey Bay Coastal OceanMicrobial Observatory (http://www.tigr.org/tdb/MBMO/),are attempting to generate vast databases that, in turn,will expand the available metagenomic information onsome of the more obscure microbial metabolic potentials.These approaches will facilitate the construction ofefficient ‘designer biocatalysts’ for bioremediation andother biotechnological applications.

Genetically engineered organisms: in situ

bioremediation

The rational combination of catabolic segments fromdifferent organisms in one recipient strain is a usefulstrategy for developing bacteria with enhanced degra-dation capabilities [2,24]. In the present era of functionalgenomics, in which complete microbial genomes andthousands of catabolic operons are known, it is easy to

TRENDS in Biotechnology

Culture-dependent Culture-independent

Efficient degradative strain

cDNA

rRNAsequence

Strainidentification

Influence of pollutants on gene expression

Whole-genomesequencing

Library construction(plasmid, cosmid and BAC vectors)

Sequence-basedscreening

Expression basedscreeningStrain

identification

rDNAsequencing

RNA–SIP and FISH Microarray analysis

RNA

Isolation of catabolicoperon(s) for pollutant(s)

DNA sequence data for constructingmicroarrays and oligo probes

DNA

Contaminated environment

Enrichment culture Direct isolation of nucleicacids (metagenome)

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; RNA–SIP, RNA-based

stable isotopic probing.

Review TRENDS in Biotechnology Vol.23 No.3 March 2005 139

construct GEMs by shuffling genes and promoters toenhance their performance in situ. Several GEMs havebeen successfully constructed and experimentally provedto have higher degradative capability and utility forbioremediation under laboratory conditions [3,4,49]. Inspite of such studies, applying GEMs in situ has beenlimited by the risks associated with horizontal genetransfer and uncontrolled proliferation of the introducedGEMs [50,51].

On release, GEMs either get eliminated from theenvironment after performing their desired task or escapeelimination and subsequently evolve and proliferate. In thepastdecade, variousattemptshavebeenmade to restrict thesurvival of the GEMs to the environment in which theyperform the desired function in such a way that any cellescaping this environment will be killed by the induction ofa controlled ‘suicide’ system. Such constructs, termed‘bacterial containment systems’, have been designed byexploring the knowledge of various ‘killer’ genes andnumerous catabolic regulatory genes [52,53].

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Several killer genes and ‘killer–anti-killer’ genepairs have been shown to be associated with variousbacterial plasmids, chromosomes and bacteriophages.Cells harbor such systems to eliminate plasmid-freecells from the population of plasmid-bearing ones. Thekilling–anti-killing mechanism essentially involves theexpression of two different genes – the toxin or poisonand a respective antitoxin or antidote – whereby thekiller toxin is stable for hours but its anti-killingcounterpart is very labile. The antidotes (either anantisense RNA or a protein) either neutralize theircognate toxins (protein) or prevent their synthesis [54].The molecular mechanisms of toxin inactivation inplasmid-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 plasmidmaintenance is evident and promotes their applicationin the construction of GEMs for bioremediation. Forexample, hok, gef, sac, nuc, relF and colE3 are some of

Review TRENDS in Biotechnology Vol.23 No.3 March 2005140

the lethal genes that have been used to constructvarious containment systems [52,54].

Molin et al. [55] proposed the first containment systemin 1987, after which the concept of a suicidal system wasmodified by researchers to create a ‘fail-safe’ system forintroducing GEMs into the environment for bioremedia-tion purposes. A generalized scheme for constructing suchcontainment systems is shown in Figure 2a. In theseconstructs, the killer gene is negatively regulated; that is,it is switched off by default and is activated only when theconcentration of the pollutant falls below detectablelimits. Streptavidin-based systems have been sub-sequently constructed in which the gene encoding strep-tavidin (stv) is controlled by a promoter that is negativelyregulated by a repressor protein synthesized inresponse to the presence of environmental contami-nants. In the absence of the pollutant the cells producestreptavidin, which can bind D-biotin (a prostheticgroup for some essential cellular enzymes) therebycausing cell death [56]. The main disadvantage of thistype of system is that random mutations in the suicidegenes or control elements can enable GEMs to escapefrom programmed killing.

