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Biotechnology and Molecular Biology Review Vol. 2 (1), pp. 001-013, February 2007
Available online at http://www.academicjournals.org/BMBR
ISSN 1538-2273 © 2007 Academic Journals
Standard Review
Molecular strategies of microbial adaptation to
xenobiotics in natural environment
Olusola Abayomi Ojo
Department of Microbiology, Lagos State University, Badagry Expressway, P.O. BOX 12142 Ikeja, Lagos, Nigeria. E-
mail: solayom@yahooo.com. Mobile: 234-8055055478.
Accepted 28 November, 2006
The unprecedented population increase and industrial development during the twentieth century has
increased conventional solid and liquid waste pollutants to critical levels as well as produced a range of
previously unknown strange synthetic chemicals for which society was unprepared. Increasing
pollution of the environment by xenobiotic compounds has provoked the need for understanding the
impact of toxic compounds on microbial populations, the catabolic degradation pathway of xenobiotics
and upgrade in bioremediation processes. Adaptation of native microbial community to xenobiotic
substrates is thus crucial for any mineralization to occur in polluted environment. Enzymes which
catalyze the biodegradation of xenobiotics are often produced by induction process and this
subsequently determine the acclimation time to xenobiotic substrates. Microbial degraders are adapted
to xenobiotic substrates via various genetic mechanisms that subsequently determine the evolution of
functional degradative pathways. The ultimate goal of these genetic mechanisms is to creating novel
genetic combinations in microorganisms that facilitates mineralization of xenobiotics. Moreover, recent
development of high-throughput molecular techniques such as polymerase chain reaction (PCR),
microarrays and metagenomic libraries have helped to uncover issues of genetic diversity among
environmentally relevant microorganisms as well as identification of new functional genes which would
enhance pollution abatement management in the twenty-first century.
Key words: Biodegradation, bioremediation, DNA, metagenomics, microarrays and xenobiotics.
TABLE OF CONTENTS
1. Introduction
2. Factors affecting xenobiotic degradation
3. Genes and degradation of aromatics
4. Strategies of adaptation to xenobiotics
4.1. Recombination and transposition
4.2. Gene duplication
4.3. Mutational drift
4.4. Gene transfer
5. New molecular techniques for detecting xenobiotic-degrader
5.1. Metagenomic libraries
5.1.1. Limitations of Metagenomics libraries
5.2. Microarrays
5.2.1. Limitations of Microarray technology
5.3. Complementary roles of function-driven and sequence-driven approaches
6. Conclusion
7. References
INTRODUCTION
Xenobiotics are chemically synthesized organic compo-
unds most of which do not occur in nature (Schlegel, 1995). Xenobiotics are defined as compounds that are
foreign to a living organism. Where these compounds are
002 Biotechnol. Mol. Biol. Rev.
not easily recognized by existing degra-dative enzymes,
they accumulate in soil and water (Esteve-Nunez et al.,
2001). Xenobiotics include fungi-cides, pesticides, herbi-
cides, insecticides, nematicides, and so on. Most of
which are substituted hydrocarbons, phenyl carbonates,
and similar compounds. Some of these substances of
which great quantities are applied to crops and soil are
very recalcitrant and are degraded only very slowly or not
at all. Therefore, the discovery of a new catabolic path-
way to complete mineralization of the pollu-tant would be
much desirable. Synthetic fibres like polyethylene and
polypropylene, though harmless, are practically non-
degradable. Whilst the plastizers and softeners contained
in textiles are gradually oxidized, the polymer skeleton
remains intact (Schlegel, 1995). Among the xenobiotics,
polyaromatic, chlorinated and nitro-aromatic compounds
were reported to be toxic, mutagenic and carcinogenic for
living organisms. However, microbial diversity and versa-
tility for adaptation to xenobiotics makes them the best
candidates among all living organisms to convey xeno-
biotic compounds into natural biogeochemical cycles.
Although, more microorganisms are being described as
able to degrade these anthropogenic molecules, some
xenobiotics have been shown to be unusually recalcitrant
(Esteve-Nunez et al., 2001).
The discovery of new catabolic pathways leading to
mineralization of this pollutant would be more valuable
and afford a better knowledge of the diversity of catabolic
pathways for the degradation of xenobiotics as well as
bring valuable information for bioremediation processes
(Black, 1999). The large majority of the earth’s micro-
organisms remain uncharacterized because of the ina-
bility to isolate and cultivate them on appropriate media.
Although, cultivation techniques are improving, the scien-
tific knowledge of their growth conditions in nature (that
is, chemistry of the original environment, life in complex
communities, obligate interactions with other organisms,
etc.) remains insufficient to cultivate most of these micro-
organisms (Leadbetter, 2003). This is particularly true in
complex biological systems like soils, where, despite a
huge bacteria diversity (up to 1010 bacteria and probably
thousands of different species per gram of soil) (Rosello-
Mora and Amann, 2001).Whereas less than 1% of bact-
eria has been cultured so far (Torsvik and Qvreas, 2002).
Several different molecular methods independent of
cultivation have been developed to explore the diversity
of microorganisms, cultivable or not, in natural environ-
ment.
Most of these methods are based on PCR amplification
and subsequent analysis of bacterial rRNA genes by
sequencing and fingerprint methods (clone libraries, res-
tricttion fragment length polymorphism (RFLP), random
amplified polymorphic DNA (RAPD), denaturing gradient
gel electrophoresis (DGGE, etc.). The discovery of many
new bacterial lineages and the reassignment to the most
ecologically significant group when using these methods
have led to a dramatic change in our perception of micro-
bial diversity and phylogenetic tree of life (Ward et al.,
1990; Amann et al., 1995).
Venter et al. (2004) using a cultivation-independent
molecular approach, found thousands of new bacterial
species and more than one million new protein-coding
genes in 2001 of Sargasso seawater. Thus, corroborating
the fact that there are millions of genes, uncharacterized
microorganisms and other protein-coding genes yet to be
discovered, thus, presenting a tremendous potential for
the discovery of new antibiotics, secondary metabolites
or xenobiotic degradation pathways. El–Fantroussi
(2000) also reported similar discovery while studying the
biodegradation of Linuron, a herbicide where the majority
of the microbial species involved in the biodegradation
were difficult to culture, but were detectable by denatu-
ring gradient gel electrophoresis (DGGE).
The development of species-specific oligonucleotide
primers and polymerase chain reaction (PCR) can now
be used as a confirmatory assay for microbial isolates,
since it’s highly sensitive if conducted with one of the
real-time technologies (Schaad et al., 1999). DNA array
technology, essentially a reverse dot blot technique, is an
emerging methodology useful for rapid identification of
DNA fragments and may be applicable for rapid iden-
tification and detection of plant pathogens (Levesque,
1997, 2001). An array of species-specific oligonucleotide
probes representing the various pathogens of potato,
built on a solid support such as a nylon membrane or
microscope slide, could be probed readily with labelled
PCR products amplified from a potato sample. This is
done by using conserved primers to amplify common
bacterial genome fragments from extracts of potato tu-
bers that might contain the bacterial pathogens, the
presence of DNA sequences indicative of pathogenic
species would be revealed by hybridization to species-
specific oligonucleotide probes within the array (Fesse-
haie, 2003). This has been done using conserved ribo-
somal primers and labelled simultaneously with digo-
xigenin-dUTP. Hybridization of amplicons to the array
and subsequent serological detection of digoxigenin label
revealed different hybridization patterns that were distinct
for each species and subspecies tested (Fessehaie,
2003).
These recently developed molecular methods have the
capacity to explore simultaneously the astonishing taxo-
nomic and functional variety among microorganisms. The
metagenomic libraries are constructed from the environ-
mental genome and clones are screened either for a
desired trait (“function –driven” approach) or for a specific
sequence (“sequence – driven’’ approach) (Schloss and
Handelsman, 2003). These molecular techniques would
enhance several aspects of environmental biotechnology,
spanning the spectrum from environmental monitoring
(Guschin et al., 1997) to bioremediation and biodegrad-
ation (Dennis et al., 2003).
The objective of this review is to emphasize the poten-
cy of application of new molecular biology techniques
such as polymerase chain reaction (PCR), micro-arrays
and metagenomic libraries for assessment of the genetic
diversity among environmentally relevant micro-organi-
sms and identification of new functional gene involved in
the catabolism of xenobiotics.
FACTORS AFFECTING XENOBIOTIC DEGRADATION
Microorganisms are ubiquitous; hence they have the
capacity to adapt to xenobiotic compounds as novel
growth and energy substrates. The pollution of the
environment with synthetic organic compounds has
become an issue of public health concern. Many harmful
synthetic organic compounds, which are slowly degra-
dable, have been identified; this includes halogennated
aromatics (such as benzenes, biphenyls and anilines),
halogenated aliphatics and several pesticides (Spain and
Van Veld, 1983). Several factors may be responsible for
the slow biodegradation of these compounds in the
natural environment; this may include unfavourable
physio-chemical conditions (such as temperature, pH,
redox potential, salinity and oxygen concentration),
presence of alternative nutrients, the accessibility of the
sub-strates, or predation (Goldstein et al., 1985). Slow
biode-gradation of xenobiotics may also occur due to
absence of genetic information coding for appropriate
catabolic enzymes or proteins in native microbiota.
However, microbial communities exposed to strange
synthetic organic compounds over a long period often
metabolize them completely (Rieger et al., 2002).
Although, adaptive acquisition of degradative abilities by
bacteria for some organic compounds or the resistance to
heavy metals induced after long acclimation period in
laboratory-simulated ecosystems has been observed
(Aelion et al., 1987). Acclimation to xenobiotics may be
due to;
(i) Induction of specific enzymes among microbial
community which enhanced the degradative capacity of
the entire community (Spain and Van Veld, 1983),
(ii) Development of a specific sub-population of microbial
community with capacity for co-metabolic process with
the main microbial population,
(iii) Adaptation can also be due to the selection of
mutants which acquired altered enzymatic specificities or
novel metabolic activities and which were not present at
the onset of the exposure of the community to the
introduced compounds (Barkay and Pritchard, 1988;
Timmis and Pieper, 1999).