Regulator

Regulator

Regulator

Repressor

Regulator Repressor

Anti-killer

Anti-killer

Pollutant

Pollutant

(a)

(b)

Figure 2. Representation of bacterial containment systems designed for the safe introduc

regulation of the killer gene by environmental signals. In the presence of the pollutant, th

the killer gene followed by cell death. (b) Proposed containment system in which the

regulated by environmental signals. In the absence of the pollutant, synthesis of anti-kille

black arrows represent positive regulation, blue arrows represent constitutive regulatio

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Very recently, a dual lethal system has been con-structed in which colicin E3 RNase and EcoRI endo-nuclease (lethal genes) simultaneously control cell deathby regulating different cellular targets, namely, RNAand DNA, respectively. This system has been developedin an attempt to reduce the chances of ‘escape’ becausesimultaneous mutations in both enzymes seem improb-able [53].

The construction strategy of containment systems forbioremediation purposes requires further refinement todecrease the probability of cells escaping programmed celldeath. We therefore envisage a novel construct in whichthe killer gene product is produced constitutively andexpression of its cognate anti-killer gene is controlled byenvironmental signals (Figure 2b). Thus, only in thepresence of the pollutant will the anti-killer be producedand counteract the suicidal effects of the killer gene. In theabsence of the pollutant, cells will be rapidly killed,thereby avoiding any chance of proliferation and uncon-trolled growth of the introduced GEMs. In addition, theuse of a dual lethal system (two killer–antikiller pairs), inwhich one of the killer genes is placed on a plasmid and theother toxin and the antidote genes are places on the

TRENDS in Biotechnology

Killer

Killer

Killer

Killer

Death

Death

Survival

Survival

tion of GEMs into the environment. (a)Model containment system showing negative

e killer gene is repressed; however, depletion of pollutant results in derepression of

killer gene is constitutively expressed and its cognate anti-killer gene is positively

r ceases, which immediately leads to cell death. Block arrows represent promoters:

n and yellow arrows represent negative regulation.

Review TRENDS in Biotechnology Vol.23 No.3 March 2005 141

chromosome, should result in a tighter regulation ofsuicidal functions, thereby stimulating immediate celldeath in the case of plasmid-mediated horizontal genetransfer. In this type of construct, the presence of morethan one promoter–antidote fusion (i.e. promoters induc-ible by different pollutants) would facilitate its use forsites that are contaminated with a heterogeneous mixtureof pollutants.

Exploiting biodiversity for enhanced bioremediationtherefore involves the elucidation of (i) efficient or novelcatabolic pathways for improved detoxification, (ii) suit-able chemotactic or biosurfactant-producing strains toaccess insoluble or ‘aged’ pollutants easily for microbialdegradation, and (iii) regulatory circuits and genes forconstructing suicidal systems. Biotechnologists should,however, always emphasize the ethical responsibilitiesbefore using such novel strategies for bioremediation. Inaddition, before releasing such an organism, its geneticmodifications should be understood in full and it should bepossible 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 researchinto various strategies that might be used to clean up thecontaminated environment. Microbial metabolism is nowaccepted as a safer and efficient tool for the removal ofmany such organic pollutants; however, some of themore toxic and recalcitrant pollutants remain a challengefor environmental biotechnologists because they are notefficiently degraded by culturable microorganisms.

The remaining unculturable microbial diversity cantherefore be exploited to identify desirable degradativegenes and/or properties via the construction of meta-genomic libraries and to transfer them subsequently toculturable bacteria for enhanced bioremediation.Although the resulting GEMs are expected to haveincreased efficiency for bioremediation, they pose a threatto the environment because of the possibility of horizontalgene transfer and uncontrolled proliferation. The con-struction of containment systems is thus a prerequisite forthe release of GEMs in situ in order to resolve theseconcerns.

AcknowledgementsThe authors are supported, in part, by the Council of Scientific andIndustrial Research (CSIR), India, and the Department of Biotechnology(DBT), India. This is IMTECH communication no. 046/2004.

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