Such a selective process (that is, induction, growth and
mutation) may be responsible for the adaptation
observed in mineralization of recalcitrant xenobiotics.
(Haigler et al., 1988; Timmis and Pieper, 1999).
Van der Meer et al. (1992) critically analyzed some of
these genetic mechanisms by which microorganisms ad-
Ojo 003
apt to xenobiotics that includes genetic recombination,
transposition, mutational drift and gene transfer. These
genetic strategies accelerate the processes of evolution
of catabolic pathways in bacteria. The analysis of seque-
nce information showed divergence of micro-organisms
isolated from geographically separated areas of the world
but harbouring the catabolic genes for xeno-biotics.
Haigler et al. (1988), Timmis and Pieper, (1999) attribu-
ted the genetically influenced selective process as being
the underlying principle behind mineralization of recalci-
trant halogenated aromatics.
GENES AND DEGRADATION OF AROMATICS
Aromatic compounds carrying substituents forms a spe-
cial class of xenobiotics because of their recalcitrance.
Often reported are the aerobic processes of mineraliza-
tion whereas anaerobic processes of biode-gradation do
occur in natural environment (Reineke, 1984; Becker et
al., 1999). A general comparison of the major pathways
for catabolism of aromatic compounds in bacteria has
revealed that the initial biotransformation steps are medi-
ated by different enzymes that subse-quently produce
limited number of central intermediates such as proto-
catechuates and substituted cathecols (Reineke, 1984).
These dihydroxylated intermediates are channelled into
either a ‘meta cleavage or ortho cleavage’ pathway
(Haigler et al., 1988).The ortho cleavage pathways are
involved in the degradation of catechol and protocatechu-
ate (Doten et al., 1987). Moreover, the enzymes involved
in the mineralization of chlorocathecols (that is, substi-
tuted catechols) have wider substrates specificities;
hence they are rather referred to as the modified ortho
cleavage pathway (Figure1). This parti-cular pathway has
been detected in Pseudomonas sp. Strain B13; Alcali-
genes eutrophus JMP134 among many others which
metabolize chlorinated benzenes (Haigler et al., 1988).
The Modified ortho cleavage pathway genes for three
bacteria species were extensively studied:
(i) The clc ABD operon of Pseudomonas putida (pAC27)
(Ghosal and You, 1989).
(ii) The tfdCDEF operon of A. eutrophus JMP134 (Pjp4)
(Don et al., 1985).
(iii) The tcbCDEF operon of Pseudomonas sp. strain P51
(pP51) (Van der Meer et al., 1991).
The outcome of these studies and many others
corroborated the fact that the genes for the modified
ortho cleavage pathways are generally located on cata-
bolic plasmids and their organization into operon struc-
tures was contrary to that of the chromosomally encoded
cat and pca genes (Don et al., 1985).The cat and pca
genes encodes for the ortho cleavage pathway
enzymes, they are located on the chromosomes (Doten
et al., 1987; Neidle and Ornston, 1986).
004 Biotechnol. Mol. Biol. Rev.
STRATEGIES OF ADAPTATION TO XENOBIOTICS
The spontaneous occurrence of DNA rearrangements in
xenobiotic-degraders that resulted in evolution of different
pathways for mineralization of synthetic compounds in
natural environment is one of the principal mechanisms
of adaptation to xenobiotic substrates. The evolution of
catabolic pathways (that is, modified ortho cleavage path-
way, meta cleavage pathway and others) in micro-organi-
sms for xenobiotic substrates often involves different
gene clusters encoding for the aromatic path-way enzy-
mes.
RECOMBINATION AND TRANSPOSITION
Recombination is the combining of genes (DNA) from two
or more different cells. This is principally based on
molecular methods involving cutting of DNA fragments
from different cells harbouring desired catabolic traits.
These DNA fragments through hybridization in host
cells that are known as recombinants, is seeded onto
polluted environment where the expression of the cata-
bolic trait is desired (Black, 1999). This strategy is often
practiced in vitro than in vivo. El-Fantroussi (2000) in the
soil enrichment method for the degradation of an herbi-
cide (Linuron) engaged a modified strategy which can be
extended for bioremediation process in soil polluted by
this herbicide.
In another biodegradation involving the use of Acine-
tobacter and Pseudomonas species, DNA rearrangement
strategy was used to achieve mineralization. The orders
of the genes encoding the ortho cleavage pathways of
Acinetobacter calcoaceticus and P. putida differ from one
another and from those of other organisms, suggesting
that various DNA rearrangements have also occurred
(Van der Meer et al., 1991). Gene rearrangements are
also evident even between the different operons for the
modified ortho pathways enzymes (Figure 1). There are
as yet no clear indications of what mechanisms may
direct these rearrangements. Rearrangement of DNA fra-
gments is believed to be due to insertion elements which
subsequently enhance gene transfer as well as activation
or inactivation of silent gene (Tsuda et al., 1989).
GENE DUPLICATION
This is an important mechanism for the evolution of
different strains of microorganisms of the same species.
Once a gene becomes duplicated, the extra gene copy
thus becomes independent of selective pressures and
subsequently imbibes mutations with speed. These muta-
tions could eventually lead to full inactivation, rendering
this copy silent. Reactivation of the silent gene copy
could then occur through the action of insertion elements.
This occurred in Flavobacterium sp. Strain K172 that pro-
duced two isozymes of 6-aminohexanoate dim-mer
hydrolase, one of the enzymes involved in the degra-
dation of nylon oligomers (Okada et al., 1983).
MUTATIONAL DRIFT
Mutational drift in terms of point mutation is of much rele-
vance in xenobiotic degradation. It is possible that a
number of stress factors such as chemical pollutants in-
duce error-prone DNA replication that subsequently acce-
lerates DNA evolution.
Point mutation involves base substitution, or nucleotide
replacement, in which one base is substituted for another
at a specific location in a gene (Black, 1999). This kind of
mutation changes a single codon in mRNA, and it may or
may not change the amino acid sequence in a protein.
Several examples have illustrated that single-site-muta-
tions can alter substrate specificities of enzymes or
effector specificities. Clarke (1984) isolated mutants with
altered substrate specificities of the AmiE amidase of
Pseudomonas aeruginosa, which were provoked by
single-base-pair changes. Sequential mutations in the
cryptic ebg genes of Escherichia coli were shown to
result in active enzymes capable of metabolizing lactose
and other sugars.
Single-site-mutations are believed to arise continuously
and at random as a result of errors in DNA replication or
repair. Although, the important effects of single-base-pair
mutation on the adaptive process have been demon-stra-
ted experimentally, the accumulation of the single-base-
pair changes may not be the main reason for the differ-
ences in the properties of the catabolic enzymes elicited
by xenobiotic-degraders. There are other factors that
would precipitate changes in DNA sequences, this inclu-
ded gene conversion or slipped-strand mispairing (Niedle
et al., 1988).
GENE TRANSFER
Gene transfer is a process of movement of genetic infor-
mation between organisms (Black, 1999). The impor-
tance of gene transfer for adaptation of host cells to new
compounds has been explicitly demonstrated in many
studies on experimental evolution of novel meta-bolic
activities. Such studies identified biochemical block-ades
in natural pathways that prevented the degradation of
novel substrates and these barriers scaled by trans-
ferring appropriate genes (Reineke et al., 1982).
Genetic interactions in microbial communities are effe-
cted by several mechanisms such as conjugation via
plasmid replicons, transduction and transformation (Saye
et al., 1990). The occurrence of plasmids in bacteria in
the natural environment is certainly a general phenol-
menon and an important pool of genetic information resi-
ding on plasmid vehicles may flow among indigenous
organisms. The self transmissible plasmids that carry
Ojo 005
Figure 1. Modified ortho cleavage pathway. Adapted from Ferraroni et al. (2004).
genes for degradation of aromatic or of other organic
compounds are known and their roles in spreading these
genes to other organisms is predictable (Assinder and
Williams, 1988).
Although, the transfer of catabolic plasmids can lead to
regulatory and / or metabolic problems for the cells and
therefore additional mutations in the primary transconju-
gants are often needed to construct strains with the desi-
red metabolic activities (Reineke et al., 1982).
NEW MOLECULAR TECHNIQUES FOR DETECTING
XENOBIOTIC- DEGRADER
There are many microorganisms in natural environment
006 Biotechnol. Mol. Biol. Rev.
that can degrade biphenyls, halogenated aromatics, nap-
hthalene and xylenes. However, the assessment of the
distribution of these microorganisms exhibiting specific
genetic traits has been handicapped due to the fact that a
large proportion are not culturable and some genes are
latent.The discovery of DNA-DNA hybridization technique
that is a relatively novel experimental app-roach in envi-
ronmental biotechnology provided the solution to the
problems of culturability and gene expression among
microorganisms (Sayler and Layton, 1990; Leadbetter,
2003). Fessehaie et al. (2003) obtained oligonucleotides
from bacteria pathogenic on potato which he designed
and formatted into an array by pin spotting on nylon
membranes. Genomic DNA from bacterial cultures was
amplified by polymerase chain reaction (PCR) using
conserved ribosomal primers and labeled simultaneously
with digoxigenin-dUTP. Hybridization of amplicons to the
array as well as detection of digoxigenin label showed
different hybridization patterns that were distinct for each
species and subspecies tested. DNA array technology is
essentially a reverse dot blot technique useful for identi-
fication of DNA fragments and this was applied for rapid
identification and detection of bacteria pathogenic on
potato. An array of species-specific oligonucleotide pro-
bes representing the various pathogens of potato was
constructed on a solid support (that is, nylon memb-
ranes). This was probed with labelled PCR products
amplified from a potato sample, mean-while, conserved
primers to amplify common bacterial genome fragments
from extracts of potato tubers that had previously been
infected by different bacterial pathogens was generated.
The presence of DNA sequences indicative of patho-
genic species would be shown by the hybridization to
species-specific oligonucleotide probes within the array.
This discriminatory technology identifies genomic DNA
fragments of bacterial pathogens via the distinct species-
specific hybridization patterns shown using the gel elec-
trophoresis. Specific DNA sequences of native micro-
organisms could also be detected in environmental sou-
rces by hybridization with probes after amplification of
those sequences using PCR technique (Bej et al., 1990;
Weisburg et al., 1991; Eyers et al., 2004). Theoretically,
the use of conserved DNA sequences in a gene family as
universal primers in polymerase chain reaction amplifica-
tion and consequent cloning of the amplified fragments
would facilitate the detection and isolation of a wider
variation of genotypes from the environment (Van der
Meer et al., 1991). Weisburg et al. (1991) used this
method for the characterization of variations in 16S rRNA
genes from microorganisms in natural communities.
METAGENOMIC LIBRARIES
Metagenomics is the culture-independent genomic analy-
sis of entire microbial communities (Schloss and Handel-
sman, 2003). In other words, metagenomics provides
access to the pool of genomes of a given environment.
While direct genomic cloning gives access to retrieve
unknown sequences or functions in a given ecosystem
that may be used for the design of primers. The PCR
amplification requires prior knowledge of the sequences
of genes for the design of primer. Total genomic DNA is
extracted from the environment (Figure 2A) before meta-
genomic libraries can be constructed. The genomic DNA
is enzymatically or mechanically fragmented. Fragments
can be separated on the basis of their size by pulsed
field gel electrophoresis (PFGE). This methodology per-
mits fragments of an appropriate size to be isolated from
the gel and inserted into host cell by cloning vectors
(bacteriophage lambda, cosmid, fosmid or bacterial artifi-
cial chromosome (BAC) vectors). BAC vectors are quite
efficient in maintaining stably large DNA inserts (up to
300 kb) in low copy numbers in the host cells (1-2 per
cell) (Shizuya and Simon, 1992; Rondon et al., 2000).
Metagenomic libraries can be screened for functional and
/ or genetic diversity. New catabolic genes for the degra-
dation of xenobiotics are discovered via the “functional
approach”. Clones are screened for a desired trait on
appropriate media. For example, haloaromatic com-
pounds could be used as sole electron acceptors since it
has been reported that bacteria can metabolize them.
(El-Fantroussi et al., 1998; Van de Pas et al., 2001).
Whereas polyaromatic hydro-carbons could be utilized as
sole C-source and energy-source, this often occurs after
several years of adaptation that has led to a selection of
a bacterial consortium cap-able of completely minerali-
zing such compounds. The biodegradation of linuron, a
commonly used herbicide was monitored by enrichment
process, using reverse transcription-PCR and denaturing
gradient gel electrophoresis (DGGE), it was revealed that
a mixture of bacterial species was involved in the mine-
ralization. Although, these bacterial species appear to be
difficult to culture since they were detectable by DGGE
but were not cultivable on agar plates (El-Fantroussi,
2000).
Consequently, growth measurements could identify
clones bearing catabolic genes. This function-driven
screening remains a straightforward and successful met-
hod for the discovery of catabolic genes, as opposed to
inferring the function of cloned genes by searching for
homologous sequences available in database (Rondon
et al., 2000). Indeed, sequences coding for important
metabolic functions are frequently poorly conserved,
making the comparison of clone sequences with homolo-
gous ones very difficult. This functional screening appro-
ach has been successfully used to identify novel and
previously undescribed genes coding for antibiotics, lipa-
ses, enzymes for the metabolism of 4-hydroxybutyrate
and genes encoding biotin synthetic pathways (Schloss
and Handelsman, 2003). Most of the catabolic genes
known to date were isolated from cultivable microorg-
anisms (Table 1) (Eyers et al., 2004). However, no
genes for TNT denitration have been discovered up till
now. But high denitration activities were obtained under
Ojo 007
Table 1. Properties of selected identified genes involved in degradative pathways of recalcitrant substituted aromatic compounds.
Target compound
Function Name Target metabolite in pathway Mode of isolation Size Microorganism of origin
References
Naphthalene,
Toluene,
Anthracene
Polycyclic aromatic
hydrocarbon(PAH)
Reductase
NL1 PAH Genomic library expressed in
Sphingomonas strains 160 –
195kb Sphingomonas
subterranean, S.
aromaticivorans
F199,B0695
S. xenophaga
BN6, S. sp. HH69
Basta et al., (2005)
2-Nitrotoluene Reductase
Ferrodoxin
Iron-sulphur protein
Iron-sulphur protein
ntdAa
ntdAb
ntdAc
ntdAd
2-Nitrotoluene Genomic library expressed in
E. coli and screening for
metabolic activities
4.9 kb Pseudomonas sp. JS42 Parales et al., (1996)
2,4-Dinitrotoluene Dioxygenase
Monooxygenase
Dioxygenase
Isomerase/hydrolase
Dehydrogenase
dntA
dntB
dntD
dntG
dntE
2,4-Dinitrotoluene
4-Methyl-5-nitrocatechol
2,4,5-Trihydroxytoluene
2,4-Dihydroxy-5-methyl-6-oxo-2,4-
hexadecanoic acid
Methylmalonic acid semialdehyde
Genomic library expressed in
E. coli and screening for
metabolic activities
27 kb Burkholderia cepacia R34 Johnson et al., (2002)
2,4,6-Trinitrophenol
Hydride transferase
Hydride transferase
NADPH reductase
npdI
npdG
npdC
2,4,6-Trinitrophenol
Hydride-Meisenheimer complex of 2,4,6-
trinitrophenol
2,4,6-Trinitrophenol and its hydride-
Meisenheimer
Genomic library and selection
of clones thanks to mRNA
differential display experiments
12.5kb
Rhodococcus erythropolis
HL PM-1 Walters et al.., (2001)
;
Heiss et al., (2002)
2,4,6-
Trinitrotoluene Reductase xenB 2,4,6-Trinitrotoluene Genomic library expressed in
E. coli and screening for
metabolic activities
1.05kb
P. fluorescens I-C Blehert et al,., (1999)
2,4,6-Trinitrophenol
NADPH F420
Reductase
Hydride transferase II
npdG
npdI 2,4,6-Trinitrophenol
Hydride-Meisenheimer complex of 2,4,6-
trinitrophenol
Genomic library and selection
of clones*** 12.5kb
Rhodococcus erythropolis
HL PM-1
Heiss et al., (2003)
008 Biotechnol. Mol. Biol. Rev.
Table 1. contd.
4-
Nitrotol
uene
Monooxygenase
Nitrobenzyl alcohol
dehudrogenase
Nitrobenzadehyde
dehudrogenase
Nitrobenzoate reductase
Hydroxylaminobenzoate lyase
ntnMA
ntnD
ntnC
pnbA
pnbB
4-Nitrotoluene
4-Nitrobenzyl alcohol
4-Nitrobenzadehyde
4-Nitrobenzoate
4-Hydroxylaminobenzoate
Genomic library screened with designated probes;
activities confirmed by cloning and expression in
Escherichia coli.
Genomic library expressed in P. putidaPaW340 and
screening for metabolic activities
14.8kb
6 kb
Pseudomonas sp. TW3
Pseudomonas sp. TW3
James and
Williams
(1998); James
et al. (2000)
Hughes and
Williams
(2001)
defined conditions with a TNT-contaminated soil sample
(Eyers et al., 2004). Further more, the presence of a speci-
fic bacterial consortium in this polluted soil was demon-
strated by DGGE (Eyers et al., 2004).Therefore, metageno-
mic libraries are particularly promising for identifying deni-
tration genes, compared with methods based on isolation of
pure cultures.
Comparatively, the sequence–driven approach of the
metagenomic libraries is based on conserved regions in
microbial genes. Clone libraries are screened for specific
DNA sequences by means of hybridization probes and
PCR primers, whose design is based on the information
available in databases. Such hybridization probes may
use, in the case of denitration of 2,4,6-trinitrophenol or
other electron-deficient aromatics, the npdG and npdI
sequences of Rhodococcus erythropolis HL-PM1(Table
1), as homologous sequences that have been identified
in other nitroaromatic compounds-degrading Rhodococ-
cus strains (Heiss et al., 2003). These sequences are
clustered separately from the related enzymes. Hence, it
was suggested that they may be suitable as genes pro-
bes for finding bacteria in the environment with the ability
to “hydrogenate” electron-deficient aromatic ring systems
(Heiss et al., 2003).
LIMITATIONS OF METAGENOMIC LIBRARIES
Although, metagenomic libraries constitute at present the
most powerful tool to assess the functional diversity of
natural microbial communities, they do not cover geno-
mes of low abundance in complex environment like soils,
which can be responsible for an important degradation or
related process. Hence, the frequency of clones of a
desired nature in a library can be very low. This implies
screening thousands of clones, which can be laborious
and time-consuming. High- throughput equipment is now
available to facilitate colony picking, inoculation in micro-
titre plates and screening numerous clones at the same
time. However, an enrichment strategy is required before
the construction of the library to select a specific feature
and in that way ensure better cover for a subset of the
community (Entcheva et al., 2001). Communities enrich-
ed naturally by long-term exposure to high concentrations
of xenobiotics may host the genes of interest at a high
frequency. In this context, metagenomics might shorten
the time required to understand the genetics of degra-
dation.
Moreover, the catabolism of specific xenobiotics may
be achieved by two or more bacteria via co-metabolic
process (Abraham et al., 2002). In this case, it is not
possible to isolate a contiguous piece of DNA containing
all the genes involved in the catabolic pathway. There-
fore, Schloss and HandeIsman (2003) suggested study-
ing multiple clones simultaneously on liquid media in
which substrates and products can diffuse freely among
members of the mixture. Finally, it is important for func-
tional screening that catabolic genes are effectively expr-
Ojo 009
essed. Hence, another challenge lies in choosing an
appropriate host of expression. Moreover, the host has to
be both relatively insensitive to the toxic xenobiotic and
unable to catabolize it in the absence of the vector.
MICROARRAYS
DNA chip is an emerging technique making it possible to
analyze hundreds and even thousand of genes at the
same time, thus getting away from the ‘’one gene at a
time’’ analysis. This allows extremely small amount of
biomolecules (RNA, cDNA, etc) to be printed at high den-
sity on a substrate. Comparatively, microarrays present
the advantage of miniaturization (thousands of probes
can be spotted on a chip), high sensitivity and rapid
detection which is not obtainable with the traditional
nucleic acid membrane hybridization. Zhou and Thomp-
son (2002) has reported that microarray –based genomic
technologies would revolutionize the analysis of microbial
community structure, function and dynamics. DNA micro-
arrays are coated glass microscope slides onto which
thousands of target DNA sample are spotted in a precise
pattern. There are two principal microarrays types based
on the nature of the target (DNA, e.g., cDNA, PCR
products, oligonucleotides or plasmids, or RNA) and on
the method of spotting (mechanical microspotting or
photolithography). In the first method, purified DNA is
spotted onto the membrane or coated glass slide.
Although, DNA will stick to glass, aminosilane-coated and
poly-L-lysine (PLL)-coated slides are predominantly used.
Silanized slides (RSiX3, where X is typically alkoxy)
attach the DNA by forming covalent bonds between pri-
mary amines on the surface and the phosphate backbone
(Celis et al., 2000; Ye et al., 2001). The photolithography
method uses an ultraviolet light source that passes throu-
gh a mask where a photochemical reaction takes place
on a siliconized glass surface (Affymetrix). This is by far
the most efficient method of generating high-density
oligonucleotide chips, but has practical limitations in
terms of fragment length and affordability (Kumar et al.,
2000). After printing the microarrays, therefore, extraction
of DNA or messenger RNA from pure cultures or the
environment (Figure 2B), labelling it with specific fluore-
scent molecules and hybridizing it to target DNA spotted
on the glass slide. The resulting image of fluorescent
spots is visualized by confocal laser scanning and it’s
digitized for quantitative analysis.
Fessehaie et al. (2003) designed and formatted into an
array by pin spotting on nylon membranes, genomic
DNA from bacterial cultures which was amplified by PCR
using conserved ribosomal primers and labelled simulta-
neously with digoxigenin –dUTP. Hybridization of ampli-
cons to the array and subsequent serological detection
distinctly confirmed the identity of each species of potato
pathogens within the mixed cultures and inoculated
potato tissues.
010 Biotechnol. Mol. Biol. Rev.
Figure 2. (A). Construction and analysis of metagenomic libraries from sampled ecosystems. (B). Construction of microarrays
and hybridization with samples from the environment or pure cultures.
DNA microarrays is applied in research for gene
expression profiling, that is, identification of changes in
mRNA expression of strains exposed to a particular subs-
trate, for example, a specific xenobiotic. Schut et al.
(2001) constructed a DNA microarray with probes target-
ing 271 open reading frames (ORFs) from the genome
sequence of the hyperthermophile Pyrococcus furiosus.
When this strain was grown with elemental sulphur (S),
two previously uncharacterized operons were identified
and their products were proposed to be part of a novel S-
reducing, membrane-associated, iron-sulphur cluster-
containing complex. DNA microarray was also used to
assess the mRNA expression levels in Bacillus subtilis
grown under anaerobic condition (Ye et al., 2000). Trans-
criptional activities of more than 100 genes affected by
the oxygen–limiting conditions were identified (Ye et al.,
2000). Microarray has also been used for physiological
studies of environment samples. Wu et al. (2001)
detected genes involved in nitrogen –cycling (nirS) using
1ng of labelled genomic DNA of a Pseudomonas stutzeri
strain and 25 ng of bulk community DNA extracted from
soil samples. Catabolic genes involved in degradation of
xenobiotics were assessed with microarrays by Dennis
et al. (2003).
In microarray technology, sequence information is
needed to design probes. However, this approach cannot
be applicable for discovering new catabolic genes for
which no sequences are available in databases. More-
over, knowledge of the entire sequence is not necessary
for the construction of microarrays; and PCR products of
a random genomic library constructed from a microorg-
anism of interest may be used. It is expected that to a
toxic substrate, differential gene expression would result
at the transcript level. This is reflected by differential
hybridization patterns in the presence or absence of the
toxic pollutant. Afterward, clones of the library associated
with differentially hybridized probes can be picked up for
sequencing. This methodology applied in identifying
genes involved in N2 –fixation in Leptospirillum ferrooxi-
dans by Parro and Moreno-Paz (2003). Sebat et al.
(2003) used metagenomes as microarrays by deriving
cosmid library from a microcosm of groundwater and
used this library as probes for microarrays. Afterwards,
they hybridized the microarrays with cDNA of individual
strains isolated from the microcosm and cDNA of the
microcosm itself. Comparisons of the hybridization
profiles of the microcosm with isolated strains, clones
were identified in the library corresponding to uncultured
members of the microcosm. Sequencing of these clones
revealed ORFs assigned to functions that have potential
ecological importance, including hydrogen oxidation,NO3-
reduction and transposition (Sebat et al., 2003).
Therefore a random genomic library approach associa-
ted with microarrays offers a high potential for the
discovery of novel genes and operons. This is desirable
since it offers additional information apart from the biolo-
gy of the microorganisms but precise knowledge of bio-
Ojo 011
degradation processes of xenobiotics for application in
pollution control and prevention under field conditions.
LIMITATIONS OF MICROARRAY TECHNOLOGY
Presence of humic matter, organic contaminants and
metals in environmental samples that may interfere with
RNA and DNA hybridization (Zhou and Thompson,
2002). Possibility of extraction of undegraded mRNA is
additional problem (Burgmann et al., 2003). In cases of
sequences of poor abundance, microarrays are not supe-
rior in sensitivity to PCR that is 100 to 10,000 fold more
than that of microarrays (Zhou and Thompson, 2002).
The success of the application of microarray technology
in a study lies in the possibility of determining, in complex
environments, the relative abundance of a microorganism
bearing a specific functional gene. Therefore, it is
important to be able to differentiate between differences
in hybridization signals due to population abundance from
those due to sequence divergence (Wu et al., 2001).
COMPLEMENTARY ROLES OF FUNCTION-DRIVEN
AND SEQUENCE-DRIVEN APPROACHES
In the case of metagenomic libraries, sequencing clones
with interesting functional properties may reveal a seque-
nce that can be used to confirm the phylogenetic
affiliation of the organism from which the DNA was isola-
ted. Where the sequencing clones harbours an rRNA
sequence in a big fragment that can lead to functional
information about the microorganism from which the
fragment originated (Beja et al., 2000; Liles et al., 2003;
Quaiser et al., 2003). Environmental systems often con-
tain a high diversity of bacteria, the use of labelled xeno-
biotics can provide information about active bacteria
within a complex environmental system such bacteria
incorporates radio labelled atoms into their DNA, making
it denser than non-labelled DNA. By centrifugation, sepa-
ration of labelled from non-labelled DNA can be achieved
(Radajewski et al., 2000) and thereby distinguish the
bacteria involved in the catabolic process from those
which are not. This labelled DNA can be used afterwards
to construct metagenomic libraries. This forms an excel-
lent method for finding clones carrying catabolic genes.
Conclusion
It is clear that polluted sites, particularly those that are or
become sources of contamination of surface and ground-
water, have to be remediated. Bioremediation can be a
cost-effective and ecologically suitable alternative to
physical methods.
The genetic characterization of an increasing number
of aerobic pathways for degradation of (substituted) aro-
matic compounds in different bacteria has made it
possible to compare the similarities in genetic organizat-
ion and in sequence, which exist between genes and pro-
012 Biotechnol. Mol. Biol. Rev.
teins of these specialized catabolic routes and more
common pathways. Sequence information provided the
scientific evidence of the occurrence of catabolic genes
coding for specialized enzymes in the degradation of
xenobiotic chemicals. Moreover, molecular biology evi-
dences corroborated the fact that a range of genetic mec-
hanisms, such as gene transfer, mutational drift, genetic
recombination and transposition can accelerate the
evolution of catabolic pathways in microorganisms.
Metagenomic libraries open access to the world of
uncultivated microorganisms and their undescribed cata-
bolic genes for the degradation of xenobiotics. Micro-
arrays are also useful for the discovering new catabolic
genes as well as provide the opportunity of easily moni-
toring catabolic genes. In both approaches, the use of
radio-labelled molecules could improve the recovery and
identification of microorganisms involved in biodegrada-
tion of xenobiotics. The technical challenges associated
with metagenomic libraries and microarrays not withstan-
ding, these methods presents an exceptional opportunity
for discovering the scientific basis of microbial degrada-
tion of xenobiotics. The application of both metagenomic
libraries and DNA microarrays in bioremediation process-
es will facilitate rapid transfer of genetic information
between axenic and native microbial communities, thus
enhancing microbial adaptation to metabolism of xeno-
biotics in the environment.
In order to minimize future environmental impact by
xenobiotics it may be economical to develop new synthe-
tic compounds that fit in the naturally existing catabolic
potential of the microorganisms.
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